JOURNALOFLIPIDRESEARCH§AsBMB
`
`In vitro effect of Triton WR-1339 on canine plasma
`high density lipoproteins
`
`Kyomhe Yamamoto,’ Robert Byrne,‘ Celine Edelstein,‘
`Betty Shen,“ and Angelo M. Scanu"l‘
`
`Departments of Medicine,‘ Biochemistry,1' and Biophysics and Theoretical Biology,"
`University of Chicago. Pritzker School of Medicine, Chicago, IL 60637
`
`Abstract We studied the effect in vitro of various concentrations
`
`of Triton WR-1339 on normolipidemic canine plasma and on
`the high density lipoproteins (HDL) isolated from this plasma
`by ulttacentrifugation. As a preamble to this study, we established
`that Triton WR-1339 has a unimer molecular weight of 4,500,
`a micellar molecular weight of 180,000, and a critical micellar
`concentration (CMC) of 0.018 mM or 0.008 g/dl. Above its
`CMC. Triton WR-1339 in concentrations between 2 and 10
`mg/ml induced concentration-dependent structural changes in
`HDL which were characterized by a progressive displacement
`of apoA-1 from the HDL surface without loss of lipids. The
`addition of Triton WR-1339 to the HDL particles modified
`their electrophoretic mobility and caused an increase in size (95
`1 5 A to 114 x 7 A). At the extreme Triton WR-1339 con-
`centrations utilized in these studies (10 mg/ml) disruption of
`the HDL particles occurred; at this stage, the original. relatively
`homogeneous, spherical HDL particles were replaced by a het-
`erogenous population ranging in size between 50 and 250 A,
`representing complexes ofTriton W R-l 339 with lipids essentially
`free of apoA-I which could be sedimentecl by ultracentrifugation.
`The effects of Triton WR-1339 on whole plasma or isolated
`HDL were comparable.II These studies indicate that Triton
`WR-1339 in vitro alters HDL in a concentration-dependent
`manner and that these changes vary from a displacement of
`apoA-l from the HDL surface to a state where all lipids are
`solubilized into the Triton WR-1389 micellar phase and are
`driven away from the protein moiety. These important structural
`changes in HDL may be responsible, at least in part, for the
`hyperlipidemia attending the intravenous administration of Tri-
`ton WR-l339 into experimental animals.—-Yununoto, K.,
`R. Byrne, C. Edeluein, B. Sben, and A. M. Scanu. In vitro
`effect of Triton WR-1839 on canine plasma high density li-
`poproteins. Lipid Res. 1984. 25: 770-779.
`
`Supplementary key words critical micelle concentration 0 detergent
`0 micelles
`
`Although the intravenous administration of Triton
`WR-1339 is known to produce hyperlipidemia and plasma
`lipoprotein changes in the experimental animal,
`the
`mechanisms whereby these intravascular changes occur
`have not been clearly defined (1-9). In earlier in vitro
`studies, Scanu and Oriente (10) showed that Triton WR-
`l339 interacts with plasma lipoproteins and causes im-
`portant physico-chemical changes in these particles, and
`
`770
`
`journal of Lipid Research Volume 25, 1984
`
`PAGE 1 OF 10
`
`the HDL particles were recognized to be particularly sen-
`sitive to the action of the detergent both in vitro and in
`vivo (1 l). The early studies in the dog, and the more
`recent reports on the squirrel monkey (9) and the rat
`(12), have led to the suggestion that the physico-chemical
`changes in HDL are responsible for the development of
`the hyperlipidemia. However, studies by Portman et al.
`(9) in the squirrel monkey, have failed to show an in vitro
`effect of the detergent on HDL. Moreover, a primary
`effect of Triton WR-1339 on lipid-modifying enzymes
`has also been suggested (13-15). Because of these un-
`certainties, we considered it of interest to explore in more
`depth the nature of the interactions occurring between
`Triton WR-1339 and the plasma lipoproteins. For this
`purpose, we have used normolipidemic canine plasma
`since it contains mainly the HDL class and we also ex-
`amined some of the physico-chemical properties of Triton
`WR-1339 in solution.
