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Heavy marijuana users show increased serum apolipoprotein C-III levels: Evidence from proteomic analyses

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Marijuana (MJ) is the most commonly used illicit drug in the United States. Its abuse is associated with cognitive dysfunctions and increased resistance to blood flow in the cerebral vasculature. In addition, MJ abuse is associated with increased risks of potentially serious cardiovascular disorders. In the present study, we used the protein chip platform based on surface-enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI-TOF-MS) to test the possibility that MJ abuse might be associated with changes in serum protein levels. Indeed, MJ users showed significant increases in three protein peaks, which were identified as three isoforms of apolipoprotein (apo) C-III. Immunoprecipitation using an apoC-III antibody also validated the identification of the proteins. Marijuana-induced increases in apoC-III levels might occur through chronic stimulation of hepatic cannabinoid receptors (CB1 and/or CB2) by its active ingredient, Delta(9)tetrahydrocannibol (THC). Thus, chronic MJ abuse might cause increased transcription and/or translation of apoC-III in the liver with corresponding changes reflected in the plasma of these patients. In any case, because apoC-III is a cardiovascular risk factor, the increased levels observed in MJ users might explain, in part, the cardiac and cerebral abnormalities reported in these patients.
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Heavy marijuana users show increased serum apolipoprotein C-
III levels: evidence from proteomic analyses
S Jayanthi
1
, S Buie
1
, S Moore
2
, RI Herning
1
, W Better
1
, NM Wilson
1
, C Contoreggi
3
, and JL
Cadet
1
1
Molecular Neuropsychiatry Branch, NIH, BRC, Baltimore, MD, USA
2
Ciphergen Biosystems, Freemont, CA, USA
3
Office of the Clinical Director, National Institute on Drug Abuse-Intramural Research Program, NIH,
BRC, Baltimore, MD, USA
Abstract
Marijuana (MJ) is the most commonly used illicit drug in the United States. Its abuse is associated
with cognitive dysfunctions and increased resistance to blood flow in the cerebral vasculature. In
addition, MJ abuse is associated with increased risks of potentially serious cardiovascular disorders.
In the present study, we used the protein chip platform based on surface-enhanced laser desorption/
ionization time-of-flight mass spectroscopy (SELDI-TOF-MS) to test the possibility that MJ abuse
might be associated with changes in serum protein levels. Indeed, MJ users showed significant
increases in three protein peaks, which were identified as three isoforms of apolipoprotein (apo) C-
III. Immunoprecipitation using an apoC-III antibody also validated the identification of the proteins.
Marijuana-induced increases in apoC-III levels might occur through chronic stimulation of hepatic
cannabinoid receptors (CB1 and/or CB2) by its active ingredient, Δ
9
tetrahydrocannibol (THC). Thus,
chronic MJ abuse might cause increased transcription and/or translation of apoC-III in the liver with
corresponding changes reflected in the plasma of these patients. In any case, because apoC-III is a
cardiovascular risk factor, the increased levels observed in MJ users might explain, in part, the cardiac
and cerebral abnormalities reported in these patients.
Keywords
cannabis; apolipoproteins; protein profiling; cerebrovascular and cardiovascular risk factors
Introduction
Marijuana (MJ) is the most commonly used illicit drug in the United States, with 4.3% of young
adults reporting daily MJ use.
1
Acute adverse effects of MJ administration include tachycardia,
2
orthostatic hypotension,
3
supine hypertension,
4
dose-dependent increase in resting heart rate,
5
increase in myocardial oxygen demand,
6
ventricular tachycardia, acute myocardial infarction
and psychosis.
7
Long-term abuse of MJ is also associated with cognitive deficits that included
impaired learning, poor retention and retrieval and perceptual abnormalities.
8-11
Chronic MJ
abusers can also experience cerebrovascular accidents including transient ischemic attacks and
strokes that have been reported in young adults with history of heavy drug use.
12-15
© 2008 Nature Publishing Group All rights reserved
Correspondence: Dr JL Cadet, Molecular Neuropsychiatry Branch, National Institute on Drug Abuse, Intramural Research Program, NIH
Biomedical Research Center, 251 Bayview Boulevard, Baltimore, MD 21224, USA. E-mail: jcadet@intra.nida.nih.gov.
NIH Public Access
Author Manuscript
Mol Psychiatry. Author manuscript; available in PMC 2010 January 1.
Published in final edited form as:
Mol Psychiatry. 2010 January ; 15(1): 101–112. doi:10.1038/mp.2008.50.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
The physiologic effects of MJ are secondary to the effects by Δ
9
tetrahydrocannibol (THC), the
active ingredient in MJ.
16
THC exerts its effects by binding to cannabinoid (CB) receptors
located on several cell types in various organs.
17
The CB receptors include CB1 and CB2
receptors, which are differentially expressed in the brain and periphery.
18
In the periphery, CB
receptors are found in heart, liver, kidney, spleen, small intestine, testis or ovary.
17
Thus, it
was not far-fetched to suggest that chronic MJ abuse might be associated with changes in the
expression of proteins that would be reflected in the serum. It is also possible that some of
these proteins might be responsible, in part, for some of the adverse effects reported as a
consequence of prolonged MJ abuse.
As a first step toward testing these ideas, we decided to make use of a proteomic approach that
has been adapted for analysis and characterization of overall protein expression in the plasma,
other body fluids and various cell extracts.
19,20
The surface-enhanced laser desorption/
ionization time-of-flight (SELDI-TOF) ProteinChip strategies consist of arrays on which a
chromatographic phase is grafted to bind different serum proteins, according to their
biochemical properties. The proteins selectively retained on the surface are then analyzed using
a SELDI-TOF mass spectrometer (MS). That approach has allowed researchers to successfully
identify unique protein biomarkers for cancers of the ovary,
21
prostate,
22,23
mammary
glands
24
and for the pathophysiology of schizophrenia.
