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CHOLESTEROL FLUX INTO ATHEROSCLEROTIC AORTA Bremmelgaard et al. 443
Methods
Patients
We studied 13 patients with aneurysms and one patientwith stenosis of the abdominal aorta who were scheduledfor arterial graft implantation. Two patients were used formeasurement of plasma contamination of the intimal layer(contamination experiment). One patient was used forcontrol of the identical behavior of 3H- and1 ̂ -cholesterolin the experiment (isotope equivalence experiment). Fivepatients were used for the investigation of the relationshipbetween exposure time and uptake of radioactive choles-terol by the aortic intima-media layer (time experiment).Finally, five patients were used for the measurement of theinflux of free and esterified cholesterol and esterificationand hydrolysis in the aortic tissue (distribution experiment)(Table 1).
Two plasma samples from each patient were labeledwith 3H- or 14C-cholesterol, respectively, and were there-after reinjected 0.08 to 541 hours before disruption of theblood supply to the aneurysmatic part of the aorta. Bloodsamples were drawn about 10 minutes, 1,2,6,24, and 48hours after the injection for determination of specific activ-ity decay curves from the time of injection of radioactivecholesterol until removal of tissue samples. During theoperation, one or several pieces of aortic tissue were re-moved from the area just proximal to the aneurysm and insome cases arterial tissue from the common iliac arterieswas also excised. Only intimal tissue free of coveringthrombus and visible fibrinous material was used for influxmeasurements.
Informed consent was given by all patients according tothe second Helsinki declaration. The injected amounts of
3H- and 14C-cholesterol were about 100 #iCi and 30respectively. The procedure for preparation of the sterileinjectates was approved by the appropriate Danish healthauthorities. The entire experimental protocol was ap-proved by the local ethics committee.
Isotope Purity
Preparations of 1 a, 2a (n)-3H-cholesterol and 4-1*C-cho-lesterol were obtained from Amersham (Amersham Inter-national Ltd., Bucks, U.K.). The isotope purity waschecked by chromatography on TLC-plates using hexane/dlethyl ether/glacial acetic acid 50:50:1 (vol/vol/vol) as themobile phase. The preparation was used only if more than95% of the radioactive cholesterol comigrated with a cho-lesterol standard (no. C-8258, Sigma Chemical Company,St. Louis, Missouri).
Preparation of Labeled Plasma
A 60 ml portion of blood was drawn from each patientinto a sterile syringe containing 6 ml of anticoagulant(CDP-A, Travenol Laboratories S.A. Castlebar, Eire). Itwas then separated into plasma and erythrocytes. Labeledcholesterol in ethanol was added to the plasma as pre-viously described7 and the erythrocytes were stored at 4° Cfor later use.
For the contamination and the time experiments, twoautologous plasma preparations were incubated for 48hours at 37° C with 3H- or 14C-cholesterol, respectively.Each of the two labeled plasma preparations were thenincubated with the patients' erythrocytes for 4 hours at 37°C. By this procedure, a considerable fraction of the labeledfree cholesterol in the lipoproteins was exchanged with
Table 1. Age, Sex, Experimental Protocol, and Plasma Cholesterol Concentrations for the Pa-tients Intravenously Injected with In Vitro Labeled Autologous Plasma before ReconstructiveArterial Surgery
Plasma cholesterol
Patient
AB
C
c
JKLM
DFGH*I
Age, Sex(yrs)
82 M58 M
72 M
69 M63 M67 F62 M54 M
62 M74 M76 M64 F63 M
Purpose ofexperiment!
II
II
IIIIIIIIIIIIIII
IVIVIVIVIV
TC(mM)
4.78 (32)$6.79 (30)
7.45 (35)
5.61 (28)5.70 (30)5.64 (33)3.88 (29)4.48 (34)
5.80 (30)5.95 (31)6.70 (32)7.65 (32)5.26 (32)
VLDLd<1.019
(mM)
0.290.65
3.58
0.320.711.480.422.37
0.820.581.490.720.85
LDL1.019<d< 1.063
(mM)
3.004.67
2.89
3.214.103.402.581.37
4.234.173.964.533.35
HDLd>1.083
(mM)
1.491.47
0.98
2.080.890.760.880.74
0.751.201.252.401.06
*This patient received an arterial graft due to stenosing atherosclerotic lesions.fRoman numbers indicate: I = plasma contamination of intimal tissue; II = isotope equivalency; III = tissue uptake
of labeled sterols after two different exposure periods (time experiments); IV = tissue uptake of labeled sterols afterexposure to differently labeled free and esterified plasma cholesterol (distribution experiment).
^Percent free of total cholesterol (TC).
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444 ARTERIOSCLEROSIS VOL 6, No 4, JULY/AUGUST 1986
unlabeled free cholesterol from the erythrocytes. The pa-tients were injected first with one preparation; then 24 to527 hours later they were given the other preparationwhich was labeled with the alternative isotope.
For the isotope equivalence experiment, a mixture of 3H-and 14C-cholesterol in ethanol was incubated with autolo-gous plasma for 1 hour at room temperature before thepreparation was injected into one patient.
