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Quantitative Analysis of Apoptotic Cell Death Using Proton Nuclear
Magnetic Resonance Spectroscopy
By Francis G. Blankenberg, Peter D. Katsikis, Richard W. Storrs, Christian Beaulieu, Daniel Spielman, James Y. Chen,
Louie Naumovski, and Jonathan F. Tait
Quantification of apoptotic cell death in vivo has become
an important area of investigation in patients with acute
lymphoblastic leukemia (ALL). We have devised a noninvasive analytical method to estimate the percentage of apoptotic lymphoblasts in doxorubicin-treated Jurkat T-cell ALL
cultures, using proton nuclear magnetic resonance spectroscopy (1H NMR). We have found that the ratio of the methylene (CH2 ) resonance (at 1.3 ppm) to the methyl (CH3 ) resonance (at 0.9 ppm) signal intensity, as observed by 1H NMR,
is directly proportional to the percentage of apoptotic
lymphoblasts in vitro. The correlation between the CH2/CH3
signal intensity ratio and the percentage of apoptotic
lymphoblasts was optimal 24 to 28 hours after doxorubicin
treatment (r2 ! .947, N ! 27 samples). There was also a
direct temporal relationship between an increase in the CH2/
CH3 signal intensity ratio and the onset of apoptosis as detected by nuclear morphologic analysis, fluorescein-annexin
V flow cytometry, and DNA gel electrophoresis. Thin-layer
chromatography confirmed that a dynamic and/or compositional change of the plasma membrane, rather than increases in lipase activity or fatty acid production, appears
to account for the increase in the CH2/CH3 signal intensity
ratio during apoptosis. 1H NMR may have clinical utility for
the early noninvasive assessment of chemotherapeutic efficacy in patients with ALL.
q 1997 by The American Society of Hematology.
A
ever, the percentage of cellularity of leukemic marrow does
not change significantly until several weeks after the start
of therapy and does not directly correlate to apoptotic cell
death, which begins to occur within the first several days of
treatment.5,6
More recently, 1H NMR spectroscopy has been applied to
the study of apoptotic cell death in vitro.19 We have found
that the onset of apoptosis is accompanied by a greater than
twofold increase in the signal intensity of the membrane
lipid methylene (CH2 ) resonance (at 1.3 ppm) as observed
by 1H NMR. We now have devised an analytical method
based on the magnitude of the CH2 resonance signal intensity
to quantify in vitro the fraction of apoptotic cells in Jurkat
T-cell cultures treated with the chemotherapeutic agent doxorubicin. We also show that increases in the CH2 resonance
signal intensity parallel the surface expression of phosphatidylserine (PS) an early marker of apoptosis,20-23 as determined by fluorescein-annexin V flow cytometry.
Our results show that 1H NMR can detect apoptosis and
also quantify apoptotic cell death in vitro. Quantification of
apoptosis in vivo by 1H NMR may prove clinically useful
in circumstances in which noninvasive methods of early determination of chemotherapeutic efficacy are required.
GROWING NUMBER of clinical investigators have
engaged in efforts to quantify apoptosis.1-10 Their ultimate goal is to show the clinical utility of quantifying
apoptosis in a number of disease states, including acute
lymphoblastic leukemia (ALL). Estimates of the degree of
cytoreduction during induction therapy using bone marrow
aspirates have provided prognostic information in children
with ALL.11,12 Other investigators have also shown a direct
relationship between the degree of apoptotic cell death and
subsequent tumor growth delay in murine models of leukemia.13,14 However, all methods currently used to quantify
apoptosis are invasive and require aspirated or biopsied material.
To date, proton nuclear magnetic resonance spectroscopy
(1H NMR) has been the only noninvasive method to quantify
the degree of cytoreduction in leukemic patients.15-17 These
measurements rely on the ratio of water to lipid proton resonance signal intensities and thus reflect the relative percentages of cellular and fatty bone marrow (percent cellularity)
within a defined three-dimensional volume (voxel).18 How-
From the Department of Radiology, Department of Genetics, Department of Pediatrics, Stanford University School of Medicine,
Stanford, CA; the Department of Chemistry, Stanford University
School of Humanities and Sciences, Stanford, CA; and the Department of Laboratory Medicine, University of Washington, Seattle,
WA.
Submitted September 9, 1996; accepted January 2, 1997.
