Antioxidants Supplementation in Acute Amitriptyline Abuse for Pain

The fundamental aim of this study is to establish the role of antioxidant supplementation in alleviating acute amitriptyline induced oxidative stress. The effect of supplementation was compared on treatment of acute amitriptyline intoxication cases for pain management, with alpha lipoic acid (ALA) alone or with vitamin C, with that of healthy individuals (group I), and those receiving only routine standard treatment (RST) as control (group II). A total of 132 human subjects divided into 5 groups were supplemented with either placebo, RST, RST with vitamin C, RST with ALA, or RST with vitamin C, and ALA. Results of this study revealed that the decrease in the level of oxidative stress and enzyme activity was observed among those supplemented with either alpha lipoic acid alone or along with vitamin C, with a slightly more decrease in the latter group. P value of < 0.001 was considered statistically significant. The percentage of benefit of treatment on supplementation with vitamin C and alpha lipoic acid showed a marked increase in group V cases after supplementation with both in combination. The results provided that the oxidative stress induced by acute amitriptyline poisoning is comparatively decreased by supplementation with antioxidants like alpha lipoic acid and vitamin C, than those only on routine standard treatment.


Introduction
approval from the ethical committee for research and experimental study and consent from the subject or care-taker, the supplementation was initiated orally after a routine supportive care was completed, and a stable general condition was established by the attending physician.

Inclusion Criteria
All patients acutely intoxicated with amitriptyline, assessed drowsy but responding to verbal commands as per the Edinburg scale of classification of the grade of coma, were selected. The subjects were divided into four groups with healthy volunteers forming the fifth group. The groups were classified as follows: • Group I: 30 healthy volunteers (15 males and 15 females) with mean age 32 years • Group II: 30 patients (18 males and 12 females) with mean age 34 years, who received only routine standard treatment (RST) • Group III: 21 patients (12 males and 9 females) with mean age 32 years, who received (RST) + vitamin C supplementation • Group IV: 27 patients (13 males and 14 females) with mean age 31 years, who received (RST) + alpha lipoic acid supplementation • Group V: 24 patients (14 males and 10 females) with mean age 34 years, who received (RST) + vitamin C and alpha lipoic acid supplementation

Exclusion Criteria
• Unconscious and not responding to verbal commands as per the Edinburg scale of classification of the grade of coma were excluded. • Age less than 18 years and more than 60 years were not included considering the confounding effect of hormonal and metabolic alterations. • Have taken other drugs along with amitriptyline were not included, to avoid crossinterference. • False-positive TLC (thin layer chromatography) and spectra (UV-Vis) negative were excluded.

Enzyme Levels Estimation
About 10 ml of venous blood from the ante-cubital vein of each subject and 50 ml of gastric aspirate were collected from all patients who are directly admitted to IMCU for thin later chromatography (TLC).

Lactate Dehydrogenase (LDH) Serum total LDH was analyzed by 'semi-auto-analyzer
Micro-lab 200' by modified IFCC method. LDH catalyzes the reduction of pyruvate with NADH to form NAD. The rate of oxidation of NADH to NAD is measured as a decrease in absorbance, which is proportional to the LDH activity in the sample. Initial absorbance A0 after 1 min and final absorbance reading after every 1, 2, and 3 min were recorded. The mean absorbance change per minute (ΔA/min) is calculated. Total LDH activity in microliter at 37 °C = ΔA/min × 8095.
Creatine Kinase (CK) Analysis of isoenzymes of CK was carried out by 'HYDRASYS system SEBIA, PN 1210.' The HYDRASYS SEBIA system is a semi-automated multiparameter electrophoresis system. Commercial kits 'HYDRAGEL 7 ISO-CK' are available for the analysis. On HYDRAGEL 7 ISO-CK and HYDRAGEL ISO-CK 15/30 gels, the BB fraction is the most anodic, the MM fraction is the most cathodic, and the MB is intermediary. All CK isoenzymes catalyze the same reaction that is utilized in their visualization.

Glutathione Peroxidase Activity
The activity of glutathione peroxidase was determined by the method of Rotruck et al. (1973) [20]. In brief, 0.4 ml of buffer, 0.1 ml of sodium azide, 0.2 ml of reduced glutathione, an aliquot of hemolysate, 0.1 ml of H 2 O 2 , and distilled water were taken to make a final volume of 2.0 ml. The tubes were incubated at 37 °C for 10 min. The reaction was stopped by adding 0.56 ml of 10% TCA. To determine the residual glutathione content, the supernatant was removed by centrifugation; 3.0 ml of disodium hydrogen phosphate and 1 ml of DTNB reagent were added and read at 412 nm. A blank was treated with only disodium hydrogen phosphate and 1.0 ml of DTNB reagent. The activity of glutathione was expressed as micrograms of GSH utilized/min/mgHb.  A blank and a standard were run simultaneously, and the initial absorbance (A1) and final absorbance (A2) were measured at 600 nm for both the blank and standard, respectively [21].
A2-A1 = DA of the sample/ blank/ standard were individually determined,

Statistical Analysis
Statistical evaluation was carried out using SPSS (14.0). Data obtained from the study groups were compared by the parametric student's t test. A correlation analysis between the variables was made by Pearson test and a P value of < 0.001 was considered statistically significant. The effect of vitamin C, ALA, and both combined were analyzed for each group and expressed as a percentage of change from baseline.