Fc. t ie distribution experiment, one plasma sample waslabeled with 14C-cholesterol as described above for thecontamination and time experiments.3H-cholesterol wasadded to another plasma sample that was incubated forabout 1 hour at room temperature, which means that verylittle 3H-cholesteryl ester was formed before the milipore-filtered mixture of the two preparations was injected intothe patients. Plasma labeled by this procedure was investi-gated by chromatography on Bio-Gel A-150 m 50-100mesh (Bio-rad Laboratories, Richmond, California) usingan elutlng buffer containing 5 mM Trls-HCI (pH 7.4), 150mM NaCI, and 0.5 mM EDTA. The flow was 10 ml/hr andthe column was operated at 4° C. Samples of the plasmapreparation were also subjected to uttracentrifugation atdensities of 1.019 and 1.063. A MSE 65 ultracentrifugewtth a 6 x 4.2 swing-out rotor was used. After centrifuga-tion at 50,000 rpm for 16 hours, the samples were removedin 0.3 ml fractions from the top to the bottom of the tubes.The amounts of 3H and 14C free cholesterol were mea-sured in all fractions.
Prior to the administration, all injectates were millipore-filtered (Millex-HA, 0.22 /iM, Millipore S.A., Zone Indus-trielle, 67120 Molsheim, France).
Tissue Specimens
Blood samples were collected from the time of isotopeinjection until the excision of arterial tissue samples withsurface areas of 8 to 31 cm2. Immediately after excision,the tissue samples were rinsed thoroughly under running0.9% saline. The area was outlined and a small piece wastaken for histological examination on the basis of hematox-yiin-eosin- and orcein-stained slices. The remaining tissuewas separated into an inner, a middle, and an outer layer,or when the atherosclerotic destruction made this impos-sible, in an inner layer and an outer layer.
Analytical Procedures
The first and last plasma samples from each patientwere kept at 4° C for up to 48 hours. Aliquots from eachsample were adjusted to d = 1.063 and to d = 1.019, andcentrifuged at 4° C in a 50.3 rotor for 16 hours at 50 x 103
rpm corresponding to gey = 1.8 x 10s. Llpids were extract-ed from all samples of whole plasma and from the variousultracentrifuged fractions, and free and esterified choles-terol in the lipid extract were separated by thin-layer chro-matography (TLC).4 The sterols were extracted from silicagel with chloroform/ methanol, the extract was saponi-fied,12 and aliquots were taken for determination of thecholesterol by the LJeberman-Burchard method and fordetermination of 3H and 14C after evaporation of the sol-vent and addition of Instafluor (Packard Instrument Inter-national S.A. Zurich, Switzerland). The vials with plasma
lipids were counted in a liquid scintillation counter (LS 233Beckman Instruments, Birkerad, Denmark) to a SD of lessthan 1%. Overlap and efficiency were controlled by refer-ence to calibrated samples of 3H- and 14C-toluene ob-tained from Amersham.
Tissue samples were minced and lipids were extractedwith 20 volumes of chloroform/methanol, 2:1 (vol/vol) dur-ing a period of 24 hours. After the addition of 1 volume ofmethanol, the tissue-residue was centrifuged down intothe bottom of conical glass tubes. The residue was washedwith chloroform/methanol, 1:1 (vol/vol). After addition of 1volume of chloroform to the combined washes in order toreestablish the original 2:1 ratio, the extract was washedby the Folch procedure.13 Aliquots of the lipid extract fromthe tissue were used for determination of total cholesterolradioactivity and mass. Another aliquot was used for TLCseparation of free and esterified cholesterol which wereextracted from the silica gel and transferred to vials forradioactivity determination and for determination of cho-lesterol by the same procedure as used for the plasmasamples. The samples were counted to a so of 1 % or for300 minutes. The radioactivities in free and esterified cho-lesterol from the tissue were corrected for recoveries afterthin-layer chromatography (90% to 100%). The amount ofalkaline-soluble protein in the tissue was determined bythe method of Lowry,14 following extraction of the lipids anddigestion of the residue for 24 hours with 5 M NaOH. Ser-onomn (Nyegaard and Company, Oslo, Norway) was usedfor calibration. The tissue digest was neutralized by theaddition of HCI and was dissolved in instagel. These sam-ples had undetectable amounts of 3H and 14C. This corre-sponds to an extraction of more than 97% of the labeledlipids from the tissue.
Calculations
In all of the patients, the average specific activities offree and esterified cholesterol in plasma were calculatedfrom the area under the specific activity versus the timecurve. For two of the patients, the average free and esteri-fied cholesterol specific activities for each lipoprotein frac-tion during the experimental period were also calculated.