Supported by the Office of Technology Licensing at Stanford University, CA. P.D.K. was supported by National Institutes of Health
Grant No. CA 42509, awarded to Leonard A. Herzenberg (Professor
of Genetics, Stanford University, Stanford, CA). C.B. was graciously
supported by the Alberta Heritage Foundation for Medical Research.
Address reprint requests to Francis G. Blankenberg, MD, Lucile
Suter Packard Children’s Hospital, 725 Welch Rd, Palo Alto, CA
94304.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
q 1997 by The American Society of Hematology.
0006-4971/97/8910-0040$3.00/0
MATERIALS AND METHODS
Cell culture technique. Jurkat T-cell ALL cultures were grown
in RPMI 1640 medium (Applied Scientific, South San Francisco,
CA) supplemented with 10% (vol/vol) fetal calf serum and 290 mg/
mL L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin.
Cells were maintained in the logarithmic growth phase at a concentration of 1 to 5 1 105 cells/mL at 377C in a 5% CO2 air incubator
under sterile conditions.24
DNA fragmentation assay. DNA ladder formation was detected
by an assay as previously described.19 Briefly, 5 1 106 cells were
harvested and washed with phosphate-buffered saline (PBS). Total
DNA was isolated by incubating washed cells in 400 mL lysis buffer
containing 50 mmol/L Tris, pH 7.5, 10 mmol/L EDTA, 0.5% sodium
dodecyl sulfate (SDS), 100 mg/mL RNase, and 4 mg/mL proteinase
K for at least 2 hours at 607C. After the addition of 75 mL of 8 mol/
L KAc, the DNA was extracted with phenol/chloroform, precipitated
with ethanol, and suspended in TE (10 mmol/L Tris-HCl, pH 7.5,
1 mmol/L EDTA) with 100 mg/mL of heat-treated RNase. The DNA
from 1 1 106 cells was then analyzed in a composite 1% agarose/
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QUANTIFYING APOPTOSIS WITH MR SPECTROSCOPY
0.5% Nusieve gel. The fraction of total DNA that underwent DNA
fragmentation (DNA ladder formation) was calculated from digitized
images using a BioRad Gel Doc System with Molecular Analyst
Software (Bio-Rad Laboratories, Richmond, CA).
Cell morphologic analysis. Apoptotic nuclei were identified
after staining with Hoechst #33342 (10 mmol/L final concentration)
as described by Dive et al.25 Cells were observed with a fluorescence
microscope with an excitation wavelength of 365 nm. Cells were
considered nonapoptotic when nuclei were morphologically normal
with homogeneously distributed blue-stained chromatin. Apoptotic
cells showed blue peripherally clumped or fragmented chromatin.
Cell viability was assessed in control and treated cultures before
and after 1H NMR spectral analysis using fluorescent microscopy of
doubly stained cell samples (with the Hoechst and propidium iodide
stains), as previously described.19 There were no changes in the
fractions of viable, apoptotic, late stage apoptotic (ie, with irreversible membrane failure) or necrotic cells after 1H NMR spectroscopic
analysis of either control or treated cultures (data not shown).
Fluorescein isothiocyanate (FITC) annexin V flow cytometry.
Recombinant annexin V was prepared by cytoplasmic expression in
Escherichia coli by modifications of a previous vector and method.26
Annexin V is an endogenous human lipocortin that has a high affinity
(kd Å 10 nmol/L) for PS, which is exposed on the cell surface
during apoptotic cell death. Recombinant annexin V was labeled
with FITC (Molecular Probes, Eugene, OR), purified to yield a
fraction containing a single mole of fluorescein per mole of protein,
and quantitated by absorbance at 494 nm, as previously described.27
For flow cytometry, 1 1 106 cells were stained with 100 nmol/L of
FITC-Annexin V in deficient RPMI 1640 with 3% fetal calf serum
and incubated for 15 minutes on ice, washed twice, and then fixed
with 0.5% paraformaldehyde. Fixed cells underwent flow cytometry
using a FACStar Plus (Becton Dickinson, Mountain View, CA)
within 6 hours of FITC-annexin V labeling.
1
H NMR spectral analysis. 1H NMR spectroscopy was performed in vitro using methods previously described by Blankenberg
et al.19 Briefly, 5 1 107 cells were harvested and washed twice with
PBS made with D20 (99.9% purity), suspended in a final volume of
500 mL, and placed immediately on ice until data acquisition. Samples were analyzed on a 400 MHz high resolution Bruker CSI Omega
spectrometer (Bruker; Karlsruhe, Germany) at 187C, pulse-acquire,
907 flip angle, repetition time 10 seconds, 64 or 128 excitations
(depending on desired signal to noise), 8k points, 5 kHz bandwidth.
