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Abstract

Introduction:

Illicit drugs abuse is associated with several clinical life-threatening consequences that are primarily mediated by oxidative damage to multiple cellular components with a subsequent cellular dysfunction and death. Primarily, this study aimed to investigate oxidative stress to protein and lipid components of circulatory platelets among chronic heroin and hashish addicts.

Methods:

Platelet lysates were prepared from 20 chronic intravenously administrated heroin addicts and 20 chronic smoked hashish addicts. For comparative purposes, two control groups of 20 cigarette smokers and 20 nonsmokers were included in the study. Oxidative stress to platelet’s proteins and lipids was investigated using carbonyl group contents assay and thiobarbituric acid reactive substances (TBARS) assay, respectively.

Results:

In comparison to control groups, carbonyl group contents and TBARS concentration were significantly higher among heroin addicts but not among hashish addicts. Both of these markers were significantly correlated to the duration of addiction but not to the daily administrated dose. While in regard of the timing of the latest administrated dose (TLAD), only carbonyl group contents were significantly correlated to the TLAD.

Conclusions:

Considering the contribution of drug’s route of administration, drug’s pharmacokinetics, and kinetics of circulatory platelet, we concluded that chronic heroin addiction is associated with significant levels of oxidative stress to platelet’s proteins and lipids. Due to the high proteomic contents of platelets, protein’s oxidative stress is more prominent compared to lipids. Chronic hashish smoking is not associated with significant levels of oxidative stress in platelet’s proteins and lipids.

Keywords

Heroin hashish oxidative stress platelets

Introduction

Opioids and cannabinoids are distinct categories of illicit drugs among which heroin and hashish, respectively, are the most commonly abused. Clinically, opiates and cannabis are widely used for the management of chronic pain.¹ However, the long-term administration of these drugs has been shown to be associated with physiological dependence and drug tolerance that are responsible for several negative health impacts and life-threatening complications.^2,3 Primarily, the toxic effects of the abuse of these drugs are the result of a progressive oxidative stress in multiple tissues and organs including central nervous system, liver, kidney, heart, and cardiovascular system.^4,5

Heroin induces the generation of reactive oxygen species (ROS) and nitrogen species that are responsible for cellular and systemic toxicity.⁶ The generation of reactive species results in the disruption of the oxidant–antioxidant homeostasis with a subsequent impairment of the total antioxidant capacity (TAC).^7,8 On the other hand, hashish is a plant-derived cannabis that comprises multiple psychoactive ingredients among which delta-9-tetrahydrocannabinol (THC) is the most potent.⁹ THC toxicity was correlated with mitochondrial dysfunction and subsequent induction of ROS formation and defective adenosine triphosphate generation.¹⁰

The impacts of heroin and hashish addictions on hemorheological properties and cellular changes of blood were investigated by several earlier studies; blood viscosity, cells counts, and indices were among the features that were investigated.^11
–13 However, oxidative damage precipitated by chronic addiction on blood cells including platelets is poorly studied. Here, in this study, we investigated oxidative damage in platelets in response to chronic heroin and hashish addiction via measuring platelets lipid peroxidation and protein carboxylation using carbonyl groups content assay and thiobarbituric acid reactive substances (TBARS) assay, respectively. Carbonyl groups are formed due to oxidation of arginine, lysine, threonine, or proline amino acids by ROS.¹⁴ On the other hand, TBARS is an assay of the oxidative damage to lipids, specifically polyunsaturated fatty acids.^15,16

Methods

Study subjects

A total of 80 male subjects were included in the study of whom 20 were chronic heroin addicts (aged 21–47 years old) and 20 were hashish smokers (aged 19–42 years old). All participants were exclusively addicts to either heroin or hashish with their latest dose of administration was within an average 10.6 ± 1.9 and 10.1 ± 1.7 days prior to sample collection, respectively. Among heroin addicts, the mean heroin abuse duration was 9 ± 6 years (1–23 years) with a mean claimed daily dose (DD) of 0.67 ± 0.40 g (0.25–2 g) while hashish addicts were having a mean hashish smoking duration of 5 ± 3 years (1–13 years) with a mean DD of 3 ± 2 cigarettes (1–8 cigarettes/day). Subject’s medical profiles were reviewed including their history of addiction, other drugs addiction, or medications uptake including antioxidants. Demographic criteria of study subjects are illustrated in Table 1.

Table 1.

Demographic criteria of study subjects.