The influx of esterified cholesterol Kg in nmol x cm"2 xday 1 was calculated by the so-called sink or integralmethod, in which the amount of radioactivity in the tissuet(E) in dpm x cm"2 is divided by the average specificactivity in plasma s(E) expressed in dpm x nmol - 1 and bythe duration of the experimental period T in days. Theinflux of free cholesterol Kp is calculated in a similarmanner:15
Kg = t(E)/(s(E) x T) and Kp = t(F)/(s(F) x T) (1)
Labeled esterified cholesterol may be hydrolyzed in thearterial tissue subsequent to its entrance, and labeled freecholesterol may be esterified. These conversions are tak-en into account in a set of simultaneous influx equationswhich have been developed and discussed in detail else-where11' 16-17 in connection with their use in hypercholes-teralemlc rabbits. These influx equations are:
t(*E) = Kg x T x s(*E) - H x s(*E) + S x s(*F) (2)
t(°E) = KE x T x s(°E) - H x s(°E) + S x s(°F) (3)
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CHOLESTEROL FLUX INTO ATHEROSCLEROTIC AORTA Bremmelgaard et al. 445
t(*F) = K j x T x s(*F) + H x s(*E) - S x s(*F) (4)
t(°F) = Kp x T x s(°F) + H x s(°E) - S x s(°F) (5)
where * = 3H, ° = 14C, and the amount of newly enteredesterifled cholesterol in nmol x cm~2thathadbeenhydro-lyzed in the aortic tissue, and the amount of newly enteredfree cholesterol in nmol x cm" 2 that had been esterifiedduring the T days-long experimental period are named Hand S, respectively. Equations 2 to 5 can only be solved ifthey are independent. This is the case when:
s(*E)/s(*F) # s(°E)/s(°F) (6)
Equations 2 to 5 were used in the distribution experiment.
Results
Plasma
The labeled preparations were intravenously injectedinto 13 patients who all were normocholesterolemic (Table1). The plasma lipoprotein fractions showed cholesterolvalues which were not elevated for the age and sex exceptin one male (Patient E) who had a relatively high concen-tration of HDL.
Plasma Radioactivity
The decline of labeled free cholesterol was always muchfaster than that of labeled esterified cholesterol (Figure 1).
Volumes of distribution were calculated for both 3H- and14C-free and esterified cholesterol by an extrapolationback to zero time. This extrapolation was based on a singleexponential function of time, defined by the plasma sam-ples obtained 10 and 60 minutes after injection. Except forpatient C, the volumes were within the range of plasmavolumes determined by other methods.18 Injection of la-beled unphysiological particles that are eliminated by theliver in a few minutes would result in a too high volume ofdistribution compared with the plasma volume. Chroma-tography on Bio-Gel A 150 m showed that labeled choles-terol of Injectates incubated for 1 hour at room temperaturecomigrated with that incubated for 48 hours at 37° C. La-beling for 48 hours at 37° C has previously been shown toresult in physiological lipoprotein preparations.7 Further-more, urtracentrtfugation of a mixture of plasma incubatedwith 3H-cholesterol for 1 hour at room temperature and ofplasma incubated with 1 ̂ -cholesterol for 48 hours at 37°C showed that 3H and 14C labeled free cholesterol wereidentically distributed between the lipoprotein fractions.
In the time experiments, the decay curves of 3H and 14Csterols had the same shape (Figure 1 A) and the distribu-tion of label between free and esterified cholesterol in plas-ma averaged over the two different, but overlapping peri-ods, were similar for the two isotopes (Table 2).
In the distribution experiments the specific activities of3H- and 14C-free cholesterol decreased to about the samedegree (Figure 1 B). The specific activity of 14C-cholesteryl
10 20 30 40 0 10 20 30 40HOURS
Figure 1 . A. Semiiogarithmic plot of plasma radioactivity in a time experiment after injections of 14C-cholesterol 43hours before operation and of 3H-cholesterol 19 hours before operation. B. Semiiogarithmic plot of plasma specificactivity in a distribution experiment. 14C predominantly in esterified cholesterol and 3H exclusively in free cholesterolwere injected intravenously 44 hours before operation. Duration of the experiments is indicated on the abscissa. FC =free cholesterol; EC = esterified cholesterol.
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446 ARTERIOSCLEROSIS VOL 6, No 4, JULY/AUGUST 1986
Table 2. Labeled Free and Esterlfled Cholesterol In Plasma and Arterial Tissue
Patient
AB
C
EJKLM
DFQH1
Injectate
FCxiOO
TC3)_|
a2833
98
2628482020
9596979798
14C
3030
98
2638392419
6133322630
Exnosiira
3H
0.10.1
44
2219141316
22192244423
time*14C
(hrs)
2652
44
9443
5414445
22192244423
TC
(dpmx/il
85.631.4
31.9
46.841.6
108.131.538.0
24.716.248.920.234.1
Plasmaf
14C~1)
6.55.0
5.0
7.119.64.7
10.89.5
4.92.47.1
11.518.6
FCxiOO
TC3H
2431
61
1318301213
6139725965
1<C
1718
62
1721281417
3121181718
No. oftissue
samples
43
1
56356
41626
Inner layer of arterial
TC3f-| 14C(dpmxcm"2)
174*42
167
493618710389276
459596942502583
157*99
26
221487344327227
8011311479
222
wall
FCxiOO
TC
2938
—
3235452434
6847776971
14C
3538
—
4642483329
4830303843
Times from injection of the labeled plasma preparations until disaipture of blood supply to trie site of arterial tissue excision.tMean values during exposure time.^Values are calculated from total area of analyzed tissue samples and the sum of the radioactivities in these samples.
ester decreased slightly, whereas that of 3H-cholestery1ester showed a pronounced increase. This difference isdue to estertfication, which is more visible In 3H-cholesterylester than in 14C-cholesteryl ester because of the initiallack of 3H-cholesteryl ester in the injectate (Table 2). Eventhough hydrolysis and esterification of the sterols tend toequalize the specific activities in plasma of free and esteri-fied cholesterol for each of the two isotopes, this was notyet accomplished after 48 hours. When calculated over thefirst 24 hours, there was a two to four times higher percent-age of 3H in free cholesterol than there was of 14C in freecholesterol (Table 2).
