Nitroxide reduction with ascorbic acid in spin labeled human plasma LDL and VLDL

Chemistry and Physics of Lipids 85 (1997) 1 – 12

Nitroxide reduction with ascorbic acid in spin labeled human plasma LDL and VLDL Marina Kvedera,*, Greta Pifata, Slavko Pec' arb,c, Milan Scharab, Pilar Ramosd, Hermann Esterbauerd a

Ru/er Bos' ko6ic´ Institute, P.O. Box 1016, 10001 Zagreb, Croatia b J. Stefan Institute, Ljubljana, Ljubljana, Slo6enia c Faculty of Pharmacy, Uni6ersity of Ljubljana, Ljubljana, Slo6enia d Institute of Biochemistry, Uni6ersity of Graz, Graz, Austria Received 20 May 1996; revised 27 September 1996; accepted 2 October 1996

Abstract The LDL and VLDL were spin labeled with Tempo which partitions both in the aqueous and lipid phase. The ESR spectra were measured in the equilibrium state as well as during the reduction of the spin label with ascorbic acid. The kinetics of the concentration decay curves was parametrized with two exponentials. The theoretical simulation of the experimental spectra revealed a drastic linewidth narrowing in the VLDL samples exposed to the ascorbic acid. Since the transport properties of the specific monolayer are reflected in the observed reaction rates, the analysis of the fatty acid composition of phospholipids, triglycerides and cholesterol esters in LDL and VLDL was performed. It is concluded that different lipid packing at the surface of LDL and VLDL might be the consequence of different intermolecular forces between phospholipids and cholesterol. This finding was connected to the experimentally detected different reaction kinetics in LDL and VLDL as well as their different susceptibility to the ESR linebroadening effects during the nonequilibrium conditions of the spin label reduction with ascorbic acid. Copyright © 1997 Elsevier Science Ireland Ltd. Keywords: ESR; Lipoproteins; LDL; VLDL; Composition of fatty acids

1. Introduction One of the central questions in studying the structure and function of biological membranes * Corresponding author. E-mail: [email protected].

considers the lipid distribution and the potential of lipid-protein interactions. Due to not ideal mixing of the lipid molecules, the phenomenon of domain formation and thus a nonrandom arrangement of membrane components has been postulated with possible functional implications

0009-3084/97/$17.00 Copyright © 1997 Elsevier Science Ireland Ltd. All rights reserved PII S 0 0 0 9 - 3 0 8 4 ( 9 6 ) 0 2 6 3 6 - 9

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(Welti and Glaser, 1994; Marsh, 1995). Although a transverse and lateral regionalization with respect to the molecular organization has been recognized, it is still not clear why biological membranes contain such a diversity of lipid species. If the single role of lipids were to maintain the fluidity and assembly of the membrane, such a variety of lipids would not be required. The involvement of lipids in providing a particular environment for the specific membrane functions is currently under extensive investigation (Tocanne et al., 1994). Especially, the influence of different phospholipid composition with respect to the presence/length of unsaturated acyl chains is in the focus of interest (Slater et al., 1993; Abadji et al., 1994; Mitchell and Litman, 1994; Barry and Gawrisch, 1995; Mattjus et al., 1994). In order to gain full control over the composition of constituents, the experiments are performed on model membranes such as multilamellar dispersions of lipids or vesicles. The model bilayer membranes are routinely prepared (Lasic, 1993), whereas monolayer membranes at the gas/aqueous or aqueous/lipid interface have been introduced only for specific studies (Slotte and Mattjus, 1995). Human plasma lipoproteins exhibit interesting structural complexity with the surface organization of molecules resembling that of the monolayer which surrounds the hydrophobic core of the particles (Gotto, 1987). Different research studies have been initiated after recent suggestions that lipoprotein modifications are essential in the development of pathological process of atherosclerosis. Since the detailed structure of the native lipoproteins still lacks thorough understanding, in this work low density lipoproteins, LDL, and very low density lipoproteins, VLDL, were studied in the native form. These two lipoprotein subpopulations were spin labeled with Tempo which partitions between the hydrophobic lipoproteins and the aqueous environment. The ESR spectroscopy in the equilibrium as well as during the nonequilibrium conditions (Schara et al., 1990) was combined and the experimental evidence for different properties of the surface molecular organization in LDL and VLDL have been observed.

