© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Quantification In Situ of Crystalline Cholesterol and Calcium Phosphate Hydroxyapatite in Human Atherosclerotic Plaques by Solid-State Magic Angle Spinning NMR
From the Departments of Medicine (W.G., J.A.H.) and Biophysics (J.A.H.), Boston University School of Medicine, Boston, Mass; the Departments of Medicine and Biochemistry (J.D.M.) and the Department of Surgery (M.E.D.B., G.M.L.), Baylor College of Medicine, Houston, Tex.
Correspondence to James A. Hamilton, PhD, Department of Biophysics, Boston University School of Medicine, Boston, MA 02118. E-mail Hamilton{at}med-biophd.bu.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Because of renewed interest in the progression, stabilization, and regression of atherosclerotic plaques, it has become important to develop methods for characterizing structural features of plaques in situ and noninvasively. We present a nondestructive method for ex vivo quantification of 2 solid-phase components of plaques: crystalline cholesterol and calcium phosphate salts. Magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of human carotid endarterectomy plaques revealed 13C resonances of crystalline cholesterol monohydrate and a 31P resonance of calcium phosphate hydroxyapatite (CPH). The spectra were obtained under conditions in which there was little or no interference from other chemical components and were suitable for quantification in situ of the crystalline cholesterol and CPH. Carotid atherosclerotic plaques showed a wide variation in their crystalline cholesterol content. The calculated molar ratio of liquid-crystalline cholesterol to phospholipid ranged from 1.1 to 1.7, demonstrating different capabilities of the phospholipids to reduce crystallization of cholesterol. The spectral properties of the phosphate groups in CPH in carotid plaques were identical to those of CPH in bone. 31P MAS NMR is a simple, rapid method for quantification of calcium phosphate salts in tissue without extraction and time-consuming chemical analysis. Crystalline phases in intact atherosclerotic plaques (ex vivo) can be quantified accurately by solid-state 13C and 31P MAS NMR spectroscopy.
Key Words: magic angle spinning NMR • crystalline cholesterol • calcium phosphate hydroxyapatite • phospholipids • atherosclerotic plaques
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
There is renewed interest in the ultrastructure of atherosclerotic plaques for several reasons. Recent work has shown that plaque stability, which is dependent on composition and ultrastructure, plays an important role in cardiovascular disease. Plaques with a lipid-rich core and a thin, fibrous cap are more likely to rupture, leading to thrombus formation.1 2 3 Some ultrastructural features underlying plaque stability are now known in sufficient detail to predict plaque stability. The thickness of the fibrous cap and the infiltration of macrophages and T cells are highly correlated with plaque vulnerability and rupture.4 Because there is new evidence that certain diets and drugs may promote regression of plaques,5 it is important to determine the relationship between the physicochemical properties of plaques and regression potential. Lipids present in different phases have different physical properties (eg, diffusion rates), which are directly related to the rate of lipid egress and plaque regression.6 Therefore, quantitative knowledge of the lipid phases in a plaque in situ is crucial.
Cholesterol is 1 of the major lipids found in atherosclerotic plaques.7 At the early stage of plaque development, cholesterol is incorporated mainly into phospholipid bilayers in the liquid-crystalline phase and, to a lesser extent, is partitioned into liquid cholesteryl esters (CEs).7 With progression of the plaque, cholesterol accumulates to higher levels, saturating the liquid and liquid-crystalline phases and forming a separate crystalline phase at body temperature. Methods to date have not succeeded in providing data about the amounts of different lipid phases, particularly the crystalline phase. The total cholesterol in a plaque can be measured by chemical analysis of extracts of homogenized tissue, but information about the physical state and molecular interactions of cholesterol, as well as other lipids, cannot be obtained with this approach. X-ray diffraction and optical microscopy are useful for identification of lipid phases8 but are usually not efficient for quantitative analysis. Raman spectroscopy has been used to quantify lipids in plaques ex vivo,9 10 but this method has not distinguished lipids in different phases.
Calcium phosphate hydroxyapatite (CPH) is another solid component often present in advanced atherosclerotic lesions. Calcification is an active, regulated process and is probably involved in the very early development of atherosclerotic lesions.11 12 Potential links between calcification and atherosclerosis have recently been proposed.13 Whereas natural or synthetic calcification can be detected by x-ray diffraction (CT),14 B-mode echography,15 or MRI,16 quantification of calcium salts in biological samples still requires that the tissue sample be delipidated and then digested with concentrated acid before standard chemical assays are performed.17 18 Therefore, introduction of simple and reliable instrumental methods to quantify the plaque composition (ex vivo) will be useful for the interpretation and calibration of the in vivo measurements.