Variable	Heroin addicts	Hashish addicts	Control smokers	Control nonsmokers
Age (years)	33.5 ± 5.7 (21–47)	24.7 ± 4.4 (19–42)	27.6 ± 5.6 (21–40)	28.4 ± 4.9 (19–37)
Gender (%)	Males (100%)	Males (100%)	Males (100%)	Males (100%)
TDCA (years)	9 ± 6 (1–23)	5 ± 3 (1–13)	—	—
TLAD (days)	10.6 ± 1.9	10.1 ± 1.7	—	—
DD (unit)	0.7 ± 0.4 (0.25–2.0) g	3 ± 2 (1–8) cigarettes	—	—
Cigarette smoker (daily average)	Yes (27 ± 11 cigarettes)	Yes (25 ± 8 cigarettes)	Yes (25 ± 5 cigarettes)	No

TDCA: total duration of chronic addiction; TLAD: timing of latest administrated dose; DD: daily dose.

Considering the fact that all addicts were cigarette smokers, two voluntary age and sex matched control groups were included: cigarette smoker group (n = 20) and cigarette nonsmokers group (n = 20). Prior to their participation in the study, all participants have approved their enrollment and filled a consent form in that regard. The study was reviewed and approved by the Institutional Review Board committee at Jordan University of Science and Technology and was conducted in collaboration with the Drug Enforcement Administration of Public Security Department/Amman.

Sample collection

Five milliliters of venous blood were withdrawn from each participant in acid citrate dextrose collection tubes. Platelets rich plasma (PRP) was obtained by an initial centrifugation at 200 × g for 15 min at room temperature. PRP was centrifuged at 800 × g for 15 min at room temperature to obtain platelets pellet, after which pellets were gently resuspended and washed with platelets washing buffer (Abcam, Cambridge, UK). Finally, platelets lysates were prepared by adding ice-cold 1:1 (v/v) mixture of Tyrode’s buffer and 2× platelet lysis buffer (Abcam, UK).

Estimation of platelet protein concentration by bicinchoninic acid protein assay

For proteins quantification, 50 μL of platelet lysates were transferred into a clear-bottom 96-well plate followed by an incubation for 60 min at 37°C with 100 mL of working solution containing copper salt and bicinchoninic acid (BCA). In the presence of proteins, copper salt will be reduced to cuprous ion (Cu+2) that will form a colored complex with BCA reagent. Protein concentration was then determined via spectrophotometry at wavelength (λ) equals 562 nm.

Detection of carbonyl group by colorimetric assay

Herein, protein carbonyl contents per one milligram of proteins were measured using commercially available kit (Abcam, UK) with a sensitivity detection limit of 0.15 nanomole carbonyl per milligram of protein. One hundred microliters of platelet lysate (optimized to a final protein concentration of 0.5 to 20 μg/μL) were incubated with 100 μL of dinitrophenyl hydrazine for 10 min at room temperature. Proteins were allowed to precipitate by incubation for 5 min on ice with 30 μL of trichloroacetic acid, after which precipitates were washed twice with 500 μL of cold acetone. Finally, precipitates were dissolved in guanidine hydrochloride and loaded into a clear-bottom 96-well plate. To determine the carbonyl group contents, the optical density was determined by colorimetric assay at 375 nm wavelength (λ).

TBARS assay

TBARS assay was conducted using commercially available kit (Cell Biolabs, San Diego, USA) that provides a detection range of 0–125 µM. Initially, 100 μL of platelets lysate was mixed with 100 μL of sodium dodecyl sulfate followed by an incubation for 5 min at 37°C. Then, 250 µL of TBA reagent was added followed by an incubation for 60 min at 95°C. To stop the reaction, the mixture was cooled followed by centrifugation at 1600 × g for 10 min at 4°C. Finally, 200 mL of each sample and standards were transferred into 96-well plate and the optical density was determined by colorimetric assay at wavelength (λ) of 530 nm.

Statistical analysis

The obtained results were compared among study groups using an analysis of variance (ANOVA). Correlation analysis was conducted using Pearson’s correlation and spearman’s (ρ) ranks correlation. Statistical package for social sciences version 23 was used to conduct statistical analysis. Graphical charts were built using GraphPad Prism 6.