2. Experimental procedures

2.1. Materials Tempo (2,2,6,6-tetramethylpiperidine-1-oxyl) was synthesized by one of the authors (P.S.) according to the published procedure (Rauckman et al., 1975). Chromium oxalate was prepared according to Berg and Nesbitt (1979). Ascorbic acid was purchased from Pliva Chemical Company and was dissolved in buffer just before the start of each ESR experiment. All dilutions as well as lipoprotein samples were prepared with 0.1 mol/l Tris–HCl buffer (pH 7.4) which contained 1 g/l EDTA except chromium oxalate which was dissolved in distilled water in order to match the physiological osmolarity.

2.2. Isolation of lipoproteins Human plasma was obtained from normolipidemic donors and lipoproteins were isolated according to the published procedures (Ju¨rgens et al., 1987; Puhl et al., 1994). To avoid the oxidation of lipoproteins the EDTA was present in all steps of preparation and all buffers were flushed with nitrogen. The purity of the lipoprotein samples was checked by electrophoresis. The concentrations of lipoprotein solutions were determined gravimetrically by dry weight estimation including the correction for the salt content of the buffer.

2.3. The compositional analysis of lipoproteins Protein quantification of VLDL and LDL was performed using the BCA assay (Pierce, Illinois). Total and free cholesterol in lipoproteins were determined with the CHOD-PAP enzymatic test kit (Boehringer-Mannheim, Germany). Cholesterol ester was calculated as (total cholesterol− free cholesterol)× 1.67. This factor represents the ratio of the average molecular weight of cholesterol ester to free cholesterol. Phospholipids were assayed by an enzymatic kit test (BoehringerMannheim, Germany) and the triglycerides with

M. K6eder et al. / Chemistry and Physics of Lipids 85 (1997) 1–12

the GPO-PAP enzymatic Mannheim, Germany).

test

(Boehringer-

2.4. Fatty acid composition of lipoproteins The analysis of fatty acid composition of phospholipids, triglycerides and cholesterol esters in VLDL and LDL was performed according to the published procedure (Sattler et al., 1991). Briefly, the lipoprotein solution was freeze dried prior to transesterification with BF3/methanol and after extraction, separated by capillary gas chromatography (Hewlett Packard 5890 serie II) using a 30 m ×0.25 mm I.D. D.B-23 column with a film thickness of 0.25 mm and a cyanopropyl/ polysiloxane phase. Helium was used as a carrier gas at a flow rate of 3 ml/min. The peak quantification was based on peak area comparison with the internal standard 17:0 (100 mg). For the analysis of the fatty acid distribution within the lipoprotein particle, the different lipid containing fatty acids were separated by thinlayer chromatography according to the standard procedure (Christie, 1982). The different bands corresponding to cholesteryl ester, triglycerides and phospholipids were scratched from the silica gel and transesterified with BF3/methanol. The methyl esters were subjected to capillary gas chromatography as previously described for the total fatty acid.

2.5. Spin labeling of lipoproteins Since it is not possible to work under the condition of equal LDL and VLDL concentrations, in this work the choice was made to match the number of lipid molecules in the surface monolayer of both LDL and VLDL samples (Shen et al., 1977). Tempo dissolved in Tris –HCl buffer was added to the lipoprotein suspension. The expected molar ratio of nitroxide dissolved in lipoproteins versus estimated surface lipid content was always kept less than 1:100. The equilibration of Tempo was reached within the time necessary to set up the ESR spectrometer. This was independently checked by incubating Tempo with lipoproteins at 37°C for 2 h and no difference in equilibration was observed.

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In order to independently prove that Tempo does partition in lipoprotein particles, the paramagnetic line broadening agent, chromium oxalate, which cannot penetrate into LDL and VLDL, was added to the spin labeled lipoprotein solution in the final concentration of 60 mmol/l.1

2.6. Nitroxide reduction with ascorbic acid In this type of experiment, the spin labeled lipoproteins were exposed to the ascorbic acid in order to follow the nitroxide reduction to nonparamagnetic form according to the protocol: lipoprotein solution was spin labeled with Tempo and divided in two equal samples. The solution of ascorbic acid was added to the one sample and decrease of the ESR signal was immediately followed with time. The same amount of buffer, instead of ascorbic acid solution, was supplied to the other sample in order to get the ESR spectrum at the very beginning of the experiment, i.e. at the time equal zero. In this way the time dependence of nitroxide reduction kinetic curves could be studied which reflects the processes occurring on the molecular level as an approach in revealing the lipoprotein complexity. In the experiments the concentration of ascorbic acid versus Tempo was kept in threefold excess. In the same concentration range, the reduction of Tempo by ascorbic acid was also followed in the pure system, i.e. the mixture of spin label and ascorbic acid was studied for comparison in the buffer solution without the lipoproteins.