`
`MATERIALS AND METHODS
`
`Separation of plasma lipoproteins
`
`Blood was obtained on several occasions by venous
`puncture from two fasting healthy male dogs (20-30 kg),
`which were fed a regular Purina Chow diet. Their serum
`cholesterol levels were 1 15 and 143 mg/dl, respectively,
`and their triglycerides were 15 and 41 mg/dl, respec-
`tively. The blood was collected into tubes containing so-
`dium citrate as an anticoagulant (0.28% by weight) and
`the plasma was separated by centrifugation at 4°C for
`30 min at 1000 g. The HDL ofd l.063—l.2l g/ml was
`separated by ultracentrifugal flotation as previously de-
`
`Abbreviations: HDL. high density lipoproteins d 1.063-1.21 g/ml;
`CMC. critical micelle concentration; EDTA, ethylenediamine tetra-
`acetic acid; apoA-I, apolipoprotein A-I derived from the high density
`lipoproteins; HPLC, high performance liquid chromatography; SDS,
`sodium dodecyl sulfate.
`' Present address: Department of Medicine, Saga Medical School.
`Sanbonsugi, Nabeshima, Nabeshima-cho, Saga City, 840-01 japan.
`
`SENJU EXHIBIT 2048
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`LUPIN v SENJU
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`IPR2015—01 105
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`go;‘atJequiatdesuo';san6Kq5J0‘J|_l'MMMwot;papaqumoa
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`JOURNALOFLIPIDRESEARCH§AsBMB
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`scribed (16, 17) and extensively dialyzed against 0.15 M
`NaCl, 10" M EDTA, pH 7.2, before use.
`
`Incubation of Triton WR-1339 with plasma or HDL
`
`Triton WR-l339 (Tyloxapol, Sigma, St. Louis, MO)
`was dissolved in 0.05 M phosphate buffer, pH 7.2. Canine
`plasma was incubated in a 10:1 volume ratio with Triton
`WR-1389 so that the mixture contained the detergent
`at concentrations varying from 0 to 10 mg/ml. In the
`case of isolated HDL, the protein concentration was se-
`lected to be similar to that present in the incubation ex-
`periments with whole plasma. The incubations were con-
`ducted at 37°C for 2 hr. In some experiments, ml-labeled
`Triton WR-1339 was used and was prepared with
`["’l]Nal by the method of McFarlane (18) and purified
`by passage through a Sepharose 413 column.
`
`Separation of the incubation products
`
`Density gradient ultracentrifugation. After incubation with
`Triton WR-I 389, the plasma was separated by the single-
`step density gradient ultracentrifugation as previously
`described (19). The efliuents were monitored at 280 nm
`and collected as 400-p.l fractions.
`
`Molecular sieve chromatography. Gel filtration was con-
`ducted in glass columns (2.5 X 70 cm) packed with Se-
`pharose 4B (Pharmacia Fine Chemicals, Uppsala,
`Sweden). The columns were eluted with 0.05 M phos-
`phate, pH 7.2, at a flow rate of 20 ml/hr at 6°C. Eluates
`were monitored at 280 nm or at 278 nm.
`
`Quantitative immunoassay of apoproteins
`
`Pure canine apoA-I was obtained from canine apoHDL
`by high performance liquid chromatography (20). An
`antiserum against apoA-I was raised in the goat. Good
`antiserum titers were obtained after injecting the animal
`intlamuscularly every 2 weeks fora total of four injections.
`For the first injection, the antigen was emulsified with
`complete Freund’s adjuvant; incomplete Freund’s adju-
`vant was used for the other injections. The immunoassay
`for apoA-I was carried out by the rocket immunoelec-
`trophoretic procedure described by Laurell (21) in the
`presence of 7 M urea.
`
`Determination of the critical micellar
`concentration of Triton WR-1339
`
`The CMC was determined by measurements of the
`surface tension of Triton WR~l339 as a function of its
`
`concentration at 25°C as described elsewhere (22). The
`surface tension was determined from the maximum pull
`exerted on a du Nouy ring attached to the "B" loop of
`a Cahn Electrobalance when the ring was in contact with
`and was raised above the aqueous surface; the force was
`recorded as a function of time. The surface tension was
`
`PAGE 2 OF 10
`
`calculated as the product of the weight times the ring
`constant, a, which was determined empirically with the
`use of pure liquids of known surface tension.
