Journals | Policy | Permission

Journal of Endocrinology & Metabolism

Original Article

Volume 14, Number 4, August 2024, pages 174-183

Intestinal Absorption and Transformation of the Increased Dose of Paracetamol in Streptozotocin-Induced Diabetic Rats

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, GSH) is one of the most important antioxidant low-molecular-weight tripeptide, which can be found in all mammalian tissues (0.5 - 10 mmol/L). The majority of GSH is present in the liver (10 mmol/L) [1-3], and it also plays an important role in the intestinal function [4, 5]. GSH serves many essential functions especially in the detoxification of exogenous compounds by conjugation as a nucleophile molecule to form GSH adducts [2, 6]. Some non-steroidal anti-inflammatory drugs decrease the level of GSH by phase II conjugation. Noteworthy examples include the metabolism of paracetamol and diclofenac [6-8].

The significance of the intestinal tract is attributed to its location because this is the site where drugs can be absorbed, and also the formed metabolites can be excreted into the circulation or back to the intestinal lumen. Therefore, the small intestine is an important site of drug absorption and metabolism, where both phase I and phase II enzymes and transporters are expressed [9, 10].

The tested compound, paracetamol (acetaminophen, 4-hydroxy-acetanilide, N-acetyl-para-aminophenol) is a weak acid with moderate water solubility and a low molecular mass. At a physiological pH, it is unionized, and therefore it is absorbed by passive diffusion theoretically along the entire gastrointestinal tract but mostly in the proximal part of the small intestine [11]. At higher dose, the surface of the membranes for passive diffusion and the available transporters can be saturated, and the pharmacokinetics of the drug can be altered [12].

Paracetamol, a frequently used analgesic and antipyretic, is mostly metabolized in the liver, but the small intestine and the kidneys are involved too. It is transformed into inactive paracetamol-β-D-glucuronide (PG) and paracetamol sulphate (PS) [13, 14]. A minor fraction is mostly oxidized by CYP3A4, CYP2E1, CYP1A2, CYP2A6 and cyclooxygenase into a highly reactive metabolites, including N-acetyl-p-benzoquinone imine (NAPQI) and 3-hydroxy-paracetamol [15-17]. NAPQI is formed through two intermediate products: the N-acetyl-p-benzoquinone imine radical and the 4-aminophenoxyl radical [18, 19]. Then NAPQI conjugates with GSH to form an inactive intermediate, which is subsequently transformed into paracetamol cysteine (PC) and with deacetylation into paracetamol mercapturate (PM) [20]. Also, one of the several important metabolites is 4-aminophenol, which plays an important role in the mechanism of action [21, 22]. It can form 4-aminophenol-glucuronide and 4-aminophenol sulfate, and it is able to form a conjugate with GSH to produce a nephrotoxic product [23, 24]. Generally, paracetamol is administered in doses of 500 - 1,000 mg. However, because of its high number of its generic forms, and numerous trade names, the duplication of the single dose might be relatively common. This can lead to elevated, but not toxic drug levels, which may provide alterations in its pharmacokinetics [25].

Diabetes mellitus is an endocrine metabolic disorder that affects a significant proportion of the population of the world and shows a growing tendency [26]. In this experiment, paracetamol metabolism was investigated under pathological condition (diabetes). Metabolites that were observable and excreted by the small intestinal enterocytes were PG, PS, PC, and PM [27].

Pathological conditions such as prolonged hyperglycemia or untreated diabetes can influence the metabolism of xenobiotics [28-30], and it is able to cause an elevation of reactive oxygen species (ROSs) and free radicals, therefore leading to oxidative stress [31, 32]. Oxidative stress plays a major role in the development of both types of diabetes mellitus and the related micro- and macrovascular complications such as retinopathy, nephropathy, neuropathy and endothelial disfunctions [33-35]. These ROSs, free radicals and endogenous compounds are eliminated by the conjugation with GSH. This process can occur spontaneously, be catalyzed by the enzyme GSH transferase [36, 37], or can be reduced with GSH either. Besides the glucose metabolic disorders, other factors that can increase the amount of the formed reactive intermediates of paracetamol and elevated doses can contribute to the formation of the oxidative stress [38].