2.7. ESR spectroscopy and analysis The experiments were performed in glass capillaries (1 mm inner diameter) on a X-band Varian E-109 ESR spectrometer. The following settings were used: microwave power, 10 mW; modulation amplitude, 0.1 mT; modulation frequency, 100 kHz and scan range 10 mT. The acquisition of ESR data was performed using EW Scientific 1

The authors are aware of the denaturing effect of chromium oxalate but the only purpose of this experiment was to check if Tempo really dissolves in the lipid phase of the sample.

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Software Services program Morse, 1987. All the ESR spectra were measured at T=37°C. Tempo dissolved in aqueous lipoprotein suspension reveals a complex ESR spectrum with a superposition of hydrophobic and hydrophilic components differing in spectral properties of each environment. The qualitative analysis, leading to the empirical partitioning of Tempo between two surroundings of different polarity, was done according to the method of Shimshick and McConnell (1973). For the quantitative analysis of such composite ESR spectra the simulation with two superimposed Gaussian type spectra was performed.2

3. Results The chemical composition of VLDL and LDL is given in Table 1 and the obtained values are in a good agreement with those reported previously (Kostner and Laggner, 1989; Esterbauer et al., 1992; Croft et al., 1995). The analysis of fatty acid composition in phospholipids, triglycerides and cholesterol esters is given in Table 2. Two different presentations are introduced: (a) For each fatty acid type its cumulative content in all the lipid classes, i.e. in phospholipids, triglycerides and cholesterol esters is presented per lipoprotein particle in terms of the fatty acid mole fraction. For instance, the mole Table 1 Compositional analysis of VLDL and LDL Molecule

VLDL (% weight) LDL (% weight)

Triglycerides Total phospholipids Free cholesterol Cholesterol ester Total cholesterol Protein

47.55 17.51 6.4 13.9 14.67 14.65

4.12 20.07 7.45 41.06 31.91 27.3

Data shown are mean values of triplicate analysis, coefficient of variation was always less than 5%. 2 Due to the unresolved superhyperfine splittings the Gaussian lineshapes resulted in a better simulation of the experimental spectra than the Lorentzian ones.

fraction of arachidonic fatty acid in VLDL is 2.89 with respect to the total number of moles of all the other fatty acids present in the particle (phospholipid+ triglyceride+ cholesterol ester data taken together). (b) For each fatty acid type its contribution in building up different lipid classes is treated separately. Namely, according to the accepted scheme of the structural organization of lipoproteins (Gotto, 1987) the fatty acid composition of triglycerides and cholesterol esters could be presented together as a fatty acid composition of the core whereas the fatty acid composition of the phospholipids as that of the monolayer of the particle. Therefore, considering separately the constituent fatty acids in the core from those in the monolayer, the comparison of their relative distribution in the lipoprotein particle is offered. The data are calculated as the mole fraction of the specific fatty acid in the core (triglyceride+ cholesterol ester data taken together) and in the monolayer (phospholipid data) with respect to the total number of moles of this very fatty acid per lipoprotein particle (triglyceride + cholesterol ester+ phospholipid data taken together). For instance, arachidonic acid contributes in the VLDL core with 41.6 mole fraction and in the VLDL monolayer with 58.4 mole fraction. When comparing data for the core and the monolayer, due to the difference in the dimensions of lipoproteins, it could be estimated (Schnitzer and Lichtenberg, 1994): VOLUMEcore (VLDL) VOLUMEmonolayer (VLDL) : 3:1

VOLUMEcore (LDL) :1:1 VOLUMEmonolayer (LDL)

(1)

So, if the distribution of the certain fatty acid type throughout the lipoprotein particle were uniform, the similar ratios as in the relation (Eq. (1)) would be expected for the fatty acid mole fractions as well. This analysis is given in the parenthesis of the Table 2b. For instance, the arachidonic acid in both LDL core and monolayer shows nearly equal distribution whereas in the VLDL monolayer is present in a twofold excess.