A coaxial tube filled with trimethysialoproponic acid (TSP), 0.1%
solution in D20 was used as reference (0.0 ppm) for each experiment.
The relative areas underneath the CH2 and methyl (CH3 ) resonances
(at 1.3 and 0.9 ppm, respectively) were calculated by integration of
the proton spectrum using the trough between the CH2 and CH3
resonances as a baseline reference. Changes during spectroscopic
analysis were minimal, as confirmed by rescanning cell suspensions
after storage on ice for 4 hours (data not shown).
Thin-layer chromatography (TLC) of lipid extracts. Samples
containing 5 1 107 cells were added to ice-cold solvent containing
5 mL methanol/chloroform (2:1, vol/vol), 2.5 mL chloroform, and
1 to 3 mL water using methods described by Mitchell et al.28 After
vortexing, suspensions were centrifuged (1,000g for 5 minutes at
room temperature), and organic (bottom) layers were collected.
Aqueous layers were extracted again by adding 2.5 mL of chloroform, centrifuging as described above, and combining organic layers
with those collected previously. After solvent removal under a gentle
nitrogen stream, extracts were suspended in chloroform/methanol
(2:1, vol/vol), and 0.05 vol of each extract was spotted on Silica
Gel HLF TLC plates (Analtech, Inc, Newark, DE). DPPC (dipalmitoylphosphatidylcholine), DSPC (distearoylphosphatidylcholine),
DOPC (dioleoylphosphatidylcholine), lyso LPC (lauroyl lysophos-
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phatidylcholine), DLPE (dilauroylphosphatidylethanolamine; Sigma
Chemical Co, St Louis, MO), and DMPS (dimyristoylphosphatidylserine; Avanti Polar Lipids, Inc, Alabaster, AL) standard solutions
were also spotted as indicated. Plates were developed in chloroform/
methanol/formic acid (65:30:8, vol/vol), treated with iodine vapor
to detect lipid components, and image-scanned (Tamarack Telecom,
Inc, Orange, CA; image resolution: 266 ppi).
Spot assignments were confirmed further by fluorescamine
(Sigma) detection of primary amine groups under long-wave UV
light.29 After development of TLC plates to detect lipid components,
the entire area of each plate was sprayed with fluorescamine. After
20 minutes at room temperature, plates were viewed under longwave UV light. Any spots containing primary amines (ie, PS, PE)
using this procedure fluoresce under observation with long-wave UV
light.
RESULTS
Time course of the spectral changes associated with the
exposure of PS (apoptosis). Jurkat T-cell cultures were
treated with 200 ng/mL of doxorubicin and harvested at 0,
6, 12, 18, 24, and 48 hours. Cells were then analyzed by
FITC-annexin V flow cytometry, DNA gel electrophoresis
nuclear morphologic analysis, and 1H NMR. Annexin V positivity, the percentage of DNA fragmentation, the percentage
of apoptosis as observed by morphologic analysis, and the
CH2/CH3 signal intensity ratio all increased to greater than
baseline values at 18 hours (Fig 1A, B, and C). Annexin V
positivity (fraction of cells expressing phosphatidylserine on
the surface of the plasma membrane) and the CH2/CH3 signal
intensity ratio showed a linear correlation (r2 Å .883) up to
24 hours, after which annexin V positivity plateaued,
whereas the CH2/CH3 signal intensity ratio continued to increase. Annexin V positivity also demonstrated an excellent
linear correlation between the percentage of DNA fragmentation (r2 Å .996) and nuclear morphologic analysis (r2 Å
.966) up to 24 hours after doxorubicin treatment. The percentage of DNA fragmentation and the percentage of
apoptosis as observed by morphologic analysis correlated to
the CH2/CH3 signal intensity ratio (r2 Å .886, and r2 Å .982,
respectively) during the entire 48 hours of doxorubicin treatment.
There was also a progressive decrease in the choline
[N(CH3 )3]/CH3 signal intensity ratio starting at 18 hours that
paralleled the increase in the CH2/CH3 signal intensity ratio
and the onset of apoptosis (data not shown). The choline/
CH3 signal intensity ratio showed a weak correlation with
annexin V positivity over the 24-hour period (r2 Å .698) of
doxorubicin treatment. The percentage of DNA fragmentation and the percentage of apoptosis, as observed by morphologic analysis, weakly correlated with the choline/CH3 signal
intensity ratio (r2 Å .76 and r2 Å .74, respectively) over the
entire 48 hours of doxorubicin treatment.