The average specific activities in the three differentlipoproteins (VLDL, LDL, and HDL) over a 48-hour peri-od determined in two of the patients were nearly thesame for free cholesterol. This was also true for esterifiedcholesterol.
Arterial Tissue
Macroscopic and microscopic examination always re-vealed heavy atherosclerotic lesions with calcification,cholesterol accumulation, and partial destruction of thenormal architecture of the tissue. The cholesterol concen-tration decreased from the luminal to the abluminal layer,whereas the middle layer contained considerably less la-beled cholesterol than the inner and the outer layers (Fig-ure 2). The high amount of radioactivity in the outer layer isdue to a high blood contamination of that layer.7
Contamination Experiment
To investigate the quantitative importance of plasma lefton the intima-media tissue, one patient (A) was first inject-ed with plasma labeled with 14C-cholesterol 26 hours be-
200-
o100
ARTERIAL WALL
I M A I M A
Figure 2. The cholesterol concentration of the arterial wall (leftside) and the amount of 3H-cholesterol radioactivity (right side)obtained from Patient H. The letters I, M, and A denote Intimal,medial, and adventitial layers, respectively. Esterified cholesterolis represented by solid bars; free cholesterol Is represented byopen bars.
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CHOLESTEROL FLUX INTO ATHEROSCLEROTIC AORTA Bremmelgaard et al. 447
fore and again with plasma labeled with 3H-cholesterol 5minutes before disrupture of the blood supply to the aneu-rysmatic part of the abdominal aorta. In another patient (B)the same injections were made at 52 hours and at 7 min-utes before removal of the tissue. The amounts of 3H-cholesterol in the tissues can be ascribed to plasma con-tamination due to the short uptake times andcorresponded to 1.7 ± 1.2^1 (mean ± SD) plasma per cm2
(Table 3). When the amounts of 14C-cholesterol present inthat plasma volume are subtracted from the amounts of14C-sterols in the tissue after 26 and 52 hours exposure,respectively, about 95% of the 14C in the tissue could beascribed to a proper uptake.
Isotope Equivalence Experiment
In Patient C the two species of labeled cholesterol weremixed before incubation with the patient's plasma. Theratio 3HP*C in free and esterified cholesterol in plasmasamples drawn 0.2 to 44 hours after the injection was 6.1to 6.3 and 5.6 to 6.4, respectively. The 3H/14C ratio in totalcholesterol in the arterial tissue obtained 44 hours after theinjection of the labeled plasma was 6.3. We therefore con-sider the two labeled cholesterol preparations to behaveidentically when they are used to trace the in vivo transferof plasma cholesterol into and out of the arterial tissue.
Time Experiment
The amounts of labeled free and esterified cholesterol inthe arterial tissue were divided by the mean values of thecorresponding specific activities in plasma. These normal-ized arterial radioactivity values for the long exposure timeand for the short exposure time were depicted in relation tothe duration of the exposure time (Figure 3). The samevalues obtained in the contamination experiment are alsodepicted. The lines that connect the values calculated foreach tissue sample intercept the y-axis close to the origin.This linear increase of labeled free and esterified choles-terol in the tissue from time 0 to 40 hours after the injection,under conditions of a fictive constant specific activity inplasma of the two sterol fractions, is in accordance with aconstant influx of free and esterified cholesterol with anegligible efflux of labeled cholesterol during an exposuretime of about 50 hours. The average accumulation rate ofradioactive free and esterified cholesterol decreased after50 hours exposure time, probably due to equilibration be-tween influx and efflux of labeled cholesterol.
The calculated influx values for the time experiment andfor the long exposure time of the contamination experimentwhen using equation 1 are given in Table 4. The influx for26 to 94 hours exposure time compared to that for the 13 to22 hours exposure time in the same tissue were: 60% to150% (99% ± 5%, mean ± SE) for free cholesterol and40% to 170% (95% ± 8%) for esterified cholesterol (pa-tients E, J, L, M). The calculated influx values are theslopes of the lines in Figure 3 from the origin to the valuesat the longer and shorter exposure times. The similaritybetween the two slopes is in accordance with a linearrelationship between the uptake of radioactivity and theexposure time when corrected for the decreasing specificactivity in plasma.
Table 3. Plasma Contamination of Aortic Intimal Tis-sue Calculated from the Amounts of Labeled Choles-terol In the Tissue and In Plasma.
Plasma contamination
FCPatient
EC(/dxcrrr2)
FC EC
A
B
1.8*22.1.04.8
1.02.61.5
1.4*1.60.73.8
0.51.81.3
2.6t1.91.06.7
2.33.12.0
6.6t4.82.0
13.0
4.911.13.2
Exposure times for Patient A were 0.08 hours (*H) and 26 hours(14C) and for Patient B 0.12 hours ( ^ and 52 hours (14C).