Results

Protein’s carbonylation

As demonstrated in Figure 1, chronic heroin addicts were having a mean carbonyl group content in platelet proteins equal 2.83 ± 0.15 nmol/mg. One-way ANOVA analysis revealed that this is significantly greater than those among control smokers as well as control nonsmokers who were having mean carbonyl group content of 1.17 ± 0.09 nmol/mg (p = 0.000) and 1.43 ± 0.09 nmol/mg (p = 0.000), respectively. On the other hand, the mean carbonyl group content among hashish smokers was 1.62 ± 0.17 nmol/mg which has been shown to be insignificantly different from its corresponding value among both control smokers and control nonsmokers groups with p values of 0.889 and 0.138, respectively.

Figure 1.

Carbonyl contents in platelets lysates from study subjects: A significantly higher carbonyl contents were evident among subjects with chronic heroin addiction in comparison to both control smoker and nonsmoker subjects. The elevated level among chronic hashish addicts has been shown to be statistically insignificant (p > 0.05). Results are presented as mean ± SEM (nmol/mg). *Significant difference with control groups (p < 0.05).

Lipid peroxidation

As demonstrated in Figure 2, platelet’s lipid peroxidation analysis has revealed a significantly higher TBARS concentration among chronic heroin addicts with a mean concentration of 7.75 ± 0.96 μmolar as compared to control smoker subjects and control nonsmoker subjects with mean TBARS levels equal 3.71 ± 0.19 (p = 0.003) and 3.02 ± 0.20 (p = 0.001), respectively. In regard to hashish addicts, the mean TBARS concentration among addicts was 4.87 ± 0.71 μmolar which is insignificantly different from that among control smoker subjects (p = 0.567) and control nonsmoker subjects (p = 0.116).

Figure 2.

TBARS concentration in platelets lysates from study subjects: A significantly higher TBARS concentration was evident among subjects with chronic heroin addiction in comparison to both control smoker and nonsmoker subjects. The elevated level among chronic hashish addicts has been shown to be statistically insignificant (p > 0.05). Results are presented as mean ± SEM (µmolar). *Significant difference with control groups (p < 0.05). TBARS: thiobarbituric acid reactive substances.

Correlation analysis

In accordance with the previous findings, correlation analysis has revealed a moderate but statistically significant correlation between protein carbonyl contents and TBARS concentration among study subjects as represented by Pearson’s correlation coefficient equals 0.326 (p = 0.003). Furthermore, correlation analysis has demonstrated a monotonic relationship of protein carbonyl contents and TBARS concentration among the study groups as represented by spearman’s correlation coefficients (ρ) equal 0.651 (p = 0.000) and 0.521 (p = 0.000), respectively.

To further investigate the contribution of chronic heroin addiction to oxidative damage in circulatory platelets, heroin addicts were defined based on the total duration of chronic addiction (TDCA), the DD as well as the timing of latest administrated dose (TLAD). As illustrated in Table 2, both of protein carbonyl contents and TBARS concentrations were moderately correlated to the TDCA (in years) as revealed by Pearson’s correlation coefficients equal 0.490 (p = 0.002) and 0.339 (p = 0.035), respectively. On the other hand, no correlation between these oxidative markers and the daily administrated heroin dose while a strong inverse relationship was evident for proteins carbonyl contents with the latest administrated dose as revealed by a correlation coefficient equals 0.643 (p = 0.002).

Table 2.

Correlation analysis of carbonyl contents and TBARS concentration in platelet’s lysate from chronic heroin addicts.

Variable	Carbonyl contents		TBARS conc.
Variable	ρ	p Value	ρ	p Value
DCA	0.490	0.002^a	0.339	0.035^b
DD	0.300	0.199	0.288	0.218
TLAD	−0.643	0.002^a	−0.156	0.512

DCA: duration of chronic addiction (in years); DD: daily dose (in mg); TLAD: timing of latest administrated dose (in days).

^a p < 0.01.

^b p < 0.05.

To validate the obtained significant correlation of protein carbonyl contents with the TLAD, heroin addicts were subcategorized based on the TLAD into three groups: recent group (with their latest dose was within up to 9 days from the sample withdrawing), intermediate (within 10 to 20 days), and distant (more than 20 days). This categorization has considered the pharmacokinetics of intravenously administrated heroin as well as the life span of circulatory platelets in the peripheral blood that is 8 to 9 days.¹⁷ Obtained results are illustrated in Figure 3.

Figure 3.