NMR spectroscopy is a nondestructive method that detects the magnetic resonances of nuclei with different chemical environments. NMR spectroscopy studies of human atherosclerotic tissues date back to the early 1970s but were limited mostly to the identification and quantification of the lipids in different liquid phases.19 20 Newer applications of solid-state NMR with magic angle sample spinning (MAS NMR) extend the feasibility of using NMR to study anisotropic biological samples, as documented by reports from our laboratory21 22 23 and others.24 Our previous study of atherosclerotic plaques focused on establishing protocols to identify different plaque components in a qualitative manner. The current study presents new and detailed protocols to quantify the crystalline cholesterol and calcium phosphate in situ by MAS NMR. The application of this method is demonstrated by using human carotid plaques as the biological sample.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Plaque Samples
Tissues obtained from endarterectomy specimens, which included atherosclerotic plaques and some adherent intima and media, were dissected, beginning at the common carotid artery and extending to 1 to 2 cm below the bifurcation and
1 cm
into the internal carotid artery. Except as noted below, tissues were
fixed in 10% formalin–Tris-buffered saline buffer immediately after
excision at The Methodist Hospital, Houston, Tex. Formalin stabilizes
the tissue by cross-linking its proteins but does not alter the
physical properties of lipids in the plaque.25 Fixed
plaque samples were shipped to Boston at ambient temperature. For NMR
analysis, samples were cut into a suitable size
(6x4x2 mm) and then transferred to a 7-mm
ZrO2 rotor without buffer. Two fresh tissue
samples were shipped in 50% glycerol/PBS with "blue ice." These
specimens were studied by 13C MAS NMR
spectroscopy before and after formalin fixation. No significant
differences in the 13C spectra of fixed and
unfixed tissue were detected.
Chemical Preparation
Cholesterol (>99% pure as checked by thin-layer
chromatography) was purchased from Nu-Chek Prep
and used without further purification. Cholesterol
monohydrate crystals were prepared by recrystallization of anhydrous
cholesterol in ethanol/water (70:30,
vol/vol).21 Synthetic CPH was purchased from Aldrich
Chemical Co and used without further purification. Chicken wings were
purchased at a local supermarket (Boston, Mass) and boiled. The cleaned
bones were ground into powder for NMR and x-ray diffraction
experiments.
NMR Spectroscopy
Experiments were performed on a Bruker AMX-300 spectrometer
equipped with solid-state and MAS accessories. A standard
cross-polarization (CP) pulse sequence or a dephased CP pulse sequence
was used for the study of crystalline cholesterol (2-ms
contact time, 45-µs dephasing delay), with a sample spinning rate of
5 kHz. High-power proton decoupling (65 W) was used in all
13C experiments to remove the C-H dipolar
couplings. A standard direct-polarization observation pulse sequence
with a moderate 1H decoupling power level (25
W) was used to study phosphorus in calcified plaques with a sample
spinning rate of 4 kHz. Although significant
1H-31P interactions have
been described in phospholipid bilayers,26 such
interactions are minimal in CPH
[3Ca3(PO4)2Ca(OH)2]
because the dipolar interaction decays in proportion to
1/r6 of the internuclear distance.
Therefore, a moderate 1H decoupling was
sufficient to remove line-broadening effects by any other nonspecific
1H-31P interactions.
To optimize the sample temperature for the NMR experiments, preliminary
tests were performed at ambient temperature (25°C), 37°C, and
50°C. Crystalline cholesterol monohydrate,
liquid-crystalline cholesterol, and CPH were
thermotropically stable in this temperature range, and the spectral
features of these components were essentially unchanged. The results
reported in this study were acquired at ambient temperature (25°C)
to maximize sample preservation. It is important to emphasize that
calibration of signal intensities must be done for each
probe/spectrometer and must be performed at constant-temperature as
well as constant-acquisition conditions.
X-Ray Powder Diffraction
X-ray powder diffraction patterns were recorded by using
nickel-filtered CuK
radiation from an Elliot GX-6 rotating-mode
generator (Elliot Automation) equipped with a camera using Franks
double-mirror optics.27 The sample (1.5 mg) was packed
into 1.0-mm-diameter Lindeman capillary tubes (Charles Super)
and sealed. Diffraction experiments were performed at room temperature.
The sample-to-film distance was calibrated by using the data of a
standard material (crystalline cholesteryl myristate).