25
Similar high-throughput proteomic
approaches are being used to study toxic effects of exposure to methamphetamine and
methylenedioxymethamphetamine.
26
Thus, the purpose of the present paper is to report the
identification of apolipoprotein (apo) C-III, as an apolipoprotein that is significantly increased
in the serum of MJ abusers.
Materials and methods
Subjects
Eighteen MJ users and 24 control subjects were used in the present study. The demographic
data for these volunteers are given in Table 1. All volunteers had undergone medical,
neurological, psychological and laboratory evaluations. Exclusion criteria that applied to all
subjects include: (1) major medical and psychiatric illnesses including history of hypertension,
(2) head injuries with loss of consciousness for greater than 5 min, (3) evidence of any
neurological abnormalities by history or examination, (4) HIV seropositivity and (5) drug
(cocaine, heroin and so on) or excessive alcohol use by DSM-IV criteria for alcohol abuse or
dependence. In addition to self report, the use of other illicit drugs was also ruled out by
obtaining at least two observed urine samples for toxicological examinations during screening
visits. These standard toxicological examinations did not document the presence of any illicit
drugs (that is, amphetamine, barbiturates, benzodiazepines, CBs, cocaine metabolites,
methadone, opiates and phencyclidine) in any study participant, except for CBs for the MJ
group. The detection threshold for a positive screen for CBs was 50 mg per 100 ml. The research
protocol was approved by the National Institute on Drug Abuse and Johns Hopkins Bayview
Medical Center Institutional Review Boards for Human Research and was carried out in
accordance with the Declaration of Helsinki. Informed consent was obtained from all subjects.
These patients have been the subject of previous papers from this laboratory.
28
In addition to the medical history, demographic information and drug use history information
were obtained from the Addiction Severity Index.
27
The metric, joints per week, was
determined from the subjects self-reported drug history. The heavy users smoked the equivalent
of 130.8±
73.0
joints per week (n = 20; range 78350). Blood samples were obtained in 7 ml
Kendal vacutainer glass purple top tubes (BD 311743) following venous blood sample
procedures. The clotted samples were centrifuged (10 min, 4 °C, 3500 × g), aliquoted in
eppendorf tubes and frozen at 80 °C within 2 h after venous puncture.
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Serum sample preparation: subcellular prefractionation
Serum samples were pre-fractionated using pH gradients prior to analysis. Pre-fractionation
has been shown to increase the number of detected peaks
29
and, thus, can increase the
probability of identifying peptides or proteins that are differentially expressed.
30
Serum
fractionation employing 96-well filtration plate filled with dehydrated Q Ceramic HyperD F
sorbent (Expression Difference mapping kit from Ciphergen Biosystems Inc., CA, USA) was
used as a first purification step. A volume of 100 μl of the serum was diluted in 150 μl of U9
and incubated for half an hour at room temperature. The samples were diluted twice in binding
buffer, and loaded on the spin column and incubated under shaking for half an hour at room
temperature. The column was centrifuged at low speed (1000 r.p.m. for 1 min), to obtain the
flow through fractions. Then, a sequential fractionation of the column with a decreasing buffer
pH (pH 9; pH 7; pH 5; pH 4; pH 3; organic solvent) was performed. Each solution was incubated
for 20 min on the column. Fractionated serum samples were then applied to Cu II IMAC30
(immobilized metal affinity capture) chips in triplicate using the Ciphergen Bioprocessor (96-
well plate). IMAC30 ProteinChip Arrays reversibly bind proteins to the surface, through a co-
ordinated metal (Cu
2+
) interaction. This spotting allows the visualization of the different
proteins in each fraction to localize the proteins of interest. Reference serum samples were also
spotted along with the subject's fractionated serum samples.
IMAC30 ProteinChip processing
The IMAC30 chips were placed in a ProteinChip bioprocessor and 50 μl of 0.1 M copper sulfate
was incubated on the chips for 5 min with vigorous shaking. The chips were then rinsed with
deionized water and neutralized for 5 min with 50 μl of 0.1 M sodium acetate, pH 4.0. After
another rinse with deionized water, the chips were equilibrated by washing twice with 150 μl
of binding buffer (0.1 M sodium phosphate and 0.5 M sodium chloride, pH 7.0). Next, the
chips were loaded with 90 μl of binding buffer to which 10 μl of the fractionated serum (Fraction
6/organic fraction) was added. The serum was incubated with the chips for 1 h with vigorous
shaking. The serum was removed after an hour, and the chips were washed three times with
150 μl of binding buffer. A final rinse with deionized water was performed, the bioprocessor
was removed and the chips were allowed to air dry for 30 min. Once the chips were dry, 1 μl
of a 50% solution of SPA (sinnapinic acid) diluted in 50% (v/v) acetonitrile and 0.5% TFA (v/
v) was applied to each spot. The SPA solution was allowed to dry completely (15 min interval)
before it was added for a second time.
SELDI-TOF mass spectrometric analysis: data acquisition and processing
After the addition of the matrix (SPA), protein chips were read by a Protein Biology System
II (PBS II) instrument equipped with ProteinChip Software Version 3.2 (Ciphergen
Biosystems). ProteinChip Reader calibration in the low to high mass range was performed
using All-in-1 protein standard II prior to obtain the most accurate mass readings possible.