To more accurately determine the correlation between
CH2/CH3 signal intensity ratio and annexin V positivity during the narrow window between induction of apoptosis and
the plateauing of the annexin V positivity, additional Jurkat
T-cell cultures were treated (200 ng/mL of doxorubicin) and
harvested at 0, 18, 20, 22, and 24 hours. Again, annexin V
positivity and the fraction of apoptotic cells determined by
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Fig 1. Time course data of doxorubicin-treated
Jurkat cultures. Jurkat cultures were incubated with
200 ng/mL of doxorubicin for 0, 6, 12, 18, 24, and 48
hours. (A) The percentage of apoptotic cells in culture as observed by nuclear morphologic analysis,
FITC-annexin V flow cytometry, and DNA gel electrophoresis from 0 to 48 hours after doxorubicin treatment. (B) FITC-annexin V flow cytometric data from
0 to 24 hours after doxorubicin treatment. FCS, forward scatter; FITC-annexin V, fluorescent intensity
at 494 nm plotted on a logarithmic scale. (C) 1H NMR
spectra obtained (with 64 excitations) at 400 MHz.
The spectral resonances of choline protons (-N(CH3 ))
at 3.2 ppm, methylene protons (-CH2-) at 1.3 ppm,
and methyl protons (-CH3 ) at 0.9 ppm are indicated.
The CH2/CH3 signal intensity ratios were 0.28, 0.56,
0.33, 0.89, 2.41, and 4.42 at 0, 6, 12, 18, 24, and 48
hours after doxorubicin treatment, respectively.
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QUANTIFYING APOPTOSIS WITH MR SPECTROSCOPY
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Fig 1 (cont’d).
nuclear morphology showed an excellent linear correlation
(r2 Å .905, N Å 13, data not shown) between 18 and 24 hours
of doxorubicin treatment. However, annexin V positivity
showed only a weak correlation with the CH2/CH3 signalintensity ratio (r2 Å .642, N Å 13) between 18 and 24 hours.
Morphologic analysis also showed a weak correlation with
the CH2/CH3 signal intensity ratio (r2 Å .764, N Å 13, data
not shown) between 18 and 24 hours.
The spectral changes seen in the doxorubicin dose response curves at 24 and 48 hours. Jurkat T-cell cultures
underwent harvesting and analysis after treatment with 0,
60, 80, 100, 200, 300, and 400 ng/mL of doxorubicin for 24
hours. There was excellent correlation between annexin V
positivity and the CH2/CH3 signal intensity ratio at 24 hours
after doxorubicin treatment (Fig 2). There was also excellent
correlation between the fraction of apoptotic cells as seen
by morphologic analysis and the CH2/CH3 signal intensity
ratio at 24 hours after doxorubicin treatment. Additional Jurkat T-cell cultures were treated with 0, 25, 50, 100, 150,
200, and 400 ng/mL of doxorubicin, after which cells were
harvested at 48 hours and analyzed. Annexin V positivity
remained linearly correlated up to a dose of 150 ng/mL
doxorubicin at 48 hours, after which the fraction of annexinpositive cells plateaued (Fig 3). However, the fraction of
apoptotic cells as seen morphologically showed an excellent
correlation throughout the dose range (25 to 400 ng/mL)
with the CH2/CH3 signal intensity ratio. Three data sets of
doxorubicin dose-response curves performed at 24 and 48
hours after treatment were combined and analyzed as a composite. There was excellent correlation between the fraction
of apoptotic cells as seen morphologically and the CH2/CH3
signal intensity ratio (Fig 4). There was a weak correlation
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between the fraction of apoptotic cells as seen by morphologic analysis and the decrease in the choline/CH3 signal
intensity ratio at 24 and 48 hours (r2 Å .304, N Å 18 and r2
Å .67, N Å 9, respectively; data not shown). All other resolvable resonances did not change with apoptotic cell death.
TLC of apoptotic cell cultures treated with doxorubicin,
serum starvation, and CH11 IgM anti-Fas antibody.