Chemical Analysis
After completion of the NMR experiments, the tissue sample was
freeze-dried overnight to constant weight. Tissue dry weight was
measured by direct weighing on an electric balance. Lipids were then
extracted by the Folch protocol.28 The total lipid was
measured as the dry weight of the lipid extract, and the quantities of
specific lipids were measured by standard methods.28 29 30 31
Folch extraction has been widely tested and accepted as 1 of the
standard extraction protocols for the analysis of lipids in
biological samples.32
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Quantification of Cholesterol Monohydrate in Human Carotid Plaques
In atherosclerotic plaques, cholesterol can exist in 3 forms: solubilized in the isotropic CE phase; a liquid-crystalline form within phospholipid bilayers (CholLiqCr); and a crystalline form (cholesterol monohydrate [CholM]). The distribution of cholesterol between the crystalline and liquid-crystalline phases that predominate in the plaque cannot be directly estimated by chemical analysis because this information is lost when the lipids are extracted into the same organic solution. Furthermore, the incorporation of cholesterol into phospholipid bilayers in atherosclerotic plaques depends on their sphingomyelin content8 33 34 as well as other (unknown) factors,35 making it difficult to predict the exact amount of liquid-crystalline cholesterol in the bilayer structures.
Previously, we demonstrated that CholM and CholLiqCr can be identified
without interference from other plaque components in separate
13C MAS NMR experiments with pulse sequences
optimized to highlight each phase separately.23 It would
be ideal if both of these phases could also be quantified in situ.
However, we were not able to calibrate the signal intensity of
CholLiqCr in plaques (see below), probably because of variations in the
phospholipids, the more heterogeneous environments that
might exist in the lamellar phase, and/or the motion characteristics of
the CholLiqCr in the plaque. On the other hand, CholM in plaques is
relatively inert and essentially the same as chemically pure
cholesterol monohydrate.34 Therefore, we have
investigated the feasibility of quantifying CholM in situ and then
estimating CholLiqCr by using the following equation: CholM+CholLiqCr
+cholesterol (iso)=total cholestero1, where
cholesterol (iso) represents the amount of
cholesterol in the isotropic CE phase. Total
cholesterol and CE can be analyzed by chemical
analysis after NMR experiments, and cholesterol
(iso) can be calculated from its solubility in CE, which has already
been established (5.2% to 5.5% by weight).21 36
Because of its low abundance, cholesterol in this pool has
not been detected in natural-abundance NMR spectra of
plaques.20 37 38
As shown in Figure 1
, the
13C CP MAS NMR spectrum of a typical carotid
plaque contains signals arising from CholM, CholLiqCr, phospholipids,
and proteins.22 39 The CP MAS efficiently filters out most
signals from noncrystalline lipids.23 In the spectrum of
Figure 1, the C5 twin peaks are well resolved from other
resonances and are also symmetrical as they are for pure
CholM,23 indicating that the signal contribution from the
CholLiqCr phase is negligible. Otherwise, the downfield peak would be
more intense because its chemical shift is close to that of the C5 peak
of cholesterol in a liquid-crystalline phase of a simple
model system.21 (It is not clear why the C5 peak from the
CholLiqCr phase in the plaque was not detected, but this is another
indication of the problems encountered when attempting to quantify
CholLiqCr as discussed above.) Therefore, quantification of CholM in
principle can be achieved by integration of the C5 twin peaks without
interference from the CholLiqCr phase. A prerequisite for
cholesterol quantification in plaque is demonstration of
the accurate quantification of CholM.
|
CH3),
and cholesterol resonances (C, followed by IUPAC
carbon numbers).
Spectra of pure CholM of varying quantities were obtained under
identical conditions. Selected spectra (C5 region) are shown in Figure 2 (left). The integrated intensities of
the twin peaks and the corresponding sample quantity (Figure 2, middle) gave a linear relationship. The linear least-squares fit of
these data can be used as a calibration curve for the quantification of
CholM in plaque samples when the NMR spectra are obtained under the
same conditions. As shown in Figure 2, the experimental data and
the calculated results agree closely with each other when samples
contained an intermediate to high level of CholM. For samples
containing less than 2 mg CholM, the low signal-to-noise
ratio (S/N) in spectra of these samples resulted in scatter of the data
points.
|
Spectra of carotid plaques from different patients were acquired under
the same experimental conditions to test the feasibility of quantifying
CholM in the heterogeneous environment of the plaque. The
C5 region of selected spectra (Figure 2
, right) exhibited the
twin peaks characteristics of CholM.1
The quantity of CholM in plaques with an intermediate to high S/N
was determined from the calibration curve after integration of the C5
peaks (Figure 2). For samples with low spectral S/N, the amount
of CholM was estimated by the peak height in comparison with spectra of
known amounts of CholM rather than by integration. After completion of
the NMR measurements, chemical analysis was used to quantify
the total cholesterol, CE, and phospholipids in these and
other plaque samples (Table 1).