Spectra were obtained at laser intensities: low intensities (185 and 195, arbitrary units) were
used for low mass proteins ( < 20 kDa) and high intensities (200 and 215, arbitrary units) were
used for high mass proteins ( > 20 kDa). For both ranges, data were collected (with detector
sensitivity = 9) by averaging at least 200 laser shots/spectra collected in the positive mode from
each spot.
The raw data were transferred to ProteinChip Software Version 3.1 (Ciphergen Biosystems)
for analysis. Spectra were calibrated with the same external calibrants (IgG; albumin; enolase;
carbonic anhydrase; myoglobin; Cytochrome c; hirudine— All-in-1 peptide standard II;
Ciphergen Biosystems) used for instrument calibration. Peak intensities were normalized to
total ion current, excluding the mass range below 3000 Da, which is composed of strong signals
from the matrix. In this study, the mass range was narrowed to 300010 000 Da based on visual
inspections of spectra. Each spectrum was rescaled by a normalization factor, which was based
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on the average total ion current. Spectra were deleted if the normalization factor was more than
two times higher or lower than the mean normalization factor. Baseline was subtracted and
‘detected’ peaks were identified as those with a peak height 3 × noise and 6 × the valley depth.
The signal-to-noise cutoff was set at 3 for the first pass of peak detection (to pick up obvious
and well-defined peaks) and 1.5 for the second pass (to detect smaller peaks) and to ensure one
does not miss a potential significant peak. Detected peaks were labeled by m/z and the total
number of detectable peaks was recorded.
After the above processing, mass spectral data were used for expression difference mapping
using the Ciphergen ProteinChip software system (Ciphergen Biosystems), which provides
rapid statistical analysis of multiple samples to identify peaks with differential intensities. The
first step in the statistical analysis of peak intensity was to carry out biomarker wizard analysis
on calibrated and normalized data. To average the triplicate data export the biomarker wizard
analysis as a .csv file and create a pivot table to average the triplicate with Excel. The program
grouped peaks or ‘clusters’ within a designated mass range (for example, 300020 000 kDa)
that were present across multiple spectra at the same (that is, within a narrow window, such as
0.3%) m/z value. Each cluster then was treated as a single protein or peptide fragment.
Intensities of clustered peaks were analyzed by Student's t-test between two sample groups.
Differences were considered significant when P < 0.05. The experiments were performed blind
to diagnosis.
Reproducibility of SELDI-TOF MS spectra
The reproducibility of spectra generated by SELDITOF MS was evaluated in two experiments.
The goal of the first experiment was to investigate whether peaks of the same m/z appeared in
the duplicate spectra measured under the identical protocol (that is, two spots on the same 96-
well plate) and the goal of the second experiment was to determine the variability of spectra
across chips and over time (experiment done in triplicate). Mass spectra were generated,
calibrated by external calibrants (All-in-1 protein standard II, Ciphergen Biosystems) and
normalized by total ion current. Three peaks from the organic fraction spectra (P < 0.05) were
chosen to analyze the variation of m/z and relative peak intensity.
Enrichment of the biomarker protein by reverse phase beads
Among the three peaks that were identified, we enriched the peak (9.4 kDa) that showed the
highest levels of significance (P < 0.002, Table 2). Organic fractions containing the proteins
of interest were equilibrated with 50 μl of RPC Poly-Bio beads (Biosepra, Cergy, France) with
10% ACN/0.1% TFA. The sample solution was adjusted to a final concentration of 10% ACN/
0.5% TFA, mixed with 50 μl of equilibrated RPC beads and incubated for 30 min at room
temperature on the rotating shaker. Samples were then centrifuged for 1 min at 5000 r.p.m.
The super-natant was transferred to a fresh tube and bound proteins were eluted using 10, 20,
30, 40 and 50% ACN in 0.1% TFA. Proteins in the eluted fractions were analyzed by profiling
aliquots of each fraction on to a NP20 ProteinChip Array. The fractions of interest were
observed in the 50% acetonitrile fraction. This fraction was further concentrated by vacuum
centrifugation and dissolved in sample buffer for sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE).
Purification by SDS-PAGE gel electrophoresis
The dried fractions were dissolved in a loading buffer (2 × Tris-glycine SDS sample buffer,
Invitrogen, Carlsbad, CA, USA), and loaded onto a 18% Tris Glycine polyacrylamide gel
(Invitrogen). This was stained overnight using colloidal Coomassie blue (Safe-stain,
Invitrogen) and then destained with deionized water. The gel spot was extracted and used for
passive elution or for in-gel digestion.
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Passive elution of proteins from SDS-PAGE and protein in-gel digestion
The gel pieces were dehydrated in 100% acetonitrile (15 min). After removal of acetonitrile,
the gel pieces were covered with 70 μl of 50% formic acid, 25% acetonitrile, 15% isopropanol,
10% H
2
O and incubated for 2 h at room temperature under vigorous shaking. Aliquots of the
samples were analyzed directly on a NP20 array. This step allows for the comparison of the
molecular mass of the passively eluted protein with the original SELDI-TOF MS spectra to
ensure the digestion of the correct marker protein.
31
For in gel digestion, aliquots of 50% acetonitrile and of 100 mM ammonium bicarbonate (pH
8) solutions were added to the gels. This was followed by shaking for 1 h, followed by the
addition of 100% acetonitrile and further shaking. After removal of acetonitrile, the gel pieces
were dried in a 70 °C heat bath (dry) for 5 min. The gel pieces were rehydrated with 50 mM
ammonium bicarbonate (pH 8) containing 1020 ng μl
1
modified Trypsin (Roche Applied
Science, Indianapolis, IN, USA). The samples were then incubated for 16 h at 37 °C. These
samples are tested further with QSTAR mass spectrometer (Applied Biosystems, Foster City,
CA, USA) equipped with a PCI-1000 ProteinChip Interface.