Apoptosis was induced in Jurkat T-cell cultures with doxorubicin (200 ng/mL for 24 hours), serum deprivation (for 72
hours), or CH 11 IgM anti-Fas monoclonal antibody (Immunotech Inc, Westbrook, ME; 200 ng/mL for 48 hours). 1H
NMR spectroscopy was performed on control, doxorubicintreated, serum-deprived, and CH11 IgM anti-Fas monoclonal
antibody-treated Jurkat cell cultures (Fig 5A, B, C, and D,
respectively). TLC was performed on the lipid extracts of
each of the four Jurkat cell cultures (Fig 6). No differences
were noted between control and apoptotic cultures with any
of the three treatments by TLC despite marked increases
in the CH2/CH3 signal intensity ratios above baseline. In
particular, there were no increases in the intensity (amount)
of lysolipid staining (Rf É 0.10) in apoptotic cultures that
would indicate lipase activity with subsequent degradation of
diacyl phospholipid. In addition, the results of fluorescamine
staining confirmed that the only spots containing primary
amines were the ones corresponding to the standards containing PS (eg, Fig 6, lane 1e, DMPS; Rf values [0.55 to
0.68]) and PE (Fig 6, lane 2a and 3a DLPE; Rf values [0.69
to 0.75]) as well as all classes of the lysolipids containing
PS and PE from the cell lipid extracts center lanes of TLC
#1, #2, and #3 (Rf values; É 0.10). The extracts of each of
the four cell cultures demonstrated that the spots corresponding to the lipid fractions containing PS (Rf values; TLC #1
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Fig 2. Dose-response data of doxorubicin-treated
Jurkat cultures at 24 hours. Jurkat cultures were
treated with 0, 60, 80, 100, 200, 300, and 400 ng/
mL of doxorubicin for 24 hours. The percentage of
apoptotic cells in each culture is plotted against the
CH2/CH3 signal intensity ratio determined by the 1H
NMR spectra obtained at 400 MHz (64 excitations).
The solid line and dashed lines are the best fit lines
determined by linear regression analyses corresponding to the percentage of apoptotic cells as
measured by morphologic analysis (r2 ! .965) and
FITC-annexin V flow cytometry (r2 ! .985), respectively.
[0.65 to 0.75], TLC #2 [0.70 to 0.88], TLC #3 [0.70 to 0.88])
and phosphatidylcholine (Rf values; TLC #1 [0.26 to 0.45],
TLC #2 [0.45 to 0.65], TLC #3 [0.45 to 0.65]) remained
unchanged in apoptotic cultures with respect to intensity
(amount) and Rf values. These data indicate a lack of significant change in lipid polarity (such as with head group
exchange, change in acyl chain length or increases in the
degree of unsaturation of acyl chains) with apoptotic cell
death.
DISCUSSION
The signal intensity of CH3 resonance does not change
with apoptotic cell death.19 The CH2 resonance signal intensity increases to a maximal value of 5 to 6 times the CH3
resonance signal intensity during apoptosis. The ratio of the
integrated areas of CH2 and CH3 resonance peaks, therefore,
can be used to estimate the fraction of apoptotic cells in
culture. We found that the CH2/CH3 signal-intensity ratio,
as measured by 1H NMR, correlated with the fraction of
apoptotic cells seen morphologically and by DNA fragmentation throughout the 48-hour time period, whereas annexin
V positivity plateaued after 24 hours with doses of 200 ng/
mL of doxorubicin or greater.
The plateauing of annexin V positivity after 24 hours may
suggest that the concentration of FITC-annexin V (100 nmol/
L) used in our assay was insufficient to saturate all annexin
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V receptors present in the later stages of apoptosis. The
incomplete saturation of annexin V receptors may have resulted in a plateauing of annexin V positivity seen by flow
cytometry. Because there were no significant changes in the
plasma membrane attributable to phospholipid degradation
or head group exchange, it is unlikely that there was a loss
or degradation of exposed PS (ie, annexin V receptors) in
the later (after 24 hours) stages of apoptosis.
The correlation between the CH2/CH3 signal intensity ratio
and the fraction of apoptotic cells as seen morphologically
was optimized at 24 and 48 hours after treatment with doxorubicin. This implies that there is a time dependence before
24 hours of the magnitude of the increase in the CH2/CH3
signal intensity ratio with doxorubicin-induced apoptotic cell
death.
The increase in the CH2/CH3 signal intensity ratio is coincident with increases in annexin V-positive cells, DNA fragmentation, and the number of apoptotic cells seen morphologically. It is unclear what event(s) in the apoptotic cascade
is directly associated or responsible for the increase in the
CH2 resonance.