There was no direct correlation between sample dry weight (30 to 100
mg) and the lipid composition or cholesterol content. The
quantity of cholesterol solubilized in the isotropic CE
phase in each plaque was calculated by assuming a solubility of 5.3
weight percent.21 36 Together with the CholM content
measured by MAS NMR, the amount of cholesterol in the
CholLiqCr phase was estimated according to the equation described
above.
|
Assuming that CholLiqCr is complexed with phospholipids, we calculated
the cholesterol-to-phospholipid molar ratio of each sample
(Table 2). For most plaques, the results
gave ratios higher than the 1:1 ratio normally obtained with a
phosphatidylcholine model membrane saturated with
cholesterol.41 However, our results are
consistent with previous estimates for lipid-rich particles
isolated from rabbit42 and human advanced
plaques33 and from the fractionation studies of Katz and
Small.35
|
Quantification of Plaque Calcium
Calcium salt deposits are often found in advanced atherosclerotic
plaques, especially in carotid plaques formed near the bifurcation. In
atherosclerotic lesions, the predominant chemical form of calcium salts
is CPH.43 The 31P nucleus
represents an intrinsic probe for detection of CPH by NMR. The
identification of CPH in carotid plaques by 31P
and chemical analysis has been described in detail in our
previous report.23
For calibration of the integrated 31P signal
intensity in plaques, we first used synthetic CPH purchased from a
commercial source as a reference compound. The
31P resonance of this CPH (Figure 3A) had approximately the same chemical
shift as that of the plaque (Figure 3B), but the peak for CPH
was narrower. CPH prepared from diluted CaCl2 and
NaH2PO4
solutions44 gave a 31P resonance
similar to that in the 31P NMR spectrum of
freshly prepared CPH (Figure 3A). 31P MAS
NMR spectra were obtained with known amounts of CPH to test the
validity of using it for the calibration of 31P
signals in plaques. The plot of peak intensity of the center band
versus phosphorus content showed a strong, linear correlation (Figure 4A). Spectra of plaque samples were then
obtained under identical conditions, and the intensities of the
31P resonances were plotted against the
phosphorus content of the corresponding samples (Figure 4A). In
both cases, the actual sample phosphorus content was determined by
chemical analysis after completion of the NMR measurements. The
data for intact tissue also showed a linear relationship but with a
different slope from that obtained for pure CPH.
|
|
), chicken bone powder (
), and human plaques
(•). The results for plaques show a close correspondence with results
for bone (bottom) rather than synthetic CPH (top).
These results indicated that synthetic CPH is clearly not an ideal
calibration compound for plaque calcification. As an alternative, we
tried animal bone powder, which is also composed mainly of CPH. The
31P spectrum of bone powder resembled that of the
plaque with respect to both chemical shift and line width (Figure 3C). A plot of the spectral intensities of the bone samples
versus mass exhibited a slope similar to that of the plaque samples
(Figure 4B). Thus, pulverized bone is an appropriate reference
for the phosphate signal in plaques. The total phosphorus contents in
selected plaques, as determined by the MAS NMR (referenced to bone) and
by chemical analysis, are presented in Table 3. The 8 samples studied contained a wide
range of phosphorus, extending from 0.60 to 15 mg, corresponding to 3.2
to 81 mg of CPH. The value of total phosphorus in each plaque sample
measured by MAS NMR was within 10% of the value determined by the
chemical analysis. The calibration accuracy in the low
concentration range was significantly higher for CPH than it was for
CholM (Figure 2), mainly because of the higher relative NMR
sensitivity of the 31P nucleus. The close
correspondence validates the use of 31P MAS NMR
as a convenient, nonperturbing method for measuring CPH in intact
plaques.
|
To understand why there were differences in the measured
31P signal intensities of CPH from different
sources, we measured the x-ray powder diffraction pattern of the
synthetic CPH samples, the bone powder, and an atherosclerotic plaque.
Synthetic samples, either freshly prepared or purchased from a
commercial source, gave the same diffraction pattern (pattern A, shown
schematically in Figure 3A). The plaque sample and the bone
sample gave the same pattern: both showed some differences from the
synthetic samples (pattern B, shown schematically in Figure 3B).