Protein identification by peptide fragmentation using a QSTAR mass spectrometer (ABI/
Sciex) equipped with a PCI-1000 ProteinChip interface
A portion of the tryptic digests was analyzed on a Q-TOF Tandem mass spectrometer (QStar
Pulsar, ABI Applied Biosystems, Darmstadt, Germany) equipped with a SELDI source: PCI
1000 ProteinChip Interface (Ciphergen Biosystems). The system was externally calibrated
using human angiotensin I (m/z 1296.6853) and porcine dynorphin A (m/z 2147.1990). Raw
data were analyzed using Analyst Software (ABI, Foster City, CA, USA). After reviewing the
spectra, we selected specific ions for CID fragmentation and submitted the CID spectral data
to the database-mining tools Mascot (Matrix Science, London, UK) for identification.
Confirmation of apoC-III protein identification using Immunocapture
To confirm the identity of the biomarker, we used immunocapture assays. The affinity-purified
anti-human apoC-III antibody (1:4 dilution; Academy Bio-medical Company, Inc., Houston,
TX, USA) and control rabbit IgG (host species) were added to protein A ceramic HyperD beads
(Biosepra). After vigorous shaking, the beads were washed 3 times with 1 × PBS. The diluted
serum sample (1:10 dilution with 1 × PBS) was then added and incubated for 90 min. The
beads were washed according to the following procedure: once with 1 × PBS; 5 times with 50
mM Tris-HCl (pH 8.0), 1 M urea, 0.2% CHAPS, 500 mM NaCl; 3 times with 1 × PBS and
once with deionized water. The washes were followed by addition of 100 mM acetic acid. An
aliquot of the sample was then profiled onto a NP20 Chip array and the peaks were compared
to an apoC-III peak.
Assays of serum lipid and protein parameters
Triglycerides, cholesterol, α-1-globulin and albumin were determined using commercial kits
(Roche, Basel, Switzerland) and Hitachi 917 autoanalyzer from Roche Company.
Statistical analyses
Potential differences between the controls and the MJ users were assessed using analysis of
covariance (ANCOVA) with gender, ethnicity, monthly alcohol use and daily tobacco use as
co-variates. These covariates were used to statistically control for small ethnic and gender
sampling differences observed between the two groups. They were used to control for slightly
heavier use of alcohol and tobacco by the MJ users. Post-hoc analyses were done using
Student's t-test. Correlations were evaluated by calculating Pearson correlation coefficients
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(using a two-tailed test; alpha = 0.05). Analyses were performed using SPSS (version 12.0)
(SPSS Inc., Chicago, IL, USA). The null hypothesis was rejected at P < 0.05.
Results
MJ users show increased abundance of a protein peak at approximately 94 kDa m/z
In this study, we profiled the fractionated sera of 18 MJ users and of 24 controls using IMAC
Cu
2+
chromatographic surface. Profiles generated on IMAC Cu
2+
arrays allowed the best
discrimination between the control and the patient groups. To choose the optimal fraction and
array, the number of significant m/z values and the cluster plots were evaluated for each
condition using the Ciphergen Express software. We found several SELDI-TOF MS peaks that
retained discriminatory values in each of the three data sets. By using Biomarkers Patterns,
decision trees were made and the decision trees generated from the fraction 6 samples resulted
in a superior classification. All data from fraction 6 were reproduced at least three times, thus,
ensuring a good level of confidence in our observations. Table 2 shows the list of peaks that
showed significantly different between the groups (P < 0.05) in the ANCOVA after controlling
for gender, ethnicity, daily tobacco use and monthly alcohol use. Gender, ethnicity daily
tobacco and monthly alcohol use did not contribute to the differences observed between the
control and MJ group as these covariates were not significant in these analyses.
Figure 1a shows representative SELDI-MS spectra of MJ users and controls obtained on IMAC
Cu
2+
array, using SPA as matrix. Variation of intensity between patients and controls are visible
at approximately 9.4 kDa m/z. The intensity values for the 9.4 kDa m/z peak were consistently
higher in the serum of MJ users. As shown in the cluster plot in Figure 1b, the 9.4 kDa m/z was
one of the three diagnostic peaks that displayed a clear discriminatory differential expression
in MJ users in comparison to control subjects, with the MJ users showing significant higher
expression of the identified peak.
Identification of apoC-III and its isoforms as the relevant proteins that are differentially
expressed between the two groups
Isolation and enrichment of the identified 9.4kDa m/z peak was done as described above. Figure
2 shows that the fraction eluted by 50% acetonitrile/0.1% TFA fraction contained the 9.4 kDa
m/z peak along with two other peaks (8.8 and 9.7 kDa) that were also observed in the profiling
process (Table 2). The selected protein fraction was also subjected to SDS-PAGE and
Coomassie staining for further isolation and purification. In each case, three protein bands with
an apparent molecular weight between 8 and 10 kDa on the SDS-PAGE were found to be
increased in sample of MJ users. The three bands are shown in Figure 3a. The three protein
bands were excised and the proteins were passively eluted from the gel pieces, and their masses
were verified by MS profiling (Figure 3b), which confirms the SELDI-TOF-protein profile
results.
Identification of the 9.4 kDa m/z peak was done after excising this peak out of the SDS-PAGE
gel and in-gel trypsin digestion. The protease digest was subjected to tandem mass
spectrometry. The tryptic digest fragment of 1716 m/z (Figure 4a) was
DALSSVQESQVAQQAR. The CID spectral data was submitted to database-mining tools
Mascot and were identified as apoC-III (P02656) with a probability Mascot score (probability
based mowse score) of 128 (Figure 4b). Protein scores greater than 67 are significant (P <
0.05). The same protein purification and identification were repeated on four control and four
MJ users serum samples, and each time the excised protein band was identified as apoC-III.