16:0

Fatty acid 18:0

18:1v9

18:2

16:1

20:4

18:1v7

0.72 1.17

22:6

100 100

Total

90.92 9.08 (2.5l) 100 79.47 20.53 (2.2l) 100

51.16 48.84 (2g) 100 22.9 77.10 (1.5g) 100

81.19 18.81 (2.4l) 100

78.91 21.9 (e) 100 91.43 8.57 (5.4l) 100

93.17 6.83 (3.7l) 100

57.6 42.4 (e) 100

41.6 58.4 (2.3g) 100

61.64 38.36 (1.2l) 100

85.92 14.08 (1.8l) 100

84.24 15.76 (2.9l) 100

89.59 10.41 (2.4l) 100

30.39 69.61 (1.5g) 100

41.6 58.4 (2.3g) 100

of fatty acids between the monolayer (phospholipid data) and the core (triglyceride+cholesterol ester data) per lipoprotein particle

0.91 0.56

18:3

The values in (a) and (b) are mole fractions of the fatty acids. In parenthesis, deviation from the uniform distribution of fatty acids in the lipoprotein particle is given with the following abbreviations: e, equal distribution; g, greater than; l, lower than uniform distribution. For instance, 2g(l) denotes two times greater (lower) contribution of a fatty acid in the monolayer as would be expected in the case of its uniform distribution throughout the lipoprotein particle.

(b) The relative distribution VLDL Core 76.92 Monolayer 23.07 (e) Total 100 LDL Core 48.06 Monolayer 51.94 (e) Total 100

(a) Composition of fatty acids (phospholipid+triglyceride+chlolesterol ester data) per lipoprotein particle VLDL 30.63 6.66 30.73 20.80 4.10 2.89 2.55 LDL 22.3 6.76 17.99 39.46 2.83 7.47 1.46

Lipoprotein

Table 2 Analysis of fatty acid composition of phospholipids, triglycerides and cholesterol esters in VLDL and LDL

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Fig. 1. The equilibrium partitioning of Tempo in LDL and VLDL solutions. The following final concentrations were used: CTempo =0.33 mmol/l, CLDL = 13.95 mmol/l, CVLDL = 1.8 mmol/l. The corresponding concentration of the lipid molecules in both the LDL and VLDL surface monolayer was estimated as 14 mmol/l. The peak heights of the hydrophobic and polar high field components in the ESR spectrum are denoted with H and P, respectively. In the insert to the figure the effect of chromium oxalate on the ESR spectra of the lipoproteins labeled with Tempo is presented. The final concentrations were: CTempo =0.18 mmol/l, CLDL =7.7 mmol/l, CVLDL = 0.98 mmol/l, Cchromium oxalate =60 mmol/l.

Measurement of the spin probe partitioning between the lipid phase and aqueous surrounding is a complex task due to different distribution, ordering and motion of spin label molecules in different environments. ESR spectra of Tempo dissolved in LDL and VLDL solutions are presented on Fig. 1. In the superimposed spectra the one with a smaller hyperfine splitting is ascribed to Tempo dissolved in lipoprotein particles whereas the other with the larger hyperfine splitting to Tempo dissolved in the buffer. That Tempo really dissolves in lipoproteins was proved in the experiment with chromium oxalate. This

fast relaxing paramagnetic agent induces in bimolecular collisions with Tempo a faster effective relaxation of the spin label leading to the linebroadening of the spectrum. Since chromium oxalate cannot penetrate into the lipoprotein particle and resides in the aqueous environment of the lipoprotein sample, the linewidth of the hydrophilic component in the spectrum is heavily broadened and, thus, removed from the spectrum. The only component in the ESR spectrum which is left can be ascribed to Tempo dissolved in the lipid phase of the sample according to its hyperfine splitting (Insert to Fig. 1).