Patterns A and B had the same diffraction bands in the short spacing
region (the outer bands), but the long spacing bands (the center bands)
were seen only in pattern A. This result agrees with a previous study
showing that x-ray diffraction patterns of mineral deposits isolated
from human aorta matched the pattern of CPH in the short spacing range
only.18 Synthetic CPH thus has a more ordered crystalline
structure than does CPH in biological samples. In the latter,
crystallization of CPH is a slow process controlled by many
pathophysiological processes and may be limited to
very small environments, such as individual cells or cell
clusters.43 Therefore, the microcrystals of CPH in plaques
are probably small and lack long-range ordering in the lattice. It has
been suggested that plaque calcification is controlled by bone-forming
proteins such as osteopontin43 ; thus, it is not surprising
that the bone powder had the same physical characteristics as the CPH
deposit in plaques, as reflected in the 31P NMR
spectra and x-ray diffraction patterns.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Until recently, quantitative analysis of plaque components without extensive sample manipulation or destruction has proven to be especially difficult. Katz and Small35 developed a discontinuous sucrose density gradient centrifugation method to separate homogenized plaque tissue into CEs, phospholipids, CholM, and pellet phases. This method is valuable in that each phase can be obtained for physical characterization and chemical analysis. However, it was concluded that the isolated fractions showed significant contamination with other phases.35 The procedure requires disruption of the tissue and is also very labor-intensive. Other investigators using Raman spectroscopy have quantified the major lipids and calcium salts in plaques.9 10 45 However, Raman studies have not distinguished the different phases of lipids, particularly crystalline from noncrystalline cholesterol, and have been applied mainly to minced or homogenized plaques9 10 rather than excised plaques in situ because of technical limitations.10
In the present study, we have demonstrated the detection and
quantification of 2 major pathological constituents of human carotid
plaques, CholM and CPH, by MAS NMR. Our results revealed a wide range
of CholM in 9 different samples (Table 2), from 1.0 mg (the
lower limit of detection) to 8.1 mg. Based on the NMR measurement and
the post-NMR chemical analyses, the amount of
cholesterol complexed with phospholipid could be estimated
(Table 2).
As cholesterol accumulates in a plaque, it partitions into
phospholipid bilayers. This lipid bilayer phase eventually becomes
saturated with cholesterol as the disease progresses. The
exact saturation ratio depends, in part, on the specific phospholipids
present. Thus, our results, showing a range of values between 1.1
and 1.7 (Table 2), reflect varying degrees of
supersaturation.35 MAS NMR provides a reliable method to
estimate this ratio in situ and to investigate the factors contributing
to its variation in future studies. In any case, the observed variation
in the amount of cholesterol complexed with phospholipids
demonstrates that the quantity of CholM in the plaque cannot be
estimated from the total cholesterol determined by chemical
analysis.
We are not aware of any independent quantification of CholM in a
plaque, and it is not practical to extract CholM selectively for
chemical quantitative analysis. Because the ratios of
cholesterol-to-phospholipid in the liquid-crystalline phase
obtained for our plaque samples (Table 2) were in the expected
range as discussed above, our quantitative measurement of CholM was
reasonably accurate. In previous studies of model bilayer systems that
were supersaturated with cholesterol, MAS NMR detected
CholM reliably, including some cases where it was not visible by light
microscopy.46 Thus, MAS NMR should be able to detect even
small crystals in tissue and potentially provide an accurate estimate
of CholM in the heterogeneous environment of the plaque.
This expectation seems to be verified by the present results.
In our experiments for calibrating the intensity of the
31P resonances from plaques, the intensity data
points from synthetic CPH and bone fell on lines with differing slopes;
a lower slope (0.045) was obtained for CPH compared with that for
bone (0.125). Therefore, if synthetic CPH is to be used as the
standard sample for quantification, then a calibration factor will be
needed (biological sample mass=0.4xits peak intensity). In a recent
report on the determination of human coronary artery
composition by Raman spectroscopy,45 a calibration factor
was also required to correctly relate the plaque calcium phosphate and
synthetic CPH (biological sample mass=0.7xRaman signal intensity).
Because animal bone is easy to obtain and prepare for NMR
analysis, it serves as a good reference for the quantification
of CPH in human plaques without the need to introduce a calibration
factor.
In situ characterization of the structures and chemical and physical properties of components in plaques can provide valuable information for understanding the pathogenesis and potential progression, stabilization, or regression of the plaque and for explaining why certain plaques are prone to rupture. The MAS NMR approaches described in this study will make it possible to determine, for example, whether the tendency of a plaque to rupture is related to its content of crystalline (or liquid-crystalline) cholesterol or calcium phosphate. Because MAS NMR is essentially nondestructive, selected samples with desired characteristics can be studied by other methods. Although MAS NMR with its rapid sample spinning is not suitable for in vivo studies, the quantification of solid phases by MAS NMR provides critical information for interpreting magnetic resonance images of the same plaque.
|
Acknowledgments |
|---|
This work was supported by American Heart Association (AHA) Fellowship 13-434-945 and AHA Grant-In-Aid No. 13519-967 to W.G.; National Institutes of Health grant RO1 HL41904 to J.A.H.; and Welch Grant No. Q-1325 to J.D.M.
|
Footnotes |
|---|
1 Because carotid plaques generally contain less lipid and more calcium salts than do typical coronary artery or aortic plaques,40 we studied a large number of tissue samples by MAS NMR to identify the limited number of cholesterol-containing plaques discussed below. The results presented here demonstrate our capability to quantify plaque cholesterol in different phases but are not intended to be representative of plaque features in the majority of carotid plaques.