*The values were calculated by dividing the amount of labeled freecholesterol fH-FC) and labeled esterified cholesterol fH-EC) in thetissue by the corresponding ^ concentrations in the final plasmasamples obtained at the end of the exposure time.
tThe values given in the two previous columns were multiplied bythe corresponding 14C concentrations in the final plasma samplesand expressed as percent of the total 14C-FC and 14C-EC found inthe tissue after 26 and 52 hours, respectively.
150
60HOURS AFTER INJECTION
80
Figure 3. The 29 tissue samples obtained from six patients (A,B, E, J, L, M) were exposed to the 3H-cholesterol for a shorter timeand to the 14C-cholesterol for a longer time in the contaminationand time experiments. A. Labeled esterified cholesterol in thetissue [t(E)] normalized with mean specific activity of esterifiedcholesterol in plasma [s(E)] expressed as nmol x cm" 2 . B.Labeled free cholesterol in the tissue [t(F)] normalized with meanspecific activity of free cholesterol in plasma [s(F)] expressed asnmol xon"'.
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448 ARTERIOSCLEROSIS VOL 6, No 4, JULY/AUGUST 1986
Table 4. Concentrations of Cholesterol and Protein in the Inner Layer of Human Arterial Tissue with AdvancedAtherosclerosis and the In Vivo Flux of Free and Esterrfied Cholesterol from Plasma into the Arterial Tissue
Experiment
No. oftissue
samples
Time and contamination experiments (6Shorter exposure fH) 22*
Longer exposure (14C)
Distribution experiments (5Injectate incubated for
Injectate incubated for48 hours (14C)
Calculation by Eq. 2-5
29
patients)§
19
19
Wetweight
(gxcn r 2 )
patients)t0.13±0.01
0.13± 0.01
0.13 ±0.01
0.13±0.01
Protein(mgxcm"2)
16±1.0
16±1.0
14±1.8
14±1.8
TC
cm"2)
11 ±0.9
11 ±0.9
13±2.4
13±2.4
FCX100TC
45±1.5
45±1.5
50 ±2.2
50 + 2.2
suretimes(hrs)
13-22
26-94
22-192
22-192
Plasmaradio-
activity*
FCx100TC
12-18
14-21
39-72
17-31
Influx
FC EC(nmolxcm"2
xday~1)
44±4
46±4
37±5
50±6
41±5|[15±1]H
37±5
35±3
56±6
36±4
45±5|[20 ±2]**
Values are means ± SE.'Mean values during the exposure times.fFirst injection of plasma labeled with 14C-cholesterol and later injection of plasma labeled with 3H-cholesterol.i l n Patients A and B, influx was not calculated for the 0.08 and 0.12 hours exposure time.§Simultaneous injection of two plasma preparations labeled with 3H primarily in free and 14C primarily in esterified cholesterol.|lnflux values which take into account that labeled sterols are hydrolyzed and esterified in the arterial tissue.lIEsterification of labeled free cholesterol in the arterial tissue as % of the amount which has entered."Hydrolysis of labeled esterified cholesterol in the arterial tissue as % of the amount which has entered.
Due to a postponement of the operation in Patient Kafter the injection of the 14C-labeled plasma preparation,the interval between the injections of 14C- and ^-choles-terol was extended to 527 hours. The influx value calculat-ed for the 541 hours exposure time was only about 30% ofthat calculated for the 14-hour period. This suggests aconsiderable loss of labeled sterols from the aortic tissuebetween Day 20 and Day 2 before the operation. In theory,however, in short term experiments the efflux mechanismalso influences the measured values which will be lowerthan the true values.
Distribution Experiment
The quantitative importance of hydrolysis and estertfica-tion in the arterial wall was evaluated in five patients whoreceived a labeled plasma preparation, which resulted in adifferent distribution of 3H and 14C between free and esteri-fied cholesterol in plasma during the exposure time (Table2). When Equation 1 was used on the data in these experi-ments the influx of free cholesterol based on 3H was 44%to 93% (72% ± 3%, mean ± SE) of that based on 14C in thesame tissue in spite of identical exposure periods. Like-wise, the influx of esterified cholesterol based on 3H was110% to 431% (168% ± 16%, mean ± SE) of that basedon 14C in the same tissue in spite of the same exposuretime for the two isotopes (Table 4). These differences canbe explained by hydrolysis and esterification of the labeledsterols in the tissue in combination with the different distri-bution of ̂ and 14C between free and esterified cholester-ol in plasma. This is taken into account by use of Equations
2 to 5. About 7% to 30% of the labeled estertfied cholester-ol that had entered the arterial tissue was hydrolized and8% to 24% of the labeled free cholesterol was esterifiedduring the exposure time.
Discussion
Assumptions for the Influx Calculation
In order to calculate an influx based on the amounts oflabeled sterols in plasma and in the arterial tissue usingEquations 1 to 5, the following assumptions have to befulfilled.
Labeled Free and Esterified Cholesterol ShouldTrace Endogenous Free and Esterified PlasmaCholesterol
The plasma samples from the patients were labeled byan in vitro procedure, which changes the composition ofthe plasma lipoproteins. Immediately after the injection theaortic tissue was thus exposed to labeled cholesterol pres-ent in these lipoproteins. The small amounts of labeledcholesterol in the intimal layer of the artery after 0.08 to0.12 hours of exposure time compared with the amount ofradioactivity in the same tissue after 26 to 52 hours ofexposure time exclude a preferential uptake of labeledcholesterol during that initial period (Table 3).