Comparison of carbonyl group contents and TBARS concentration among chronic heroin addicts based on the TLAD: Heroin addicts were categorized based on TLAD as subjects with recent (≤9 days), intermediate (10–20 days), and distant (>20 days) TLAD. (a) Carbonyl group contents: A significant difference was evident only between heroin addicts with recent and distant TLAD (p < 0.035). However, addicts with recent and intermediate TLAD were significantly different from control smoker subjects with p values <0.001 for both groups. (b) TBARS concentration: There were no significant difference between heroin addicts with recent, distant, and intermediate TLAD (p > 0.05). However, addicts with recent and intermediate TLAD were significantly different from control smoker subjects with p values of 0.029 and 0.002, respectively. *Significant difference between the specified group to control smoker group at p < 0.05, **significant difference between specified group to control group at p < 0.005, and ***significant difference between specified group to control group at p < 0.001. TLAD: timing of latest administrated dose; TBARS: thiobarbituric acid reactive substances.

In comparison to control smoker group, we reported significantly increased carbonyl contents (Figure 3(a)) and TBARS concentration (Figure 3(b)) among subjects with recent and intermediate TLAD but not among subjects with distant TLDA. Furthermore, we have noticed that the highest protein carbonyl content was among addicts with recent dose (p = 0.000), after which it was gradually decreasing among addicts with intermediate and distant TLAD. On the other hand, TBARS concentration was significantly increased among addicts with recent TLAD and maintained increasing among those with intermediate TLAD and markedly decreased among addicts with distant TLAD.

Discussion

Initially, we have demonstrated that carbonyl contents and TBARS concentrations are significantly correlated with heroin addiction. This suggests an induced toxicity in platelet proteins and lipids in response to heroin abuse. Comparative analysis indicates that only chronic heroin addicts were characterized by having a statistically significant higher carbonyl contents and TBARS concentration in comparison to both the control groups. On the other hand, the induced carbonyl’s contents and TBARS concentration among chronic hashish addicts have been shown to be statistically insignificant when compared to both control groups.

The insignificant oxidative markers levels among hashish addicts might be a result of the contribution of multiple factors that represent various aspects of hashish cultivation, preparation as well as hashish pharmacokinetics through smoking route. Collectively, these factors may contribute into limited and unpredicted plasma levels of potent THC metabolites and subsequently a limited exposure of platelets to the oxidative stress mediated by hashish abuse. These factors include, firstly, the low peak of delta-9-THC and its metabolites in the plasma following smoking hashish; it has been shown that smoked hashish is associated with a lower peak of potent metabolites in comparison to intravenous injection.¹⁸ Secondly, a variable annual average potency of cannabinoids was defined as a consequence to variation in plant cultivation climate and plant’s parts that were used for drug extraction.¹⁹ Thirdly, the exposure of cannabis plants to heat and light during drying should result in the decomposition of variable amount of THC into much less potent cannabinoids.¹⁸ Fourthly, systemic THC bioavailability is subjected to “smoking dynamic” variables including number, duration, and spacing of puffs in addition to inhalation volume and holding time.¹⁸ Finally, plasma THC concentration reaches peak levels during smoking, after which it decreases rapidly and progressively post-smoking due to the rapid distribution into highly perfused lipophilic tissues reaching a 20% of the plasma peak levels within up to half an hour and a detection limit levels within up to 27 h.¹⁸

In regard of chronic heroin addicts, there were significantly greater carbonyl content and TBARS concentration in platelets isolates which indicates significant levels of oxidative stress in platelet’s proteins and lipids, respectively. Heroin administration induces oxidative stress through a reduction in the TAC, induction of nitric oxides (NO) and ROS generation, disruption of oxidation and antioxidation balance as well as induced oxidative damage to lipids and proteins in the brain and liver.^7,8 In a study where human platelets were used as a neuronal model, Authors have concluded that platelets from chronic heroin addicts are characterized by significantly reduced NO levels and nitric oxide synthase activity which is indirectly responsible for vascular dysfunction among drugs abusers.²⁰ Later, it has been postulated that ROS can reduce NO availability and therefore the quantification of NO level is a cellular marker of oxidative damage induced by ROS.²¹ Conclusively, these findings and ours are suggestive of heroin-mediated oxidative damage in platelet’s proteins and lipids.