Although the purification technique and further identification using tandem MS analysis
showed consistently that the putative protein marker was apoC-III, we chose to further confirm
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its identity by specific immunoaffinity capture on ProteinChip arrays containing polyclonal
anti-sera recognizing apoC-III (Figure 5). The peak at 9420 Da was specifically captured by
anti-apoC-III from both controls and MJ users serum samples (Figure 5). Isoforms of apoCIII
(8751 and 9751 Da) were also detected.
Effect of MJ on serum triglyceride levels
Because apoC-III and triglyceride levels are known to show positive correlations, we decided
to measure triglyceride levels in the serum collected from these participants. Table 3 shows
that the triglyceride levels showed non-significant increases in the serum of MJ users.
Nevertheless, Figure 6a shows that there were significant positive relationships between serum
levels of apoC-III and the concentration of serum triglycerides (r = 0.507, P < 0.001). The
levels of apoC-III also correlated with those of α-1-globulin and cholesterol (Figures 6b and
c) but not with those of serum albumin (Figure 6d). Table 3 also shows values for the levels
of α-1-globulin and albumin measured in the same serum samples. Gender, ethnicity, daily
tobacco and monthly alcohol use did not contribute to these findings as these covariates were
not significant in these analyses.
Discussion
The main finding of the present study is that chronic MJ abuse is associated with significant
increases in serum apoC-III levels. In addition, there were significant correlations between
apoC-III and triglyceride levels. Because of the relationship between apoC-III levels and
vascular risk factors, the observations in MJ abusers will be discussed in terms of the potential
relevance to MJ-induced adverse vascular events, which include coronary abnormalities.
32
These vascular dysfunctions are thought to be secondary to complex interactions between the
various metabolic and physiologic effects of chronic MJ use. The present study, which profiled
proteins in the serum of these patients using SELDI-TOF-MS, has now identified ApoC-III as
a possible agent in this complex network of MJ-induced effects in humans.
ApoC-III is a 79 amino-acid glycoprotein that contains galactose, as its sugar moiety.
33
ApoC-
III resides on the surface of very low density lipoprotein and low density lipoprotein and
influences their metabolism.
34
There are three isoforms of the protein, designated by the
number of sialic acid residues are called C-III
0
, C-III
1
and C-III
2
.
35,36
ApoC-III plays a
regulatory role in the catabolism of triglyceride rich lipoproteins by inhibiting the activity of
lipoprotein lipase, which hydrolyzes lipids in lipoproteins.
37
It also blocks the activity of
hepatic triglyceride lipase, which metabolizes triglycerides
38
that are independent risk factors
for cardiac vascular disorders.
39
Several lines of evidence have documented positive
correlations between plasma apoC-III levels and elevated levels of plasma triglycerides.
40-42
Animals studies have indeed shown that overexpression of human apoC-III in mice causes
severe hypertriglyceridemia,
43
whereas disruption of the endogenous apoC-III gene prevents
postprandial hypertriglyceridemia in mice.
44
In addition to its effects on triglycerides, apoC-
III can contribute to atherosclerosis by recruiting monocytes onto vascular endothelium.
45
The increased serum apoC-III levels in MJ users and the positive correlations between serum
apoC-III and triglyceride levels observed in the present study are consistent with the findings
of a previous paper that had reported significant increases in serum HDL-triglyceride
concentrations in MJ users in comparison to controls.
46
Thus, when taken together with
apoCIII-mediated decreases in the rate of mitochondrial respiration,
47
the observed increases
in apoC-III in the MJ users hint to the possibility that chronic MJ abuse could lead to
impairments of cellular energetics and mitochondrial function, which are critical events
associated to myocardial infarction, stroke and ischemic/reperfusion damage.
48
This
discussion might offer a partial explanation for the chronic effects of the drug on cardiac and
cerebrovascular systems.
49-51
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The manner by which chronic MJ smoking might cause these increases in serum apoC-III levels
remains to be clarified. However, recent clinical data have demonstrated significant effects of
CB1 receptor signaling on lipid parameters. Specifically, treatment of obese patients with the
CB1 receptor blocker, rimonabant, caused significant improvements in the levels of
triglycerides.
52,53
These results provide further support for a role of CB receptors in the
mediation of the cardiovascular adverse effects observed during chronic MJ consumption,
49-51
which are mediated through actions of the actions of Δ
9
-THC, the major psychoactive
compound in MJ. After inhalation, Δ
9
-THC is rapidly absorbed and distributed throughout the
circulation; it is then metabolized, in the lungs and liver, to the more potent, 11-hydroxy- THC,
which crosses the hematoencephalic barrier very easily.
54
In the periphery, THC exerts its
effects by binding to CB receptors located in many peripheral tissues, including adipose tissue
and liver, which are key organs involved in carbohydrate and lipid metabolism. The possibility
that MJ exerts its actions via stimulation of CB1 receptors is supported by experiments
documenting promotion of lipogenesis in the rodent liver by the CB1 agonist, HU-210 and its
reversal by pretreatment with the CB1 blocker, rimonabant.
55
Moreover, in adipose tissue, the
CB1 agonist has been shown to influence activity of lipoprotein lipase,
56
an enzyme whose
activity is also regulated by apoC-III.
37
A role for peripheral CB receptors in the actions of MJ
is also supported by the report that the levels of CB1 receptor mRNA are significantly higher
in peripheral mono-nuclear cells of MJ users than in those of control subjects.