Labeled free and esterified cholesterol from injected li-poproteins are rapidly transferred into the much larger poolof endogenous lipoproteins by an exchange of free and
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CHOLESTEROL FLUX INTO ATHEROSCLEROTIC AORTA Bremmelgaard et al. 449
esterified cholesterol. Furthermore, appreciable amountsof labeled esterified cholesterol in plasma are formed by invivo esterification of labeled free cholesterol (Figure 1 B).Due to this exchange and esterification, the aortic tissue isexposed to labeled cholesterol in intact lipoproteins for themajor part of the experimental period.8'1920
Same Specific Activities of Free and of EsterifiedCholesterol in All Lipoprotein Fractions
The exchange of free and of esterified cholesterol be-tween the various lipoprotein fractions in plasma keeps thepool of free cholesterol and of esterified cholesterol rela-tively well stirred. In the hours after injection of the labeledplasma, there are still considerable differences in specificactivities of cholesteryl ester between VLDL and HDL7
This can be ascribed to a preferential exchange of choles-teryl ester between these two lipoprotein fractions.21 Overa period of more than 24 hours, these differences disap-pear.7 When patients are intravenously injected with all thecholesteryl ester label in one lipoprotein species, it usuallytakes less than 24 hours to reach equilibrium between theother lipoprotein fractions in plasma (unpublished data). Inthe present study the aortic influx of free and esterifiedcholesterol may be derived from only one or two of theplasma lipoprotein fractions without invalidation of the in-flux calculation.
The Amounts of Labeled Sterols in ContaminatingPlasma on the Tissue Should Be Small Comparedwith the Amounts of Labeled Sterols in the Tissue
By intravenous injection of a labeled plasma preparation5 to 7 minutes before the removal of the aortic specimens,the amount of contaminating plasma was estimated to 1 to5 fi\ per cm2, which could account for 1% to 13% of thelabel that had entered during the preceding period of 1 to 2days (Table 3).
777e Two Species of Labeled Cholesterol Should BeTreated Identically by the Arterial Tissue
A decreasing ratio between 3H and 14C in plasma hasbeen observed over a period of 24 hours when a mixture of3H- and 14C-cholesterol was given orally to humans.22 Inthe present experiment (Patient C) and in another similarexperiment7 there were no significant differences betweenthe 3H/1*C ratios in plasma and in arterial tissue after intra-venous injection of a mixture of the two species of radioac-tive cholesterol.
7776 Amount of Labeled Sterols Leaving the Tissueduring the Exposure Time Should Be SmallCompared with the Amount Entering the Tissue
This was studied in the time experiment by the injectionof a plasma preparation labeled with 1 ̂ -cholesterol andthen 1 to 3 days later with a plasma preparation labeledwith 3H-cholesterol. If all labeled free cholesterol remainedin the arterial wall as free cholesterol during the longer
exposure time, the influx calculated for the longer timewould be identical with the influx calculated for the shortertime. If some of the labeled free cholesterol in the arterialtissue disappeared either by efflux or by esterification, theinflux calculated for the longer time would be lower thanthe influx calculated for the shorter time. In contrast, hy-drolysis of labeled esterified cholesterol would falsely in-crease the calculated influx of free cholesterol. The sameconsiderations apply to the influx of esterified cholesterolexcept that hydrolysis and esterification in the arterial wallact in the opposite directions compared with the calcula-tion of influx of free cholesterol.
The nearly identical values obtained for an influx periodof 13 to 22 hours and for a period of 26 to 94 hours in thesame tissue are in accordance with the notion that themajor part of the labeled free and esterified cholesterolremains in the tissue during the first 2 to 3 days. This doesnot exclude the possibility that unlabeled free and esteri-fied cholesterol continuously leave the tissue during theexperimental period.
Hydrolysis and Esterification
The ratio between the influx of free and esterified choles-terol was 1.5 ± 0.1 and 1.4 ± 0.1 (mean ± SE) for thelonger and the shorter exposure times, respectively. Theinflux of free cholesterol is thus too high to be explainedexclusively as an influx of plasma lipoproteins, which hadratios of 0.4, 0.4, and 0.9 between free and esterified cho-lesterol for HDL, LDL, and VLDL, respectively. Since thespecific activity of esterified cholesterol in plasma in thetime experiments was several times higher than the specif-ic activity of free cholesterol for both isotopes (Table 2),hydrolysis in the arterial wall of a small fraction of theesterified cholesterol from plasma could result in a falselyhigh influx value for free cholesterol. The occurrence oflabeled esterified cholesterol in human atherosclerotic le-sions has been ascribed9 mainly to in situ esterification byan extrapolation from in vitro studies of foam cells.23 In thepresent study, we demonstrate that the occurrence of la-beled esterified cholesterol in the plaque can only to aminor extent be explained by in situ esterification. Esterifi-cation of free cholesterol in the aortic tissue can be due toboth ACAT formed in the aortic cells and to LCAT whichenters the tissue together with other plasma macro-molecules.