Furthermore, we have reported that carbonyl contents and TBARS concentrations are strongly correlated with the duration of chronic heroin addiction of study subjects. These results are similar to what has been reported by an earlier study that heroin-mediated oxidative damage to tissues among chronic heroin addiction is correlated to the duration of heroin addiction in parallel with a gradual decrease in the plasma levels of antioxidants.²² Despite the fact that potent metabolites are rapidly distributed into target site upon administration, still a prolonged half life time (i.e. plasma bioavailability) is observed among chronic addicts as a result of enterohepatic cycling, which ultimately result into a slow and continuous release of these metabolites at low concentrations in plasma.²³ It has been proposed that this release of metabolites from their storage sites is responsible for the long-term observed toxicities in other tissues and subsequent continuous oxidative damage.^6,18

The lack of correlation between carbonyl contents and TBARS formation with the DD of heroin is probably a result of the variable and unpredictable purities of administrated heroin illicit “street heroin” that may range from low (2–3%) purity to high purities of more than 50%.²⁴ Therefore, even with the proposed dose of street heroin that our study subjects were abusing, still the exact dose and potency is actually unknown and unpredictable. Herein, it is worthy to mention that heroin quantity or quality (purity) that exceeds addict’s tolerance was proposed to be the most common and widely accepted cause of overdose fatality among heroin addicts.²⁵

In regard to the timing of the latest dose, only protein carbonyl contents among chronic heroin addicts were found to be significantly inversely correlated. The inconsistent trend in TBARS concentration subjects with recent, intermediate, and distant TLAD may explain the observed lack of correlation of with TLAD subgroups. Proteins are major structural and functional compartments of most biological systems including eukaryotic cells. In vivo, proteins are major target of oxidative damage mediated by intra or extracellularly formed ROS.²⁶ The extent of oxidative damage to specific cellular targets depends on several factors including the concentration of that specific target and the rate constant of oxidization reaction.^27,28 Therefore, the prominent carbonyl contents compared to TBARS formation might be a result of the dominant protein constituent in platelets cytoskeleton. It has been shown that platelets proteome encompasses an approximately over 4000 unique proteins.²⁹ Furthermore, platelets cytoskeleton is characterized by lipid/protein ratio of 0.321 and phospholipids/protein ratio of 0.253.³⁰

The inverse relationship of protein carbonyl contents in platelet’s lysate with the TLAD could be explained by the generation of new platelets that were only exposed to low levels of potent metabolites that were released from their storage sites resulting in lesser extent of oxidative damage to newly generated platelets. This is compared to platelets from addicts with recent dose which were exposed to multiple peaks of heroin metabolites prior to subject’s hospitalization for treatment with their latest peak being within up to few days from sample collection. This should result in a higher exposure of living circulatory platelets to potent metabolites upon drug administration as well as a higher levels of metabolites in the storage sites which ultimately may result into higher oxidative damage to circulatory platelets.

To further validate our latter correlation analysis, heroin addicts were subcategorized according to the TLAD with consideration of platelet’s life span and heroin pharmacokinetics. Our findings were confirmative to the inverse correlation analysis and were supportive to our proposed explanation of that observed correlation. As illustrated in Figure 3(a), we have demonstrated a significant gradual reduction in carbonyl contents between subjects with recent, intermediate, and distant TLAD. On the other hand, the inconsistent rhythm in TBARS concentration between study groups is mostly a result of the observed lack of correlation with TLAD. In a study where the influence of oxidative stress on stored platelets was investigated, an incoherent elevation in the levels of carbonyl contents and TBARS concentration were reported. Authors have reported an increased ROS generation during platelets storage and a subsequent disturbance of oxidative and antioxidative balance that ultimately result in an early elevation of carbonyl contents within stored platelets in comparison to TBARS concentration.³¹

Conclusion

In conclusion, chronic heroin addiction is associated with a significant oxidative damage in protein contents of platelets. The variable purity of illicit heroin, pharmacokinetics of intravenously administrated heroin, and the life span of circulatory platelets are determinants of potent metabolites bioavailability and subsequently the exposure and the extent of associated oxidative damage in protein and lipid contents of platelets. On the other hand, no significant oxidative damage to circulatory platelets in association with hashish chronic addiction which is also relevant to the pharmacokinetic of smoked hashish where the uptake of potent harmful metabolite by other tissue may reduce the extent of damage on circulatory platelets. Further analysis is required to validate our results through evaluating antioxidative competency of platelets from chronic addicts as well as investigating the clinical impacts of heroin-mediated oxidative damage on other structural and functional features of circulatory platelets.

Footnotes

Acknowledgements

The authors would like to thank the Drug Enforcement Administration of Public Security Department/Jordan and the Deanship of Research at Jordan University of Science and Technology for their valuable contribution and participation in this work.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

MA Bani-Ahmad

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