Received February 3, 2000; accepted February 25, 2000.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Berliner JA, Navab M, Fogelman AM, Frank JS, Demer LL, Edwards PA, Watson AD, Lusis AJ. Atherosclerosis: basic mechanisms: oxidation, inflammation, and genetics. Circulation. 1995;91:2488–2496.
2.
Guyton JR, Klemp KF. Development of the lipid-rich
core in human atherosclerosis. Arterioscler
Thromb Vasc Biol. 1996;16:4–11.
3. Fuster V, Badimon J, Chesebro JH, Fallon JT. Plaque rupture, thrombosis, and therapeutic implications. Haemostasis. 1996;26(suppl 4):269–284.
4. Carr S, Farb A, Pearce WH, Virmani R, Yao JS. Atherosclerotic plaque rupture in symptomatic carotid artery stenosis. J Vasc Surg. 1996;23:755–765, discussion 765–766.[Medline] [Order article via Infotrieve]
5. Kostulas K, Huang WX, Crisby M, Jin YP, He B, Lannfelt L, Eggertsen G, Kostulas V, Hillert J. An angiotensin-converting enzyme gene polymorphism suggests a genetic distinction between ischaemic stroke and carotid stenosis. Eur J Clin Invest. 1999;29:478–483.[Medline] [Order article via Infotrieve]
6. Katz SS, Shipley GG, Small DM. Cholesterol turnover in lipid phases of human atherosclerotic plaque. J Lipid Res. 1982;23:733–737.[Abstract]
7.
Small DM. George Lyman Duff memorial lecture:
progression and regression of atherosclerotic lesions: insights from
lipid physical biochemistry.
Arteriosclerosis. 1988;8:103–129.
8. Katz SS, Shipley GG, Small DM. Physical chemistry of the lipids of human atherosclerotic lesions: demonstration of a lesion intermediate between fatty streaks and advanced plaques. J Clin Invest. 1976;58:200–211.
9. Weinmann P, Jouan M, Dao NQ, Lacroix B, Groiselle C, Bonte J-P, Luc G. Quantitative analysis of cholesterol and cholesteryl esters in human atherosclerotic plaques using near-infrared Raman spectroscopy. Atherosclerosis.. 1998;140:81–88.[Medline] [Order article via Infotrieve]
10.
Romer T, Brennan JF, Fitzmaurice M, Feld ML, Deinum G,
Myles J, Kramer JR, Lees RS, Feld MS. Histopathology of human
coronary atherosclerosis by quantifying its
chemical composition with Raman spectroscopy. Circulation. 1998;97:878–885.
11.
Kramsch DM, Chan CT. The effect of agents interfering
with soft tissue calcification and cell proliferation of calcific
fibrous-fatty plaques in rabbits. Circ Res. 1978;42:562–571.
12.
Kramsch DM, Aspen AJ, Rozler LJ.
Atherosclerosis: prevention by agents not affecting
abnormal levels of blood lipids. Science. 1981;213:1511–1512.
13. Watson KE, Demer LL. The atherosclerosis-calcification link? Curr Opin Lipidol.. 1996;7:101–104.[Medline] [Order article via Infotrieve]
14. Wimpfheimer O, Haramati LB, Haramati N. Calcification of the ligamentum arteriosum in adults: CT features. J Comput Assist Tomogr. 1996;20:34–37.[Medline] [Order article via Infotrieve]
15.
Simon A, Giral P, Levenson J.
Extracoronary atherosclerotic plaque at multiple sites and
total coronary calcification deposit in
asymptomatic men: association with coronary risk
profile. Circulation. 1995;92:1414–1421.
16. Moore JR, Garrido L, Ackerman JL. Solid state phosphorus-31 magnetic resonance imaging of bone mineral. Magn Reson Med. 1995;33:293–299.[Medline] [Order article via Infotrieve]
17. Seeger JM, Barratt E, Lawson GA, Klingman N. The relationship between carotid plaque composition, plaque morphology, and neurologic symptoms. J Surg Res. 1995;58:330–336.[Medline] [Order article via Infotrieve]
18. Schmid K, McSharry WO, Pameijer CH, Binette JP. Chemical and physicochemical studies on the mineral deposits of the human atherosclerotic aorta. Atherosclerosis. 1980;37:199–210.[Medline] [Order article via Infotrieve]
19.