Results
The samples along with the controls were run on the thin layer chromatograph, and the following results were obtained based on the R f values. The presence of oxidative stress was confirmed by the elevation of the LDH 5 and CK-MM fractions of LDH and CK enzymes, respectively (Fig. 1). The mean levels of superoxide dismutase on admission compared to that on discharge in all the groups showed a significant reduction in the activity in groups  supplemented with either of the antioxidant or their combination. The reduction in levels was highest in the latter ( Table 1). The return to levels closer to baseline values was observed in group V supplemented with a combination of vitamin C and alpha lipoic acid, viz., 4.85µmoles/mg hemoglobin. Though supplementation with vitamin C alone was beneficial, the additive effect was more beneficial (Fig. 2). The mean levels of catalase enzyme levels on admission compared to that on discharge showed clearly in group V that the combination was much more effective ( Table 2). The combination provided a dramatic reduction in the enzyme level in group V to 2.31µmoles/ mg hemoglobin (Fig. 3).
Furthermore, the mean level of the glutathione on admission and discharge showed combined supplementation has maximal effect. The levels of glutathione peroxidase showed maximum reduction of 0.97 µg/mg hemoglobin, with a percentage difference of 17.6% (Table 3) (Fig. 4). The level of total serum antioxidant levels was maximal in group V followed by group III (Table 4) (Fig. 5).
The percentage change from baseline values of all the enzymes showed that maximum benefit of decrease in enzyme activity was found in group V (Fig. 6). The demographic distribution showed a slight male preponderance of 55.3% against 44.7% female population, with a mean age distribution of 33 for the males and 32 for the females.

Discussion
Amitriptyline abuse either with suicidal intention for unrelenting pain or want of better pain relief has been widely observed. Though routine treatment in an intensive medical care unit does provide symptomatic relief and recovery, the long-term sequel of this intoxication in the form of oxidative stress needs to be addressed. Though in vivo antioxidants and free radical scavenging mechanisms do provide considerable protection against free radical-induced tissue damage, their effects fall well short of the amount of protection needed for the tissues, culminating in measurable tissue damage. The external supplementation of antioxidants directly provides for free radical scavenging and potentiating of in vivo protection mechanisms. A highly water-soluble antioxidant like vitamin C along with a versatile lipid soluble antioxidant like alpha lipoic acid do adequately provide for    this. Vitamin C as a free radical scavenger has been widely studied [22,23]. Alpha lipoic acid on the other hand, in addition to having its own antioxidant effect, also replenishes other antioxidants like vitamins C and E. The proof of oxidative stress in amitriptyline intoxication was established by the rise of acute phase reactants such as lactate dehydrogenase and creatine kinase. We observed that muscle-and liver-specific LDH fraction, LDH 5 , and CK-MM were predominantly seen on gel electrophoresis. It was also observed that supplementation with vitamin C and alpha lipoic acid produced a dramatic decrease in the activity of enzymes like superoxide dismutase, catalase, and glutathione, indicating that free radical damage was well counteracted by these ex vivo antioxidants leaving only a little role for these in vivo antioxidants to act upon. Though oral supplementation was initiated immediately upon completion of the routine standard treatment, it is not clear as to whether an intravenous administration of antioxidants could be more beneficial.
A rise in serum total antioxidant levels also supported the fact that antioxidants potentiate each other. Despite these, a direct cause-effect relationship could not be established between the levels of amitriptyline that could trigger the oxidative stress and cause derangement in the levels of free radical scavenging enzymes. Though the supplemented antioxidants potentiated their in vivo counterparts, a dose-effect relationship could not be established, to cap the maximum permissible doses of these supplements. The effect of rebound in the oxidative stress on discontinuation of the antioxidant supplementation also needs to be studied. Yet another vaguely understood part is the role of chronic intoxication with deranged drug clearance leading to drug build-up in actively metabolizing tissues such as the muscles and liver.

Conclusion
Antioxidant supplementation is an inevitable need in drug intoxication-induced oxidative stress. Vitamin C and alpha lipoic acid have profound effect in protecting against free radical-induced tissue damage, especially in acute intoxication with pain medications such as amitriptyline. A long-term supplementation is recommended to protect against free radical damage sequel. Yet again, drug-induced systemic derangements should be cautioned in persons on supplementation like any other medication.   This study provides a quantitative recommendation of supplementation with vitamin C and alpha lipoic acid for an accelerated recovery in subjects with pain medication abuse. Oral supplementation has considerably reduced oxidative stress-induced systemic damage and could be provided as an adjunct. A delay in supplementation could cause lack of harnessing the maximum potential of these antioxidants in providing superior protection to these intoxicated subjects.
Author Contribution S. Hameed Kadar Ali-conceptualized the work. K. Wasim Ali Raja-performed the work. N. Irfan-wrote the manuscript. Mohammad Habeeb-statistical analysis. Y. Ismail-guidance and analysis.

Data and Materials Availability
The authors confirm that the data analyzed and generated from this research findings are provided within this article.

Competing Interests
The authors declare no competing interests.