`
`Unimer molecular weight of Triton WR-1339
`
`The unimer’ molecular weight of Triton WR-1339
`was determined by high pressure liquid chromatography
`(IBM Model LC 9533) using an IBM GPC Type C column
`(7.0 X 250 nm; 5-» pore size) equilibrated in either chlo-
`roform or tetrahydrofuran. Both of these solvents were
`HPLC grade (Burdiclt 8: jacltson Laboratories) with a
`cutoff of about 240 nm to allow readings at 278 nm. The
`retention time for Triton WR-1339 was determined from
`
`the elution time of its peak maximum. The detergent
`solution was applied to the column in a 10-ul volume
`(concentrations between 0.1 and 10.0 g/dl). The unimer
`molecular weight was obtained by comparing the re-
`tention time of the detergent to that of polystyrene
`standards of 1900 to 7600 daltons (gift from IBM In-
`struments, Inc.).
`
`Electron microscopy
`
`Fractions from the single spin ultracentrifugation of
`mixtures of Triton WR-1339 and plasma were dialyzed
`against 0.005 M NI-l,HCO3 buffer, pH 8.2, and then
`negatively stained with 1% sodium phosphotungstate, pH
`7.0, after deposition onto a thin carbon film supported
`on a copper grid. The specimens were examined in a
`Phillips EM 300 microscope with condenser and objective
`apertures of 100 um and 50 pm, respectively. The ac-
`celeration voltage was 80 ltV and all specimens were ex-
`amined at 55,000X magnification.
`
`Electrophoretic analyses
`
`Before electrophoretic analysis, all the samples were
`extensively dialyzed against 0.005 M NHJ-{C0, buffer,
`pH 8.2. Agarose gel electrophoresis was carried out on
`Agarose Universal Electrophoresis film (AC1-Corning,
`Palo Alto, CA) using an AC1 electrophoresis apparatus.
`After electrophoresis, the lipoproteins were fixed and
`stained with Fat Red 7B or Amido Black 10B. Electro-
`
`phoretic separation ofapoproteins was performed on 10%
`polyacrylamide gels containing 0.1% SDS (23). Gradient
`gel electrophoresis was carried out on a Pharmacia Elec-
`trophoresis Apparatus GE-4 loaded with gradient gels
`PAA 4/30 at l4°C, 125 V for 20 hr. The gels were then
`fixed and stained overnight in 0.04% Coomassie Blue G-
`250 in 3.5% perchloric acid followed by destaining in
`5% acetic acid. Molecular weight standards were run in
`each gel slab.
`
`I HCKCTOEQHCOUS monomer.
`
`Yamamota er al. Triton elect on canine HDL
`
`771
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`gtoz‘gtJeqwaidasuo';san6Kq5J0'.l|_l'MMMtum;papaqumoa
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`Chemical and radioactivity analyses
`
`Protein content was determined by the method of
`Lowry et al. (24) except that 0.5% sodium dodecyl sulfate
`was added to the reagents. Total and free cholesterol
`were determined enzymatically according to the modified
`procedures of Allain et al. (25) and Gallo et al. (26).
`Lipid phosphorus was measured according to the
`method of Bartlett (27) and triglycerides were measured
`by the Technicon AutoAnalyzer 11 after isopropanol ex-
`traction of zeolite-treated preparations. Triton WR-l 339
`measurements were carried out in isopropanol extracts
`of plasma or lipoproteins at 278 nm (4). Radioactivity
`measurements were carried out in a Tracor Analytical
`Model 1190 Automatic Counter (Elk Grove Village, IL).
`
`Reagents
`
`All of the chemicals were reagent grade. Thyroglob-
`ulin, catalase, and aldolase (Pharmacia, N_]) were used as
`the molecular weight standards. ["5I]NaI (carrier free)
`was purchased from Amersham Corp. Triton WR-1339
`(Tyloxapol) was purchased from Sigma. Its structure is
`shown in Fig. 1.