Hamilton JA, Oppenheimer N, Cordes EH. Carbon-13
nuclear magnetic resonance studies of cholesteryl esters and
cholesteryl ester/triglyceride mixtures. J Biol
Chem. 1977;252:8071–8080.
20.
Hamilton JA, Cordes EH, Glueck CJ. Lipid dynamics in
human low density lipoproteins and human aortic tissue with fibrous
plaques: a study by high field 13C NMR
spectroscopy. J Biol Chem. 1979;254:5435–5441.
21. Guo W, Hamilton JA. 13C MAS NMR studies of crystalline cholesterol and lipid mixtures modeling atherosclerotic plaques. Biophys J. 1996;71:2857–2868.[Medline] [Order article via Infotrieve]
22. Guo W, Hamilton JA. Molecular organization and motions of cholesteryl esters in crystalline and liquid crystalline phases: a 13C and 1H magic angle spinning NMR study. Biochemistry. 1993;32:9038–9052.[Medline] [Order article via Infotrieve]
23. Guo W, Morrisett JD, Lawrie GL, DeBakey ME, Hamilton JA. Identification of different lipid phases and calcium phosphate deposits in human carotid artery plaques by MAS NMR spectroscopy. Magn Reson Med. 1998;39:184–189.[Medline] [Order article via Infotrieve]
24. Husted C, Montez B, Le C, Moscarello MA, Oldfield E. Carbon-13 "magic-angle" sample-spinning nuclear magnetic resonance studies of human myelin, and model membrane systems. Magn Reson Med. 1993;29:168–178.[Medline] [Order article via Infotrieve]
25. Pearse AGE. Histochemistry: Theoretical and Applied, Volume 1: Preparative and Optical Technology. 4th ed. London, England; Churchill Livingstone; 1980:250.
26. Yeagle PL, Hutton WC, Huang C, Martin RB. Phospholipid head-group conformations: intermolecular interactions and cholesterol effects. Biochemistry. 1977;16:4344–4349.[Medline] [Order article via Infotrieve]
27. Frank A. Some developments and applications of microfocus X-ray diffraction techniques. Br J Appl Phys. 1958;9:349–352.
28.
Folch J, Lees M, Stanley GHS. A simple method for the
isolation and purification of total lipids from animal tissues.
J Biol Chem. 1957;226:497–509.
29. Tercyak AM. Determination of cholesterol and cholesteryl esters. J Nutr Biochem. 1991;2:281–292.
30. Pinter JK, Hayashi JA, Watson JA. Enzymic assay of glycerol, dihydroxyacetone, and glyceraldehyde. Arch Biochem Biophys. 1967;121:404–414.[Medline] [Order article via Infotrieve]
31.
Bartlett GR. Phosphorous assay in column
chromatography. J Biol Chem. 1959;234:466–468.
32. Nolte CJ, Tercyak AM, Wu HM, Small DM. Chemical and physiochemical comparison of advanced atherosclerotic lesions of similar size and cholesterol content in cholesterol-fed New Zealand White and Watanabe Heritable Hyperlipidemic rabbits. Lab Invest. 1990;62:213–222.[Medline] [Order article via Infotrieve]
33. Chao FF, Blanchette-Mackie EJ, Chen YJ, Dickens BF, Berlin E, Amende LM, Skarlatos SI, Gamble W, Resau JH, Mergner WT. Characterization of two unique cholesterol-rich lipid particles isolated from human atherosclerotic lesions. Am J Pathol. 1990;136:169–179.[Abstract]
34. Shipley GG, Avecilla LS, Small DM. Phase behavior and structure of aqueous dispersions of sphingomyelin. J Lipid Res. 1974;15:124–131.[Abstract]
35.
Katz S, Small DM. Isolation and partial
characterization of the lipid phases of human atherosclerotic plaques.
J Biol Chem. 1980;255:9753–9759.
36. North BE, Katz SS, Small DM. The dissolution of cholesterol monohydrate crystals in atherosclerotic plaque lipids. Atherosclerosis. 1978;30:211–217.[Medline] [Order article via Infotrieve]
37.
Hamilton JA, Oppenheimer NJ, Addleman R, Clouse AO,
Cordes EH, Steiner P-M, Glueck CJ. High-field 13C
NMR studies of certain normal and abnormal human plasma lipoproteins.
Science. 1976;194:1424–1427.