Influx of Free and Esterified Cholesterol
The mean values for influx of free cholesterol and ofesterified cholesterol are similar whether the calculationsare based on a long (3 to 4 days) or a short (1 to 2 days)exposure time or whether it is based on labeled cholesterolprimarily present in free cholesterol or in esterified choles-terol (Table 4). These influx values are therefore a goodestimate of the true influx of free and esterified plasmacholesterol into arterial tissue with severe atherosclerosis.
Studies in perfused liver and heart preparations fromanimals, have suggested that cholesteryl ester can movefrom plasma lipoproteins into the tissues by a processwhich does not involve a transfer of the entire lipoprotein
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450 ARTERIOSCLEROSIS VOL 6, No 4, JULY/AUGUST 1986
particles.24'x In the hypercholesterolemic rabbit, however,it has been shown that the simultaneously measured invivo influx of apoproteins and cholesteryl ester into aortictissue is in accordance with an influx of plasma lipoproteinparticles.4 If influx values recently reported for radio-iodin-ated apoprotein B in human atherosclerotic aortic tissue28
occurred as part of lipoprotein particles, the accompanyinginflux of cholesteryl ester could be calculated to about 17nmol x cm"2 x d a y 1 . This Is about 40% of the choles-teryl ester influx reported in the present study (Table 4).However, the 17 nmol x cm" 2 x day"1 does not includethe influx of cholesteryl ester from HDL. Furthermore, thevalues for the apoprotein B influx are minimal values, be-cause efflux or degradation of iodinated apoproteins in thearterial tissue occur during the first 20 hours.26 The influx ofcholesteryl ester in the present study was up to 100 timeshigher than in human ascending aorta without atheroscle-rosis,7 with the mean value being about 10 times higher.Nearly the same 10-fold difference in influx of apoprotein Bin VLDL, IDL, and LDL between aortic tissue with andwithout atherosclerosis was reported by Nicoll et al.26
These Influx values for apoproteins and cholesteryl esterare thus in accordance with a transfer of these two lipopro-tein constituents into human atherosclerotic tissue as partof a transfer of lipoprotein particles.
An influx of intact lipoprotein particles is also supportedby the isolation of LDL- and VLDL-like particles in aortictissue homogenates26-28 or in interstitial fluid extractedfrom the intima-media tissue with small pieces of filterpaper.29
These observations indicate that the difference in mor-phology between atherosclerotic and nonatherosclerotictissue is associated with a marked difference in uptake ofplasma lipoproteins. However, the influx of esterified cho-lesterol into atherosclerotic tissue did not con-elate with thecholesterol content of the tissue specimens (Figure 4 B).This was also true when the data from patients with non-atherosclerotic aortas were included in the calculation.Similar experiments including hypercholesterolemic sub-jects may have different results.
In these normocholesterolemic subjects, the influx wasnot significantly correlated to the concentration of plasmacholesterol (Figure 4 A) or to the concentration of choles-terol in any lipoprotein fraction. This does not exclude cor-relation between the cholesterol influx into the artery walland the cholesterol concentration in plasma within thesame patient or if patients with hypercholesterolemia wereincluded in the study.
The influx of free cholesterol was strongly correlatedwith the simultaneously measured influx of esterified cho-lesterol (Figure 5). Since esterified cholesterol apparentlyenters the arterial tissue as part of a lipoprotein influx, it isaccompanied by the free cholesterol of the lipoprotein.This influx of free cholesterol with the lipoproteins canaccount for only 54% ± 4% (mean ± SE) of the total influxof free cholesterol. The excessive influx of free cholesterolcan probably be ascribed to the exchange of free choles-terol between plasma lipoproteins and the blood/arterysurface like the flux of free cholesterol between red bloodcells and the lipoproteins.30 This nonlipoprotein-associat-ed influx of free cholesterol showed a weak positive corre-lation with the influx of esterified cholesterol.
25 50 75NM0LxCM'2xDAY"1
INFLUX OF CHOLESTERYL ESTER
100
Figure 4. A. Aortic influx of esterified cholesterol in relation tothe total concentration of cholesterol in plasma. B. Aortic influxof esterified cholesterol in relation to the total content of choles-terol In the aortic tissue.
25 50 751 NMOL xCM" 2 x DAY"1
INFLUX OF CHOLESTERYL ESTER
Figure 5. Aortic influx of free cholesterol in relation to the aorticinflux of esterified cholesterol. Equation of the regression line: y =0.87x + 9.40 (r = 0.82). o indicates values without correction forhydrolysis and esterification obtained in contamination and timeexperiments. • indicates values corrected for hydrolysis and es-terification obtained in the distribution experiments.
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CHOLESTEROL FLUX INTO ATHEROSCLEROTIC AORTA Bremmelgaard et al. 451
Efflux of Aortic Cholesterol
If the influx of free and esterified cholesterol is constantwith time, about 30 /tmol of cholesterol enters 1 cm2
atherosclerotic tissue surface per year. This is nearly twiceas much as the mean value for the cholesterol contentbehind 1 cm2 of aortic surface and indicates that the influxprovides enough cholesterol for a considerable monthlyaccumulation if the cholesterol remains in the tissue. Fromthe amounts of radioactive apoprotein B in the aortic tissue5 hours and 20 hours after its injection in plasma, it can becalculated that at least 70% of the apoprotein that hasentered during 20 hours has become degraded.28 Duringthe same period of time, we found that about 20% of thenewly entered sterols were hydrolyzed and esterified. Itmeans that lipoproteins from plasma are subject to a con-siderable intra- or extracellular enzymatic degradationafter they have entered the intima.