`
`RESULTS
`
`Properties of Triton WR-1339 in solution
`
`Molecular weight in organic solvents. The HPLC elution
`profile of Triton WR-1339 solubilized in tetrahydrofuran
`in shown in Fig. 2. An identical profile was obtained with
`the detergent dissolved in chloroform. Some heteroge-
`neity was noted, likely due to different numbers of eth-
`ylene oxide units in the ether side chain and/or in the
`
`
`
`
`
`JOURNALOFLIPIDRESEARCH§‘AsBMB
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`E,278ntn
`ABSORBANC
`
`I2
`I0
`8
`6
`RETENTION TIME (mill
`
`Fig. 2. HPLC elution profile of Triton WR-1339 (0.1 mg. injection
`volume, 10 ill). The eluting solvent was tetrahydrofuran, flow rate.
`0.5 ml/min. The effluent was continuously monitored at 278 nm.
`Chart speed 1 cm/min. The temperature of fractionation was 23°C.
`The arrows indicate the elution position of polystyrene standards: a.
`7600'. b. 3650; c, 1900. which were run separately under identical
`column conditions. The negative peak eluting at 12.7 min is the buffer
`breakthrough.
`
`number of octylphenol groups. The same elution profile
`was obtained with preparations of Triton WR-I339 di-
`luted up to 200-fold in tetrahydrofuran and analyzed
`under the same chromatographic conditions. The major
`peak was attributed to unimeric Triton WR-1339 with
`some side chain heterogeneity leading to molecular weight
`polydispersity. Based on the retention times of the poly-
`styrene standards (Fig. 2) and on the retention time of
`the major peak constituent of Triton WR-1339, the un-
`imer molecular weight of the detergent was estimated to
`be 4500.
`
`Molecular weight in phosphate bufler. Triton WR—l339
`applied to a Sepharose 4B column at 5 mg/ml in 0.05
`M phosphate solution, pH 7.4, eluted as a single sym-
`metrical peak (data not shown). Aldolase, catalase, and
`thyroglobulin were applied to the same column. From
`the log molecular weight versus Kd plot it was estimated
`that the Triton WR-1339 micelle had a molecular weight
`of 180,000.
`
`Critical micellar concentration. The plot of values for the
`surface tension, 7, of Triton WR-1339 in 0.05 M sodium
`phosphate, pH 7.2, against the natural logarithm of each
`concentration (mol/liter) generated a curvilinear relation
`whose break at 0.018 mM defined the CMC of the Triton
`
`WR-1339 in solution (Fig. 3). The data were further
`analyzed according to the equation describing the con-
`centration of the detergent at the surface monolayer:
`
`5.).
`_l
`F=E'"Fa1nc
`
`Eq. I
`
`where I‘ is the surface concentration of Triton WR-1339
`
`
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`go;‘atJequratdesuo';san6Kq5J0'.I|_l'MMMwot;papaqumoa
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`TRITON WR I339
`
`(Non-ionic surfactant)
`
`
`
`e"I7
`
`R
`
`
`
`2s
`
`HI7
`
`oxyethyloted tert- oetyl phenol formaldehyde
`polymer
`
`[R is cuzcnzotcn-:2 CH2O)m CH2 CH 20H],
`m is 6to83
`nlsis
`
`Fig. 1. Structure of Triton WR-1839. This non-ionic surfactant is
`a p-(l.l.3,3-teuamethylbutyl) phenol polymer with ethylene oxide and
`formaldehyde. R = CH,CH,0(CH,CH,O).,.CH,0H; m = 6 to 8; n is
`not more than 5.
`
`772
`
`Journal of Lipid Research Volume 25, 1984
`
`PAGE 3 OF 10
`
`

`
`TABLE 1.
`
`Physical properties of Triton WR-I339
`and Triton X-100
`
`Parameters
`
`Triton WR-I339
`
`Triton X-I00
`
`Molecular weight. unimer
`
`Molecular weight. micelle
`
`1,500
`
`180.000
`
`Number of unimers/micelle
`
`40
`
`613
`
`90.000‘
`31,250‘
`
`H0“
`125‘
`
`CMC
`
`0.008 g/dl
`0.018 mm
`
`0.0l6 g/dl‘-"
`0.249 mM
`
`Limiting area. A’/molecule
`
`62.5
`
`53.0‘
`
`‘ Kushner. L. M.. and W. D. Hubbard. (28).
`‘ Biaselle. c. 1.. and D. 3. Millar. (29).
`' Ross. S.. and P. Olivier. (30).
`' Hsiao. 1... H. N. Dunning. and P. B. Lorenz. (31).