57
Furthermore,
molecular characterization of transcriptional regulatory elements in the apoC-III promoter has
also revealed binding sites for the inducible transcription factor, NF-kB.
58,59
Because Δ
9
-THC,
the active ingredient in MJ can induce NF-kB activation via increased phosphorylation of I-
KB,
60
chronic MJ abuse might also affect apoC-III expression via THC-mediated NF-KB-
induced hepatic inflammation that is known to cause substantial changes in the production and
utilization of triglyceride rich lipoproteins.
61
Thus, the collected evidence suggests that MJ
abuse may lead to sustained changes in THC-induced signaling pathways that promote the
increased apoC-III levels that we are now reporting in the serum of MJ users.
Conclusion
Marijuana is the most commonly abused drug in the USA and evidence exists that its abuse is
a risk factor for cardiovascular and cerebrovascular disorders. Our present observations that
MJ abusers show significant increases in serum apoC-III levels suggest that THC-induced
apoC-III upregulation might be a significant player in MJ-mediated vascular and neuroimaging
abnormalities.
28,62-65
The idea of a possible involvement of an apolipoprotein in a
neuropsychiatric disorder is consistent with the long history of the involvement of
apolipoproteins (for example, ApoE) as etiological factors in neurodegenerative disorders such
as Alzheimer's disease.
66,67
Verification of these ideas will await further studies that will seek
to elucidate the role of apoC-III in mediating some of the long-term neuropsychiatric and
neurological effects of MJ.
Acknowledgments
This research was supported by the Intramural Research Program of the NIH, NIDA.
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Figure 1.
SELDI-TOF-MS protein spectra of marker of m/z value around 9.4 kDa. (a) Representative
protein spectra of seven control subjects and seven MJ (marijuana) users. The marker is
indicated with an arrow. The mass is given as m/z values on the x-axis and the intensity is
displayed along the y-axis. Note the different sizes of the peaks in the control and MJ groups.
The bands at 9.4 kDa were significantly increased in the MJ patients (P < 0.002, Student t-
test). (b) Range of values for individual MJ users (on top of line) and controls (bottom of line).
Note the lack of overlap between the two groups, with the MJ group showing higher values.
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Figure 2.
The flow through from the Q fractionation was bound to the reverse phase beads and then
eluted with increasing concentrations of ACN/0.1% TFA. The biomarker was found in the 50%
ACN/0.1% TFA fraction.
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Figure 3.
Confirmation of the identity of the 9.4 kDa putative biomarker. (a) Coomassie blue stained
band pattern from samples of one healthy control sample and one of MJ patients separated by
SDS-PAGE. The left lane shows the marker, the middle lane shows the control sample and the
right lane shows the MJ sample. The bands which were excised are indicated by arrows. (b)
Profile of the biomarker on a NP-20 array after elution in formic acid, acetonitrile and 2-
propanol. These results confirm the identity of the excised proteins. SDS-PAGE, sodium
dodecyl sulphate-polyacrylamide gel electrophoresis.
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Figure 4.
The sequence of the most abundant discriminating peptide (m/z 1716 Da) was defined by TOF/
TOF sequencing as DALSSVQESQVAQQAR, which matches with high probability
(MASCOT score 128) the sequence of apolipoprotein C-III.
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Figure 5.
Confirmation of biomarker using anti-apoC-III antibody. Protein A Hyper D beads were
incubated with rabbit anti-human antibody and control rabbit IgG. After washing, serum was
incubated with serum diluted in PBS. The beads were washed followed by elution with 1215
μl of 0.1 M acetic acid. Eluted fractions were profiled on NP20 arrays. Note the differences in
the scales representing the control and MJ (marijuana) groups. In a fashion similar to the
observation in Figure 1, the peaks in the MJ group were higher than those of the control group.
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Figure 6.
Correlation analyses of apoC-III levels with (a) triglycerides, (b) cholesterol, (c) α-1-globulin
and (d) albumin in control subjects and MJ (marijuana) abusers.
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Table 1
Demographic measures and drug history
Demographic measures Controls (n = 24) mean ±
s.d.
Marijuana users (n = 18)
mean ± s.d.
Age: years 21.8 + 6.3 21.4 + 2.7
Education: years 12.2 + 1.3 11.5 + 1.2
Shipley: IQ 102.2 + 10.2 95.2 + 9.6
Women
a
72% 47.4%
African Americans
a
68% 94.7%
Drug history measures
Alcohol: days per 30 days
b
0.73 + 1.8 5.6 + 6.4
*
Alcohol: years
c
0.48 + 2.0 2.4 + 3.8
Marijuana: days per 30 days 29.1 + 1.7
Marijuana: years
c
6.2 + 1.6
Cigarettes per day 2.0 + 5.7 5.79 + 6.99
*
Cigarettes: years 0.52 + 1.6 6.37 + 15.6
Data are shown as mean ± s.d.
a
Data are shown as percentage.
b
The number of days of substance use in the last 30 days from the ASI.
27
c
Years of substance use calculated from ASI.
27
*
Mean for MJ users greater than controls at P < 0.05 using Student's t-test.
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Table 2
Biomarkers from univariate cross-site sample comparison of marijuana users vs control
individuals after controlling for gender, ethnicity, daily tobacco use and monthly alcohol use
showing m/z ratio, direction of change and P-value for each peak
m/z
a
Protein chip array type Fraction used Direction of change
in marijuana users
P-values
b
8764 IMAC30 Cu Fraction 6 < 0.014
9447 IMAC30 Cu Fraction 6 < 0.002
9750 IMAC30 Cu Fraction 6 < 0.004
The spectra were calibrated with the protein low calibration on the NP-20 array.