In one patient from whom tissue was removed 541 hoursafter the first injection, the isotope data were in accordancewith an efflux of about 70% of the cholesterol that hadentered during that time. The ratio between labeled freeand esterified cholesterol in the tissue after 14 hours andafter 541 hours is the same (Table 2). A similar unchangedratio was also observed in monkeys killed from 1 to 52weeks after injections of the labeled sterols.10 These ob-servations are compatible with an efflux of esterified cho-lesterol without prior hydrolysis to free cholesterol.
Accumulation of Aortic Cholesterol
The accumulation of free and esterified cholesterol Inthe abdominal aorta was recently published based on au-topsy studies of men from New Orleans.31 The intimal cho-lesterol content in the fifth decade was 10 ± 2 /imol xcm"2, which is similar to the cholesterol content in thepatients of this study. The accumulation of free and esteri-
fied cholesterol was found to be 0.39 and 0.43 nmol xcm"2 x d a y 1 from the second through the fifth decadeand 4.7 and 4.6 nmol x cm" 2 x day"1 from the fourth tothe fifth decade. This average accumulation rate of freeand esterified cholesterol is much lower than the choles-teryl ester influx of 45 nmol x cm"2 x day"1 reported inthe present study. The difference suggests a considerableefflux of cholesterol from the arterial tissue. The lack ofcorrelation between influx of cholesteryl ester and contentin the aortic tissue (Figure 4 B) also suggests that thecholesterol content in the tissue is determined by some-thing other than the influx, and that is probably efflux. Theconsistently higher values for cholesterol influx in our studycompared to the investigations by Field et al.8 and byJagannathan et al.9 on human atherosclerotic abdominalaortas (Table 5) can be explained by the shorter exposureperiods during which efflux of the tracer from the intimaltissue is minimal. The nearly identical concentrations ofcholesterol in the aortic tissue together with the differencesin influx between the present study and that of Jagan-nathan et al.9 suggest that about half of the cholesterol thatenters the atherosclerotic intimal tissue of abdominal aortais retained.
In the study by Un et al.10 of hypercholesterolemic mon-keys, the influx of cholesterol per unit area was ratherclose to that of humans when they used a similar tech-nique,9 and with shorter exposure periods the influx valueswould presumably increase.
In conclusion, we have obtained data that are in accor-dance with an influx of esterified cholesterol into athero-sclerotic tissue as part of a lipoprotein influx that is up to100 times higher than that found in aortic tissue withoutatherosclerosis. The cholesterol influx was so high that theatherosclerotic lesions can be prone to fast progression.Whether this progression occurs seems to depend on themagnitude of the cholesteryl ester efflux from the lesion.
Table 5. Four Studies of the In Vivo Flux of Cholesterol Into Atherosclerotic Tissue from Abdominal Aorta UsingRadioactive Cholesterol
Author(ref)
Retd et al.(8)
Jagannathanet al. (9)
Un et al. (10)
Present
No. ofpatients
8
6
6
12
Age(yre)
61 (26-71)
51 (40-63)
Adult
66(54-82)
Genus
Human
Human
Monkey |
Human
No. ofspeci-mens
8
—
51
Plasmacholes-
terol(mM)
4.9 ±0.3
7.0 ±1.0
16±2
5.7 ±0.3
49
75
40
Expo-sure
period(days)
(2.5-137)
(61-96)
(7-70)
1.6 (0.5-8)
Aortic cholesterol
Concen-tration
Oomolxg~1)1-r
44±15t86±25$
176±31§103±11 (33)*
40 ±6 (54)*
101 ±10 (48)*
(nmolxcm"2xday"1)
1 1 "
34 ±3.411
30 ± 5 "
85±6
(intima)
Influx(nmolxg - 1 *day"1)
225(43)*
282±28
485 ±84 (59)*
723 ±62 (53)*
Values are means ± SE. Numbers In parentheses indicate range except as indicated by asterisk.'Values in parenthesis are free cholesterol as percent of total.tNormal appearing.^Thickened intima (plaques).§Free lying liptd material.|Cholesterol fed rhesus monkeys.lAssuming tissue thickness of 0.12 cm."Assuming tissue thickness of 0.05 to 0.06 cm.ttAssuming 60% water content of the tissue (wet weight).
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A Bremmelgaard, S Stender, J Lorentzen and K Kjeldsenatherosclerosis.
In vivo flux of plasma cholesterol into human abdominal aorta with advanced
Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 1986 American Heart Association, Inc. All rights reserved.
Avenue, Dallas, TX 75231is published by the American Heart Association, 7272 GreenvilleArteriosclerosis, Thrombosis, and Vascular Biology
doi: 10.1161/01.ATV.6.4.4421986;6:442-452Arterioscler Thromb Vasc Biol.
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