Prior work has shown that the prominent lipid spectra in
malignant cell lines are due to dynamic changes in microviscosity of the plasma membrane.30,31 Our current study has
shown no gross changes in the lipid profiles of cells undergoing apoptosis, as observed by TLC. If activation of phospho-
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QUANTIFYING APOPTOSIS WITH MR SPECTROSCOPY
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Fig 3. Dose-response data of doxorubicin-treated
Jurkat cultures at 48 hours. Jurkat cultures were
treated with 0, 25, 50, 100, 150, 200, and 400 ng/
mL of doxorubicin for 48 hours. The percentage of
apoptotic cells in each culture is plotted against the
CH2/CH3 signal intensity ratio determined by the 1H
NMR spectra obtained at 400 MHz (64 excitations).
The solid line represents the best fit line determined
by linear regression analysis corresponding to the
percentage of apoptotic cells as measured by morphologic analysis (r2 ! .964). The dashed line is the
best fit line determined by linear regression analysis
corresponding to the fraction of apoptotic cells determined by FITC-annexin V flow cytometry assay
up to a dose of 150 ng/mL (r2 ! .978).
Fig 4. Composite of dose-response data from 24 and 48 hours.
Data from 24 and 48 hours as shown in Figs 2B and 3B were combined
with an additional dose-response experiment in which Jurkat cultures were treated with 0, 90, 110, 120, 140, 160, 180, and 200 ng/
mL of doxorubicin for 24 hours. The CH2/CH3 signal intensity ratio
was plotted against the percentage of apoptotic cells as seen by
nuclear morphologic analysis (r2 ! .947, N ! 27 samples). The solid
line is the best fit line determined by linear regression analysis;
dashed lines mark the upper and lower limits of a 95% confidence
interval plotted over the entire range of data.
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lipase A2 was responsible for our spectroscopic observations, one would expect to see increased expression of the
lysolipid spot (Rf É 1.0) with apoptotic cell death. Furthermore, if phospholipase D (and probably C as well) were
being activated, one would also expect to see enhanced fluorescamine staining closer to the origin. In addition, we
showed no significant changes in fatty acyl chain length
(longer chain lengths have higher Rf values; see TLC #2
and #3, DPPC v DSPC) or degree of unsaturation (unsaturated fatty acids tend to have increased Rf values with respect
to saturated fatty acids of the same chain length; see TLC
#3 DOPC v DSPC). Therefore, a dynamic change of the
plasma membrane that may reflect a change in lipid bilayer
composition rather than lipase activity, increases in fatty acid
production, or degree of fatty acid unsaturation would be a
more plausible explanation for the increase in the methylene
resonance occurring with apoptotic cell death.19
Of note, the CH3 resonance at 0.9 ppm did not change
with apoptotic cell death. The CH3 resonance is well defined
(relatively mobile protons) even in control (untreated) cell
cultures and also arises in part from non–lipid-related CH3
protons such as nucleic acids and other compounds.32,33
Therefore, the CH3 resonance is not likely to be greatly
affected by dynamic changes of the plasma membrane. Similarly, the N(CH3)3 protons from the phospholipid head
groups are also well defined because these head groups are
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Fig 5. Spectral changes associated with apoptotic
Jurkat cultures with different therapies. The 1H NMR
spectra (at 400 MHz) corresponding to the Jurkat cultures before lipid extraction and TLC shown in Fig 6.
(A) Control culture. The resonance peaks for choline,
CH2 , and CH3 protons are shown. (B) Jurkat cells
treated with 200 ng/mL of doxorubicin for 24 hours.
(C) Jurkat cells deprived of serum for 3 days. (D) Jurkat
cells treated with 200 ng/mL of CH 11 IgM Fas antibody for 48 hours. The CH2/CH3 signal intensity ratio,
the percentages of apoptotic cells seen by nuclear
morphologic analysis, and FITC-annexin V flow cytometric analysis for each culture were as follows:
control (0.29, 5%, and 9.8%, respectively), doxorubicintreated (2.55, 51%, and 47%, respectively), serumstarved (1.82, 54%, and 46.8%, respectively), and Fas
antibody (1.63, 69%, and 71%, respectively).
also relatively mobile (on the outer or inner leaflet of the lipid
bilayer) and also are not likely to be affected significantly
by dynamic changes of the plasma membrane. The gradual
decrease in choline/CH3 signal intensity ratio that begins at
the onset of apoptosis (and increase in the CH2/CH3 signal
intensity ratio) may be due to the loss of choline moieties not
related to the lipid bilayer. These small molecule (choline)
metabolites may not be replenished as the cell prepares to
Fig 6. Thin-layer chromatograms of lipid extracts
of apoptotic Jurkat T-cell cultures with different therapies. TLC was performed using the lipid extracts of
untreated (control) and treated (apoptotic) Jurkat Tcell cultures. Three separate TLC are shown (ie, TLC
#1, #2, and #3) each performed on different dates.