`
`WR-1339 of 10 mg/ml, it remained close to the origin
`or partly migrated toward the cathode. This shift in elec-
`trophoretic mobility was particularly evident in the studies
`with isolated HDL (Fig. 4B). As a consequence of the
`addition of Triton WR-1339, the Amido Black-stained
`
`Triton
`(mg/ml)
`
`0
`
`2.0
`
`5-0
`
`I0-0
`
`o
`
`I .0
`
`5.0
`
`Io.o
`
`A
`
`B
`
`Lip
`
`Prol
`
`O 9
`
`°‘
`
`o
`
`J,
`
`°‘
`
`2 3 —Il
`
`._
`
`lj I
`
`Li
`
`. j I
`
`I j I
`(-)
`(H l-)
`(+)
`
`IX
`
`I
`
`'
`
`0
`
`;
`
`I
`
`0
`_
`
`l
`
`o(
`
`|
`
`3
`
`3
`
`I-)
`
`(+)
`
`(-)
`
`l+)
`
`
`
`
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`Surfacetonalon,(dynes/cm)
`
`
`‘IO
`'9
`‘l5
`‘l4
`‘l3
`‘I2
`‘II
`
`In C, (moles/I)
`Fig. 3. Plot of surface tension of Triton WR-1339 versus concentration
`in moles/liter. The values of surface tension. 1. were determined
`using the du Nouy ring at 25°C as described in Methods. The solid
`line drawn from 3.0 X 10" to 1.8 X 10" moles/l represents the best
`fit to the experimental data (0 — 0) according to the equation: Y
`l264.l6
`X
`
`I l57.53 +
`
`.
`
`in mol cm", R is the gas constant in dyne-cm deg"
`mol", T is the absolute temperature, 1 is the surface
`tension in dynes cm" , and C is the molar concentration.
`From the value obtained for I‘, the area A, occupied at
`each concentration of Triton WR-1339, was calculated
`
`according to the relation:
`
`Eq.2
`
`lF
`
`.
`
`A =
`
`From the force-area curve, the straight line portion of
`the curve extrapolated to zero surface pressure gave a
`value of 62.5 A’ which defined the limiting area in A”
`per molecule of Triton WR-1389 (data not shown). A
`summary of the physical properties of Triton WR-1339
`is presented in Table 1. For comparison. literature data
`(28-31) for another non-ionic detergent (Triton X-100)
`are also tabulated.
`
`
`
`
`
`JOURNALOFLIPIDRESEARCH§A5BMB
`
`Agarose gel electrophoresis of canine
`plasma and Triton WR-1339
`
`The effect of Triton WR-1339 was dose-dependent.
`In the case of plasma (Fig. 4A). Triton WR-1339 caused
`the lipid-stained band in the a region to progressively
`decrease in mobility until at a concentration of Triton
`
`Fig. 4. Triton WR-I359 effect on the agarose gel electrophoretic
`profile of whole plasma (A) and HDL (B). After incubation of whole
`plasrm or HDL (4 mg of protein/ml) with various concentrations of
`Triton for 2 hr at 37°C. 2 ul of each sample was applied to Agarose
`film in duplicate. After electrophoresis. one film was stained with Fat
`Red 73 and the other with Amido Black 10B. Lip. lipid staining: prot.
`protein staining: 0. origin. The arrow indicates the area where the
`new band appeared.
`
`Yamamolo at al. Triton elect on canine HDL
`
`773
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`PAGE 4 OF 10
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`gtoz‘gtJoquiotdosuo‘tunaKq5.lO'.||['MMMwot;popaquma
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`EQ.
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`
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`
`A
`:3.
`
`5
`
`Triton
`(m9Im|)
`
`O
`
`2
`
`5
`
`I0
`
`(‘l
`-lO|23
`
`Distance (cm)
`
`Fig. 6. Distribution of '”l-labeled Triton WR-l 339 in whole plasma.
`After incubation of ml-labeled Triton WR-l 339 with whole plasma.
`the samples were applied to Agarose gel electrophoresis films in du-
`plicate. One gel was stained with Fat Red 78. the other was cut into
`0.5-cm pieces and their radioactivity content was determined.