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The empirical partitioning of Tempo between lipoproteins and buffer can be deduced directly from the spectra (Shimshick and McConnell, 1973). The underlying assumption of this approach is that the relative amplitudes of the high field peaks are proportional to the number of spin label molecules in hydrophobic, H, and polar, P, environments (Fig. 1). In this way the empirical partition parameter, f, can be expressed as: f=

H H+P

S = fTSH + (1 − fT )SP

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(3)

where fT denotes the theoretical partition parameter as the contribution of the hydrophobic component in the total ESR spectrum. This analysis resulted in the theoretical partition parameters, fT, for LDL (0.35 9 0.01) and for VLDL (0.409 0.02) in the equilibrium state. For the reaction of spin labeled lipoproteins with the ascorbic acid the simulations of ESR are presented in Fig. 3. It

(2)

Following this approach the empirical partition parameter, f, was derived for LDL (0.24 9 0.01) and VLDL (0.2990.01) in the equilibrium state. In the nonequilibrium experiments, the spin labeled lipoproteins were exposed to the ascorbic acid which reduces the paramagnetic nitroxide to the nonparamagnetic hydroxylamine form. Decrease of the total intensity of the ESR signal was followed with time (Fig. 2). The analysis of the reduction kinetic curves shows at least a biphasic behavior which can be parameterized with a two exponential decay. For comparison, the reduction of Tempo by ascorbic acid in the pure buffer solution was also measured and could be described with a monoexponential decrease of the ESR intensity. The results indicate that the leading term in the fitting function for both LDL and VLDL data corresponds to the same one for the pure system. Differences between the lipoproteins start to show up with later time period when the reduction process slows down. It could be observed that the overall kinetics of Tempo reduction proceeds faster in LDL than in VLDL samples. During the reaction process the increase of the empirical partition parameter, f, which now describes the instantaneous distribution of Tempo, was detected and presented in the Insert to Fig. 2. In VLDL samples this increase was more pronounced as compared to the LDL samples. In the detailed evaluation of the experimental intensity data the ESR spectra were simulated assuming two superimposed Gaussian type of spectra: SH, for the hydrophobic and SP, for the hydrophilic component. So, the total ESR spectrum, S, is given in the expression:

Fig. 2. The nonequilibrium experiments of nitroxide reduction with ascorbic acid in spin labeled lipoproteins. The following symbol legend is used: the reduction of Tempo by ascorbic acid in the pure buffer solution (“), in the spin labeled LDL suspension () and in the spin labeled VLDL suspension ( ). The ordinate represents intensities of the ESR spectra in arbitrary units after double integration. The following final concentrations were used: CTempo =0.3 mmol/l, CLDL =13.95 mmol/l, CVLDL =1.8 mmol/l, Cascorbic acid =0.9 mmol/l. The best fit of the kinetic curves is presented with the full (LDL, VLDL) and dotted lines (pure system). For the two exponential decay, y =bexp( −ax)+ dexp( −cx), the following paramters were obtained: a = 0.2 90.007, b = 3040 980, c = 0.02 90.01, d = 250 9 80 (LDL), a =0.2 90.07, b = 3010 9 90, c = 0.037 9 0.005, d =750 990 (VLDL) and a =0.2 9 0.002, b =3580 9 40 for the monoexponential fit of the reaction in the pure system. In the insert to the figure the apparent partition parameter, f =H/(H+ P), calculated according to the approach of Shimshick and McConnell (1973), is given during the reaction process.

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Fig. 3. The theoretical simulations of the ESR spectra of spin labeled lipoproteins. Two representative ESR spectra are shown: the one in the equilibrium state, i.e. without the ascorbic acid (t = 0 min) and the other corresponding to the nonequilibrium state in the reaction with the ascorbic acid (t = 28 min for LDL and t = 31 min for VLDL). The experimental data are depicted with dotted lines whereas the theoretically fitted ones are given with the full lines. The polar component in the spectrum of both LDL and VLDL spin labeled samples was fitted with the unique parameters: rotational correlation time, 0.01 ns; peak-to-peak linewidth in the derivative Gaussian lineshape for the central peak, 0.13 mT; hyperfine splitting, 1.72 mT; and g-factor, 2.0023. (a) For LDL, the hydrophobic component in the spectrum was fitted with the following parameters: rotational correlation time, 0.03 ns; hyperfine splitting, 1.56 mT; g-factor, 2.0028; and the peak-to-peak linewidth in the derivative Gaussian lineshape for the central peak, 0.17 mT (t= 0 min) and 0.165 mT (t = 28 min). The contribution of the hydrophobic component to the total spectrum was fT =0.34. (b) For VLDL, the hydrophobic component in the spectrum was fitted with the following parameters: rotational correlation time, 0.03 ns; hyperfine splitting, 1.56 mT; g-factor, 2.0028; and the peak-to-peak linewidth in the derivative Gaussian lineshape for the central peak, 0.165 mT (t=0 min) and 0.135 mT (t= 31 min). The contribution of the hydrophobic component to the total spectrum was fT = 0.42.