38.
Toussaint JF, Southern JF, Fuster V, Kantor HL.
13C-NMR spectroscopy of human atherosclerotic
lesions: relation between fatty acid saturation, cholesteryl ester
content, and lumina1 obstruction. Arterioscler Thromb.. 1994;14:1951–1957.
39. Guo W, Hamilton JA. Phase behavior and crystalline structures of cholesteryl ester mixtures: a C-13 MASNMR study. Biophys J. 1995;68:2376–2386.[Medline] [Order article via Infotrieve]
40. Fuster V. Mechanisms of arterial thrombosis: foundation for therapy. Am Heart J.. 1998;135:S361–S366.[Medline] [Order article via Infotrieve]
41. Bourges M, Small DM, Dervichian DG. Biophysics of lipidic associations, II: the ternary systems: cholesterol-lecithin-water. Biochim Biophys Acta. 1967;137:157–167.[Medline] [Order article via Infotrieve]
42. Chao F-F, Blanchette-Mackie EJ, Dickens BF, Gamble W, Kruth HS. Development of unesterified cholesterol-rich lipid particles in atherosclerotic lesions of WHHL and cholesterol-fed NZW rabbits. J Lipid Res. 1994;35:71–83.[Abstract]
43. Anderson HC. Calcific diseases: a concept. Arch Pathol Lab Med. 1983;107:341–348.[Medline] [Order article via Infotrieve]
44. Tiselius A, Hjerten S, Levin O. Protein chromatography on calcium phosphate columns. Arch Biochem Biophys.. 1956;65:132–155.[Medline] [Order article via Infotrieve]
45.
Brennan JF, Romer TJ, Lees RS, Tercyak AM, Kramer JR
Jr, Feld MS. Determination of human coronary artery composition
by Raman spectroscopy. Circulation. 1997;96:99–105.
46. Guo W, Hamilton JA. A multinuclear solid-state NMR study of phospholipid-cholesterol interactions: dipalmitoylphosphatidylcholine-cholesterol binary system. Biochemistry. 1995;34:14174–14184.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  F. L. Ruberg, J. Viereck, A. Phinikaridou, Y. Qiao, J. Loscalzo, and J. A. Hamilton Identification of cholesteryl esters in human carotid atherosclerosis by ex vivo image-guided proton MRS J. Lipid Res., February 1, 2006; 47(2): 310 - 317. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Higgins, S. A. Marvel, and J. D. Morrisett Quantification of Calcification in Atherosclerotic Lesions Arterioscler. Thromb. Vasc. Biol., August 1, 2005; 25(8): 1567 - 1576. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Nadra, J. C. Mason, P. Philippidis, O. Florey, C. D.W. Smythe, G. M. McCarthy, R. C. Landis, and D. O. Haskard Proinflammatory Activation of Macrophages by Basic Calcium Phosphate Crystals via Protein Kinase C and MAP Kinase Pathways: A Vicious Cycle of Inflammation and Arterial Calcification? Circ. Res., June 24, 2005; 96(12): 1248 - 1256. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M. Doherty, L. A. Fitzpatrick, D. Inoue, J.-H. Qiao, M. C. Fishbein, R. C. Detrano, P. K. Shah, and T. B. Rajavashisth Molecular, Endocrine, and Genetic Mechanisms of Arterial Calcification Endocr. Rev., August 1, 2004; 25(4): 629 - 672. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. McIntosh, A. M. Gallegos, B. P. Atshaves, S. M. Storey, D. Kannoju, and F. Schroeder Fluorescence and Multiphoton Imaging Resolve Unique Structural Forms of Sterol in Membranes of Living Cells J. Biol. Chem., February 14, 2003; 278(8): 6384 - 6403. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Chao, M. Zhou, A. McIntosh, F. Schroeder, and A. B. Kier ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT J. Lipid Res., January 1, 2003; 44(1): 72 - 83. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Schroeder, A. M. Gallegos, B. P. Atshaves, S. M. Storey, A. L. McIntosh, A. D. Petrescu, H. Huang, O. Starodub, H. Chao, H. Yang, et al. Recent Advances in Membrane Microdomains: Rafts, Caveolae, and Intracellular Cholesterol Trafficking Experimental Biology and Medicine, November 1, 2001; 226(10): 873 - 890. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Peng, W. Guo, J. D. Morrisett, M. T. Johnstone, and J. A. Hamilton Quantification of Cholesteryl Esters in Human and Rabbit Atherosclerotic Plaques by Magic-Angle Spinning 13C-NMR Arterioscler. Thromb. Vasc. Biol., December 1, 2000; 20(12): 2682 - 2688. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||