`
`acrylamide gel electrophoresis. agreed with the immu-
`nochemical and radioactive results (Fig. 8). By gradient
`gel electrophoresis. HDL increased in size as a function
`of Triton concentration (Fig. 9). The chemical analyses
`of the ultracentrifugal peaks in Fig. 7 showed that peaks
`a through d had the same lipid composition as control
`HDL peaks (Table 2), indicating that Triton WR-1339
`had no effect on the lipid matrix of the lipoprotein par-
`ticle. In contrast. the minor peak e was composed mainly
`of Triton and protein. The chemical analyses also showed
`that over 90% of Triton WR-l 339 that was subjected to
`ultracentrifugation was recovered with the lipoprotein
`peaks.
`When ultracentrifugal peaks d and e were passed
`through a Sepharose 48 column (Fig. 10). peak cl eluted
`as a major peak and a shoulder, whereas peak e was
`symmetrical eluting in the same position as the shoulder
`of peak d and the Triton WR-I389 peak when this de-
`tergent was run alone.
`By electron microscopy. the control H DL particles had
`a diameter of 95 I 5 A (Fig. Ila). After incubation with
`
`band in HDL decreased in mobility and became less stain-
`able; at the same time the protein-stained band shifted
`from the a to the 5 position (Fig. 4B). As shown in Fig.
`5, the B position is where apoA-l was found to migrate.
`The electrophoretic mobility of Triton WR-I339 was
`also affected by the amount of Triton WR-1339 applied
`to the agarose film (Fig. 6). When using radiolabeled
`detergent, the band that was detected both by staining
`with Amido Black and by radioactivity measurements de-
`creased in mobility from the at position to the cathode
`where the Fat Red 7B-stained band of HDL treated with
`
`10 mg of Triton WR-1339 moved (see Fig. 4B).
`
`Eflect of Triton WR-1339 on the distribution of
`
`plasma llpoproteins as assessed by density
`gradient ultraeentrifugation
`
`As shown in Fig. 7. Triton caused marked changes in
`the density gradient lipoprotein profile of whole canine
`plasma. The changes were concentration-dependent. In-
`creasing the concentration of Triton WR-I339 from 2
`to I0 mg/ml resulted in a progressive shift of the HDL
`peak to a lighter density peak (Fig. 7, peaks. at. b, c. d)
`associated with an increase in the absorbance at 280 nm
`
`which was partially contributed by Triton WR- I 339. The
`distribution of radioactivity of "°l—|abeled Triton followed
`essentially the 280 nm absorbance readings, both in po-
`sition and intensity. When this detergent was studied
`alone. its peak of maximal absorbance was at 278 nm.
`At a Triton WR-I339 concentration of IO mg/ml of
`plasma. a new shoulder within the density range of d
`l.l0 and l.l5 g/ml appeared (Fig. 7, peak e). At this
`detergent concentration, the study of immunoassayable
`apoA-l showed the displacement of apoA-l from the HDL
`peak (Fig. 7. peak d) to a new position (Fig. 7, peak e).
`The apoprotein distribution, as assessed by SDS poly-
`
`
`
`
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`JOURNALOFLIPIDRESEARCH§AsBMB
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`‘? goat serum
`
`1...‘
`(—l
`(+)
`
`(-)
`
`(+)
`
`lmmunoelectrophoretic analysis of an incubated HDL-Triton
`Fig. 5.
`WR-] 339 mixture against an amianine apoA-I goat serum. Purified
`apoA-I and an incubated mixture of HDL and Triton WR- l 339 (final
`concentration: Triton. 10 mg/ml. 2.5 ug of protein) were applied to
`Aprose films in duplicate. After electrophoresis. one (left column)
`was stained with Amido Black IOB. the other (right column) was reacted
`against a goat antiserum raised against canine apoA-l. a. apoA-I; b.
`HDL; c. HDL-Triton mixture.