should be stressed that the very beginning of the experiment (t= 0 min, Fig. 3a, b) corresponds to the equilibrium state where the same amount of buffer instead of the ascorbic acid was added to the sample. The other spectra (t = 28 min for LDL and t = 31 min for VLDL, Fig. 3a, b) represent the nonequilibrium states in the process of nitroxide reduction to nonparamagnetic form. It is interesting to note that the parameters in the simulation which differ between the LDL and

VLDL samples are the theoretical partition parameter and the linewidth of the hydrophobic component in the spectrum. All the other parameters in the simulation such as the rotational correlation times, g-factor, hyperfine splittings for polar and hydrophobic components and the linewidth of the polar component are the same for both lipoproteins. The simulated spectra of nitroxide reduction by ascorbic acid revealed that the linewidth corresponding to Tempo dissolved

M. K6eder et al. / Chemistry and Physics of Lipids 85 (1997) 1–12

in VLDL particles decreases during the experiment whereas for LDL such a pronounced change in the linewidth was not observed (Fig. 4).

4. Discussion In this study human plasma LDL and VLDL were spin labeled with Tempo which distributes between the aqueous phase and the lipoprotein particles as shown in the experiments with chromium oxalate (Fig. 1). The results of the spin labeled lipoproteins exposed to the ascorbic acid and the accompanying nitroxide reduction with time is presented on Fig. 2. The parametrization of the kinetic curves revealed that the initial reduction rate in both LDL and VLDL samples is comparable to the one in the pure system consisting of only Tempo dissolved in the buffer and ascorbic acid. Therefore, this rapid phase of the reaction could be ascribed to the process taking part in the polar environment of the lipoprotein samples governed by the

Fig. 4. The change of the peak-to-peak linewidth in the hydrophobic part of the spectrum during the kinetic imaging experiments. One set of the simulated experimental data for LDL and VLDL was chosen for the presentation. The changes of the peak-to-peak linewidth, h 0H, of the derivative Gaussian lineshape corresponding to the hydrophobic component in the spectrum (hH ) during the kinetic imaging experiments is calculated and presented for the central peak (I= 0).

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free diffusion of the reactants. Since the depletion of nitroxide by ascorbic acid in the polar surrounding drives the system to the nonequilibrium state, the efflux of Tempo incorporated in the lipoprotein appears in order to sustain the equilibrium partitioning of the spin label between the two environments. This effect shows up in the slowing down of the reaction process leading to the at least biphasic feature of the kinetic curve. So, the transport properties of the specific monolayer are reflected in the observed reaction rates. From the fitting procedure (Fig. 2), it could be concluded that the overall nitroxide reduction proceeds faster in LDL than in VLDL samples. However, in explaining the reduction kinetic curves two comments should be added: (1) It is assumed that the nitroxide reduction by ascorbic acid is mainly limited to the aqueous phase and the lipid-protein interface. Namely, the ascorbate content present in the native lipoproteins is washed out during the procedure of lipoprotein isolation/preparation and does not interfere with the exogenously added one. (2) In the previous studies of nitroxide interaction with ascorbate Prelesnik et al., 1986 it has been experimentally verified that even in a pure systems the extremely complex redox reaction cycles take place in the presence of oxygen leading possibly to the reoxidation of hydroxylamine. So, it is worth stressing that such a simple parametrization of the kinetic curves in this work should not be overinterpreted bearing in mind that a complex kinetics lays behind this simple exponential decays. Namely, the samples were measured in the aerobic conditions and the solubility of oxygen is much higher in the lipid as in the aqueous phase. So, besides diffusion and transport limited reaction of nitroxide with ascorbic acid also the possibility for the reoxidation processes should not be a priori excluded in the spin labeled lipoproteins. In order to explain, why during the nitroxide reaction with ascorbic acid, the empirical partition parameter of Tempo dissolved in lipoprotein samples increases during the experiment (Insert to Fig. 2) the theoretical simulations of the ESR