The peak information presented in bold was the peak selected for further enrichment.
a
m/z mass/charge.
b
P-values calculated from Student's t-test.
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Table 3
Serum concentration of triglycerides, cholesterol, α-1-globulin and albumin in controls and
heavy marijuana users
Controls (n = 24) Marijuana users (n = 18) P–value
Triglycerides 91.17 ± 12.49 122.00 ± 14.43 0.09
Cholesterol 175.33 ± 5.75 167.33 ± 6.64 0.36
α-1-globulin 0.213 ± 0.021 0.233 ± 0.024 0.51
Albumin 4.258 ± 0.147 4.25 ± 0.17 0.97
BMI 25.711 ± 1.255 25.863 ± 1.449 0.94
Abbreviation: BMI, body mass index.
Triglyceride and cholesterol concentrations are expressed in milligram per 100 ml and represent mean ± s.e.
α-1-globulin and albumin concentrations are expressed in gram per 100 ml and represent mean ± s.e.
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... In a proteomic analysis of serum samples from cannabis users, all 3 isoforms of ApolipoproteinC-III (ApoC-III) were enhanced in cannabis users relative to controls. ApoC-III regulates the catabolism of lipoproteins rich in triglycerides, and increased levels of this protein was proposed as a risk factor for cardiovascular disorders (Jayanthi et al., 2010). ...
... Proteomic analysis on the serum from cannabis users described alterations to proteins and pathways involved in lipid metabolism (Jayanthi et al., 2010;Alasmari et al., 2021). Specifically, increased ApoA-I was evident in cannabis users (Jayanthi et al., 2010). ...
... Proteomic analysis on the serum from cannabis users described alterations to proteins and pathways involved in lipid metabolism (Jayanthi et al., 2010;Alasmari et al., 2021). Specifically, increased ApoA-I was evident in cannabis users (Jayanthi et al., 2010). Interestingly, changes in ApoA-I expression were also found in blood from BDI patients, although in the opposite direction to cannabis users (Song et al., 2015). ...
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... However, HDL-C levels in chronic smokers vs. controls were somewhat lower in an agematched, race-matched and BMI-matched analysis: 1.30 AE 0.40 vs. 1.40 AE 0.30 mmol/l (P ¼ 0.02). In an investigation by Jayanthi et al. [47], chronic smokers (N ¼ 18), defined as having had a mean smoking duration of 6 years with a range of 78-350 marijuana cigarettes per week, had significantly higher serum levels of apolipoprotein (apo) C-III vs. nonsmoking controls (P < 0.002), after controlling for sex, ethnicity, tobacco use and alcohol use. Apo C-III is a marker for hypertriglyceridemia and coronary artery disease [47][48][49]. ...
... In an investigation by Jayanthi et al. [47], chronic smokers (N ¼ 18), defined as having had a mean smoking duration of 6 years with a range of 78-350 marijuana cigarettes per week, had significantly higher serum levels of apolipoprotein (apo) C-III vs. nonsmoking controls (P < 0.002), after controlling for sex, ethnicity, tobacco use and alcohol use. Apo C-III is a marker for hypertriglyceridemia and coronary artery disease [47][48][49]. In addition, mean TG levels trended nonsignificantly higher in the chronic user vs. nonuser group: 112.0 AE 14.4 vs. 91.2 ...
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... Patients were excluded if they had taken any anti-inflammatory analgesic within the 2 preceding weeks; had a history of neurosurgery, central nervous system (CNS) diseases, cancer, and tuberculosis; had a substance use disorder other than alcohol, caffeine, and nicotine; were currently lactating or pregnant; and/or were predicted to have an intellectual disability or difficulty in understanding Korean. These exclusions were predominantly based on previous evidence of known associations between these conditions and protein expression [11][12][13][14][15][16][17][18][19]. Patients with a history of neuromodulation or intensive psychotherapy for the past 2 months were also excluded to confine the effect of treatment to psychotropic medications. ...
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... Indeed, protein metabolism via AKT/mTOR, cytoskeleton organization, and calcium signalling are affected in human samples from SCZ patients [2], and after cannabinoid exposure in cultured cells [21,22] and mice models [23,24]. Moreover, plasmatic samples from heavy cannabis users and SCZ patients share an effect on oxidative stress response [7,25]. However, whether the coexistence of both factors has equal or distinct consequences is less clear. ...
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... Although some early experimental animal data had suggested that cannabinoid therapy may reduce the progression of atherosclerosis [44], real-life human data have been contradictory, showing a high risk of CV events in marijuana users. Even in the atherosclerosis field, subsequent human data showed that marijuana induces increases in apolipoprotein (apo) C-III levels, probably occurring through chronic stimulation of hepatic cannabinoid receptors (CB1) by its active ingredient, Δ 9 tetrahydrocannibol (THC) [45]. As apoC-III is a CV risk factor, it has been suggested that the increased levels observed in marijuana users might explain, in part, the cardiac and cerebral abnormalities reported in these patients. ...