`
`774
`
`journal of Lipid Research Volume 25. 1984
`
`PAGE 5 OF 10
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`Absorbonce M
`(zoom)
`
`8)
`I5
`I0
`5I0l5&a$5
`Froction Number
`Fraction Number
`
`25
`
`30
`
`
`L5
`LQ L04
`LCS L0
`L15
`L03
`L04
`L06 L0
`
`Density
`
`(9/ml)
`
`Density (9/mll
`
`Fig. 7. Distribution of apoA-I and "*1-labeled Triton WR-I339 after single-spin density gradient ultracen-
`trifugation of whole plasma. Canine plasma was incubated in vitro with various concentrations of "51-labeled
`Triton WR-1339 (final concentration of Triton, 0. 2. 5. and 10 mg/ml) for 2 hr at 37°C. Then one ml of
`each sample was separated by single-spin density gradient ultracemrifugation. The efiluents were continuously
`monitored at 280 nm in an ISCO UA-5 unit and collected in 0.4-ml fractions. The concentration of apoA-I
`was measured by electroimmunoassay and the radioactivity of '”l-labeled Triton WR-I339 was measured in
`a gamma counter. The combined HDL peaks were named as a. b. c, d, e. The number of the tubes collected
`was a. tubes l9-23: b, I6-2|: c. l2-18; d. I2-19', and e. 2|-25.
`
`Triton WR-1339 (2 mg of Triton WR-I339/ml), the
`HDL particles were spherical and larger (I14 :t 7 A)
`than control HDL (Fig. llb). At 5 mg of Triton WR-
`l339/ml. the particles were heterogeneous varying in
`size between 50 A and 250 A (Fig. us). At 10 mg of
`Triton WR-I339/ml. there was a granular pattern with
`small particles in the 50 A range (Fig. Ild).
`
`DISCUSSION
`
`The current studies have established that Triton WR-
`
`l339 forms micelles when dissolved in aqueous solutions
`at concentrations of 0.008 g/dl or above. These micelles
`are relatively homogeneous, have a micellar number of
`40, and a molecular weight of l80.000 (Table 1). By
`
`Yamanwto at al. Triton elect on canine HDL
`
`775
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`PAGE 6 OF 10
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`go;'9;Jeqtuatdasuo';san6Kq5JO'.l|l‘MMMmoi;papaqumoa
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`JOURNALOFLIPIDRESEARCH§AsBMB
`
`TABLE 2.
`
`Physicochemical parameters of Triton WR-I339-HDL complex
`b
`c
`d
`
`Radius (nm)"
`Hydrated density (g/ml’)
`Molecular weight X 10
`Triton WR-1339 molecules per
`particle
`
`Composition
`Protein
`Total cholesterol
`Phospholipid
`Triglyceride
`Triton WR-1339
`
`4.40
`1.100
`200
`
`5.25
`1.086
`275.3
`
`0
`
`40.2
`30.9
`28.5
`0.4
`0
`
`18.5
`
`22.4
`25.7
`21.4
`0.4
`30.1
`
`6.10
`1.068
`425.6
`
`52.1
`
`wright ‘I
`6.8
`21.1
`16.7
`0.3
`55.1
`
`6.10
`1.068
`n.d.‘
`
`n.d.
`
`1.8
`14.4
`12.2
`0.3
`71.3
`
`e
`
`3.85
`1.1 19
`n.d.
`
`n.d.
`
`27.7
`6.3
`1.8
`0
`64.2
`
`" Radii and molecular weights of molecules were obtained by gradient gel electrophoresis.
`'5 n.d.. Not determined.
`
`comparison, the commonly used Triton X-100 has smaller
`unimer and micellar molecular weights and higher CMC
`values (Table l). The current studies have also shown
`that at concentrations of Triton WR-1339 above the
`
`CMC, this detergent interacts with HDL and causes con-
`centration-dependent structural changes in this lipopro-
`tein class. This finding is in accord with earlier studies
`in the dog (10, 1 1) and other animal species (1, 3-5, 12).