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spectra were performed (Fig. 3). This analysis of the experimental intensity data demonstrated that in the nonequilibrium conditions the narrowing of the linewidth of the ESR component belonging to Tempo dissolved in the lipoprotein particles occurs (Fig. 4). Namely, the lineshape of Tempo dissolved in VLDL is exchange broadened (0.165 mT) and narrows upon ascorbic acid reduction to the nonbroadened value (0.135 mT). Consequently, the ratio of the respective amplitudes in the relation Eq. (2) increases due to the apparent increase of the amplitude, H, corresponding to the spin label molecules in the hydrophobic environment. On the other hand, the lineshape of Tempo dissolved in LDL (0.17 mT) is nonhomogeneously broadened and changes only slightly upon the reaction with ascorbic acid (Fig. 4). The explanation for these differences between LDL and VLDL samples was searched in the comparative properties of their respective surface monolayers which directly mediate the transport of the spin label molecules in the nonequilibrium conditions. The surface monolayer of lipoproteins is stabilized by intermolecular forces between the principal constituents such as protein(s)3, phospholipids (mostly phosphatidylcholine) and free cholesterol Kostner and Laggner, 1989. At the level of phospholipid head groups a network of hydrogen bonded water molecules extends between the lipids (Slater et al., 1993; Boggs, 1987) thus creating a ‘hydration layer’. The contribution of van der Waals’ forces is important for the assembly of cholesterol with phospholipid acyl chains (Mattjus et al., 1994). This interaction strongly depends on the length and degree of unsaturation of fatty acid building blocks of phospholipids. Comparison of fatty acid composition of phospholipids, triglycerides and cholesterol esters in VLDL and LDL is given in Table 2. Deviation from the uniform distribution of the certain fatty acid throughout the lipoprotein (surface monolayer versus core data) was checked according to the 3

The influence of proteins, possibly inducing packing defects in the monolayer, or the presence of lipid-protein interactions cannot be discarded, but as these hypothesis were not experimentally verified in this study the explanation of the results is searched for in the arguments dealing with the properties of lipid assemblies.

relation Eq. (1). It follows from these data that the percentage of unsaturated fatty acids (18:2, 16:1) in the surface monolayer of LDL is significantly shifted towards much smaller values as compared to the VLDL monolayer. Moreover, the arachidonic acid, with its extremely important physiological role, is present in VLDL monolayer in twofold excess. So, the property of more fluid monolayer in VLDL can be ascribed to the weaker van der Waals’ forces between cholesterol and unsaturated phospholipids as compared to LDL and as a consequence, the ESR linewidth changes could be observed more readily (Fig. 4). Considering the length of the unsaturated fatty acids in both LDL and VLDL the predominance of those with longer acyl chains ( ] 18 C-atoms) can be observed. It has been reported that the mismatch in phospholipid acyl chain length and the length of cholesterol could give rise to the formation of lateral domains with non-uniform cholesterol concentration (Mattjus et al., 1994). These findings confirm previous conclusions about the coexistence of different lipid domains with respect to the mobility constraints in the surface region of lipoproteins Kveder et al., 1994. Therefore, different molecular packing would cause non-random lipophilicity distribution for the spin label partitioning in the monolayer of LDL and VLDL and influence the reaction kinetics with ascorbic acid through different susceptibility to the linebroadening effects. In explaining the slower overall reduction of nitroxide by ascorbic acid in the spin labeled VLDL as compared to LDL (Fig. 2) one tentative explanation based on the arguments of Subczynski et al. (1994) could be offered dealing with the depth of the lipid-water interface necessary for the reaction to take place. Namely, a higher hydrophobic barrier to the permeation of the ascorbic acid into the monolayer could be expected in VLDL with respect to the unsaturated fatty acid content. So, the nitroxide reduction would proceed less effectively and together with the smaller curvature of VLDL particles the whole process would be slower in VLDL than in LDL. So, this study reveals different behaviour of the spin labeled LDL and VLDL under the nonequilibrium conditions which can be connected to

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their respective composition of lipids. The chemical analysis provides the information on non-homogeneous distribution of lipid molecules within the lipoprotein particles. The functional consequences are illustrated by ESR where the line shape analysis and the kinetics of the chemical reaction provides an insight into the molecular exchange and mobility differences of such nonhomogeneous distribution of lipids. This approach should help to understand the physiologically relevant LDL and VLDL transport properties as well as different susceptibility to modifications and/or interactions with the cell membranes.

Acknowledgements This work was in part supported by the Ministry of Science of the Republic of Croatia (project number 1-03-065), by the Ministry of Science and Technology of the Republic of Slovenia and by the Austrian Science Foundation (project SFB 709).

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