Lipid standards (outside lanes) and lipid extracts
from cell cultures (inner 3 lanes) were spotted and
developed as described. TLC #1 (lanes 1A through
1E): 1A, standard; 1B, doxorubicin-treated cells; 1C
untreated (control) cells; 1D, doxorubicin-treated
cells; 1E, standard. TLC #2 (lanes 2A through 2E):
2A, standard; 2B, untreated cells; 2C, serum-starved
cells; 2D, CH11 IgM anti-Fas antibody-treated cells;
2E, standard. TLC #3 (lanes 3A through 3E): 3A, standard; 3B, untreated cells; 3C, serum-starved cells; 3D,
CH11 IgM anti-Fas antibody-treated cells; 3E, standard. Rf scale is the fractional height of spots relative
to the total length of TLC plates from origin. Rf ! 0.0
origin; Rf ! 1.0 solvent front. Doxorubicin treatment
(200 ng/mL for 24 hours); serum-starved cells (for 72
hours); CH11 IgM anti-Fas antibody treatment (200
ng/mL for 48 hours). Standards: Lyso LPC ! lyso
(mono) [12:0] PC; DMPS ! di [14:0] PS; DOPC ! di
[18:1] PC; DLPE ! di [12:0] PE; DPPC ! di [16:0] PC;
DSPC ! di [18:0] PC. NB, [n1 :n2], where n1 is the chain
length of fatty acyl group and n2 is the number of
unsaturations of fatty acyl group. di, mono ! number
of acyl chains. PC, PS, and PE refer to type of head
phospholipid group.
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QUANTIFYING APOPTOSIS WITH MR SPECTROSCOPY
die. The variability in the rate of loss of choline metabolites
(in treated cells) may in part explain the poor correlation
between the fraction of apoptotic cells and the choline/CH3
signal intensity ratio.
It is possible that the loss of cytoskeletal architecture is
partly responsible for the surface expression of PS seen with
apoptosis.34 The loss of cytoskeletal architecture may also
allow for increased mobility of the plasma membrane lipids.
The increase in extracellular PS exposure during apoptosis
also appears to involve a downregulation of the aminophospholipid translocase that translocates PS from the outer face
to the inner face of the plasma membrane35 to maintain normal plasma membrane structural asymmetry. There is also an
increase in the activity of a phospholipid scramblase during
apoptosis35 that allows all phospholipid classes to equilibrate
rapidly across the plasma membrane. Both these changes
may allow for increased lipid mobility with a subsequent
decrease in the microviscosity of the plasma membrane during apoptosis.36 There also may be a time dependence on
the degree of cytoskeletal architectural destruction and/or
loss of aminophospholipid translocase function that would
in part explain the time-dependent changes in the methylene
signal before 24 hours with doxorubicin therapy, particularly
during the critical 18- to 24-hour window after treatment
in which the CH2 signal increases to greater than baseline
values.
Our study provides the actual analytical methods with
which to estimate the percentage of apoptotic leukemic cells
in vivo. Efforts at studying tumor-bearing animals and patients with leukemia are ongoing.
ACKNOWLEDGMENT
The authors thank Dr Linda Brunauer (Stanford University, Stanford, CA) for helpful discussion about the TLC studies, particularly
in regard to the composition of the TLC solvent system; and Dr
Wray H. Huestis (Stanford University) for helpful discussion and
the use of reagents and facilities for TLC and lipid extractions.
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1997 89: 3778-3786
Quantitative Analysis of Apoptotic Cell Death Using Proton Nuclear
Magnetic Resonance Spectroscopy
Francis G. Blankenberg, Peter D. Katsikis, Richard W. Storrs, Christian Beaulieu, Daniel Spielman,
James Y. Chen, Louie Naumovski and Jonathan F. Tait
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