`However, in this work we have defined more precisely
`the nature of the structural changes induced by Triton
`WR—1339. At low Triton WR—1339 concentrations (2 mg
`of Triton WR-1339/ml of plasma), the action of this
`detergent appears to be limited to the lipoprotein surface
`and to affect the specific displacement of apoA-1. Under
`these conditions an HDL-Triton WR-1339 complex is
`
`produced having all of the components of authentic HDL
`except that apoA-1 is replaced by Triton WR-1339. This
`indicates that the apparent afiinity of Triton WR-1339
`for the HDL surface is greater than that exhibited by
`apoA-1. A quantitative assessment of the apparent afiinity
`would require a thermodynamic treatment of the data
`which would require some unjustified assumptions (82)
`that I) micelles form a phase that is distinct from the
`aqueous solution; 2) a minimum number of amphiphile
`molecules (m = 50) would have to associate with each
`other, which is a number larger than that determined
`for Triton WR-1339; and 3) homologous aggregates
`would have to occur in aqueous media. which is not the
`case in our studies. Our results indicate that after HDL
`
`has reached maturation, apoA-1 is not essential for the
`
`
`
`
`
`abcde
`
`Fig. 8. SDS polyacrylamide gel electrophoresis of the ultracentrifugal
`peaks in Fig. 7. After dialysis of HDL—peaks (a—e. see Fig. 7) against
`0.005 M NH.HCO, buffer. pH 8.2. 100 pl ofeach sample was analyzed
`by SDS polyacrylamide gel electrophoresis.
`
`776
`
`Journal of Lipid Research Volume 25. 1984
`
`PAGE 7 OF 10
`
`--/‘ —ThyrogIobulln
`
`--4 - Apoferritin
`
`-It - Catolase
`
`- Lactate
`dehydrogenose
`l'j""BSA
`
`I
`
`~
`
`a bcdel
`std
`Fig. 9. Polyacrylamide gradient gel electrophoresis of ultracentrifugal
`peaks in Fig. 7. One hundred ul of each peak was applied to gradient
`gels at 14°C at 125 V for 20 hr. The radii of the particles were
`calculated from the calibration curve of standards: thyroglobulin (8.50
`nm). apoferritin (6.10 nm).
`lactate dehydrogenase (4.08 nm). and
`bovine serum albumin (3.55 nm).
`
`
`
`
`
`gto:‘atJOql.UO)dOsuo‘teensKq§10'.I|['MMMuroupapaqumoa
`
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`

`
`its concentration. Triton WR-1339 can either act as a
`
`Klauda, H. C., and D. B. Zilversmit. 1974. Influx of cho-
`
`
`
`
`
`JOURNALOFLIPIDRESEARCH§AsBMB
`
`surface stabilizer of HDL or penetrate the HDL particle
`and cause its disruption. In this context, we would like
`to acknowledge that Portman et al. (9) reported no effect
`of Triton WR-1339 on the HDL of the squirrel monkey.
`Based on our results and as also suggested by those au-
`thors, it is likely that the mass ratio of detergent to li-
`poprotein was too low to elicit an action.
`Overall, our studies show that Triton WR-1339 pro-
`vides a useful probe for the study of the structure of
`HDL and for the production of HDL derivatives of de-
`fined physico-chemical properties. Moreover, the knowl-
`edge of the properties of the Triton WR-1339-HDL
`complexes in vitro should provide a better basis for un-
`derstanding the effect of Triton WR-1339 in vivo. Triton
`WR-1339 has been shown to produce important changes
`in plasma lipoproteins when injected intravenously into
`animals (10-12). Because HDL plays a key role in the
`many lipoprotein interconversions occurring in plasma
`(34) and in view of the apparent high aflinity of Triton
`WR-1339 for the lipoprotein particles, we may postulate
`that one of the primary events, if not the primary event,
`in Triton WR-1339-induced dyslipoproteinemia is at the
`level of HDL. The replacement of apoA-I by Triton WR-
`l339 at the HDL surface could lead to an inability by
`this lipoprotein to act as an acceptor of the surface lipids
`generated by the lipolysis of the triglyceride-rich particles,
`decrease the efficiency of the LCAT-cholesteryl ester
`transfer/exchange system, and also affect the uptake of
`HDL by tissues. Since HDL has also been implicated in
`the cellular effiux of cholesterol (35), an impairment of
`this process may also be anticipated. All of these possi-
`bilities are amenable to experimental testing.“
`
`We gratefully acknowledge Dr. Ferenc Kezdy for his aid and
`advice on the surface tension measurements, and Rose E. Scott
`
`for the typing and preparation of the manuscript. The work
`was supported by grant USPHS-HI. 18577.
`Manuscript received 21 February 1984.
`
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`journal of Lipid Research Volume 25. 1984
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`PAGE 9 OF 10
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`lesterol into plasma in rabbits with fasting hyperbetalipo-
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