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Objective. —To test whether a predominance of small, dense low-density lipoprotein (LDL) particles and elevated triglyceride levels are independent risk factors for myocardial infarction (MI).Design. —Nested case-control study with prospectively collected samples.Setting. —Prospective cohort study.Participants. —Blood samples were collected at baseline (85% nonfasting samples) from 14916 men aged 40 to 84 years in the Physicians' Health Study.Main Outcome Measurements. —Myocardial infarction diagnosed during 7 years of follow-up.Results. —Cases (n=266) had a significantly smaller LDL diameter (mean [SD], 25.6 [0.9] nm) than did controls (n=308) matched on age and smoking (mean [SD], 25.9 [8] nm; P<.001). Cases also had higher median triglyceride levels (1.90 vs 1.49 mmol/L [168 vs 132 mg/dL]; P<.001). The LDL diameter had a high inverse correlation with triglyceride level (r=-0.71), and a high direct correlation with high-density lipoprotein cholesterol (HDL-C) level (r=0.60). We observed a significant multiplicative interaction between triglyceride and total cholesterol (TC) levels (P=.01). After simultaneous adjustment for lipids and a variety of coronary risk factors, LDL particle diameter was no longer a statistically significant risk indicator, with a relative risk (RR) of 1.09 (95% confidence interval [CI], 0.85-1.40) per 0.8-nm decrease. However, triglyceride level remained significant with an RR of 1.40 (95% CI, 1.10-1.77) per 1.13 mmol/L (100-mg/dl) increase. The association between triglyceride level and MI risk appeared linear across the distribution; men in the highest quintile had a risk about 2.5 times that of those in the lowest quintile. The TC level, but not HDL-C level, also remained significant, with an RR of 1.80 (95% CI, 1.44-2.26) per 1.03-mmol/L (40-mg/dL) increase.Conclusions. —These findings indicate that nonfasting triglyceride levels appear to be a strong and independent predictor of future risk of MI, particularly when the total cholesterol level is also elevated. In contrast, LDL particle diameter is associated with risk of MI, but not after adjustment for triglyceride level. Increased triglyceride level, small LDL particle diameter, and decreased HDL-C levels appear to reflect underlying metabolic perturbations with adverse consequences for risk of MI; elevated triglyceride levels may help identify high-risk individuals.
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Context Cognitive impairments are associated with long-term cannabis use, but the parameters of use that contribute to impairments and the nature and endurance of cognitive dysfunction remain uncertain.Objective To examine the effects of duration of cannabis use on specific areas of cognitive functioning among users seeking treatment for cannabis dependence.Design, Setting, and Participants Multisite retrospective cross-sectional neuropsychological study conducted in the United States (Seattle, Wash; Farmington, Conn; and Miami, Fla) between 1997 and 2000 among 102 near-daily cannabis users (51 long-term users: mean, 23.9 years of use; 51 shorter-term users: mean, 10.2 years of use) compared with 33 nonuser controls.Main Outcome Measures Measures from 9 standard neuropsychological tests that assessed attention, memory, and executive functioning, and were administered prior to entry to a treatment program and following a median 17-hour abstinence.Results Long-term cannabis users performed significantly less well than shorter-term users and controls on tests of memory and attention. On the Rey Auditory Verbal Learning Test, long-term users recalled significantly fewer words than either shorter-term users (P = .001) or controls (P = .005); there was no difference between shorter-term users and controls. Long-term users showed impaired learning (P = .007), retention (P = .003), and retrieval (P = .002) compared with controls. Both user groups performed poorly on a time estimation task (P<.001 vs controls). Performance measures often correlated significantly with the duration of cannabis use, being worse with increasing years of use, but were unrelated to withdrawal symptoms and persisted after controlling for recent cannabis use and other drug use.Conclusions These results confirm that long-term heavy cannabis users show impairments in memory and attention that endure beyond the period of intoxication and worsen with increasing years of regular cannabis use.
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: Cognitive, cerebrovascular, and psychiatric impairments have been documented with chronic marijuana users. To better understand the nature and duration of these neurocognitive changes in marijuana abusers, we recorded the resting EEG of 29 abstinent chronic marijuana abusers and 21 control subjects. The marijuana abusers were tested twice: the first evaluation occurred within 72 hours of admission to the inpatient research unit; the second evaluation occurred after 28 to 30 days of monitored abstinence. A three-minute period of EEG was recorded during resting eyes-closed conditions from eight electrodes (F3, C3, P3, O1, F4, C4, P4, and O2). The artifacted EEG was converted to six frequency bands (δ, θ, α1, α2, β1, and β2) using a fast Fourier transform. During early abstinence, absolute power was significantly lower (p < 0.05) for the marijuana abusers than for the control subjects for the θ and α1 bands. These reductions in θ and α1 power persisted for 28 days of monitored abstinence. These EEG changes, together with cerebral blood flow deficits, might underlie the cognitive alterations observed in marijuana abusers. Additional research is needed to determine how long these deficits persist during abstinence and if treatment with neuroprotective agents may reverse them.
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Overproduction of Apo CIII causes elevated plasma triglyceride levels in transgenic animals and is associated with hypertriglyceridemia in humans. The regulation of apo CIII production is likely to play an important role in controlling plasma triglyceride levels. As an initial step in determining the role of transcriptional regulation in the production of apo CIII and in triglyceride metabolism, we have begun to characterize the activity of specific transcriptional regulatory elements in the CIII promoter. In the current study, we have identified and characterized an NF-χB regulatory element located 150 nucleotides upstream from the transcriptional start site of the apo CIII gene. Purified NF-χB, as well as an NF-χB protein in HepG2 cell nuclear extracts, bound specifically to this sequence element. The hepatic protein was induced by phorbol ester (PMA), and reacted with antibodies to the p50 and p65 subunits of NF-χB. The NF-χB element conferred PMA and IL1-β inducible transcriptional activity to a heterologous promoter/reporter construct when transfected into HepG2 cells. Analysis of the full length CIII promoter demonstrated that the inducible activity of the NF-χB element was suppressed by sequences in the apo CIII enhancer element located approximately 500 nucleotides upstream of the NF-χB binding site. A deletion removing the enhancer restored the PMA inducible activity of the NF-χB binding site. These results indicate that apo CIII gene expression is regulated by NF-χB, and suggest that apo CIII production may be modulated by cellular signals, like inflammatory cytokines, that activate NF-kB.