The present investigation was designed to study the effect of hyperglycemia and higher concentration of paracetamol on its metabolism and the level change of GSH.

Paracetamol, sodium citrate dehydrate, citric acid, Tris hydrochloride, streptozotocin (STZ), reduced GSH, DL-dithiothreitol (DTT), triethylamine, formic acid and urethan were purchased from Sigma Aldrich (Budapest, Hungary); potassium chloride (KCl), calcium chloride (CaCl₂·2H₂O), sodium chloride (NaCl), glucose, sucrose, sodium hydroxide (NaOH), perchloric acid (HClO₄) and disodium hydrogen phosphate (Na₂HPO₄·2H₂O) were purchased from Molar Chemicals Kft. (Halasztelek, Hungary); magnesium sulfate (MgSO₄); theophylline, acetonitrile and methanol were purchased from VWR International (Radnor, PA, USA); potassium dihydrogen phosphate (KH₂PO₄), oxidized glutathione (GSSG) and mannitol were purchased from Reanal Labor (Budapest, Hungary). Hepes was purchased from Alfa Aesar (Haverhill, Massachusetts, USA). Phosphoric acid was purchased from Thomasker (Budapest, Hungary). The solvents were on high performance liquid chromatography (HPLC) grade. The used standard isotonic perfusion medium had the following decomposition: 96.4 mM NaCl, 7.0 mM KCl, 3 mM CaCl₂·2H₂O, 1 mM MgSO₄, 0.9 mM phosphate buffer (pH = 7.4), 29.5 mM Tris buffer (pH = 7.4), 14.0 mM glucose, and 14 mM mannitol. Distilled water was purified using a Purelab Option Q7 Water System (ELGA LabWater, Woodridge, IL, USA) at the Department of Pharmaceutical Chemistry, University of Pecs. A Sanxin MP512 Precision pH meter and a Mettler Toledo LE410 electrode were used to adjust the pH. Blood glucose level was controlled using an AccuChek blood glucose meter (Roche, Basel, Switzerland). Homogenizer (witeg Labortechnik GmbH, Wertheim, Germany) was used to prepare the homogenates.

Four treatment groups were established: a control group, an STZ-pretreated group, a paracetamol-perfused group, and a group that was both STZ-pretreated and paracetamol-perfused.

Male Wistar-Hanover rats (weighing 240 - 300 g) were used. The rats were anesthetized with urethane (1.2 g/kg, intraperitoneal (IP) [39]. The abdomen was opened by a mid-line incision, and approximately 10 cm of the jejunal loop was isolated and cannulated. The lumen of the jejunal loop was flushed with warmed isotonic solution (30 - 40 mL) to remove digesta and food residues, and then blown empty with 4 - 5 mL air. Perfusion through the lumen of the jejunal loop with 500 µM solution of paracetamol was performed at the rate of 12 mL/min in a recirculation mode for 90 min (Fig. 1). The initial perfusion volume was 16.5 mL. During the 90-min experiment, 250 µL samples were collected 14 times (0, 0.5, 1, 3, 5, 10, 15, 22, 29, 36, 45, 60, 75, 90 min). The temperature of perfusion was maintained at 37 °C.

Click for large image

Figure 1. Time course of the disappearance of paracetamol in rat’s small intestinal perfusate during perfusion of the jejunal loop with isotonic medium containing 500 µM paracetamol. Each value represents the mean of five independent experiments of five rats. No statistical significance was found within the tested parameters. STZ: streptozotocin.

Experimental diabetes was induced by intravenous administration of STZ at a dose of 65 mg/kg (dissolved in 0.1 M citrate buffer, pH 4.0) 1 week before the experiment [40]. Control and STZ-treated rats were treated with a vehicle. Blood glucose levels were measured before the STZ treatment and 1 week prior to the start of the experiments. Animals were considered diabetic from a value of 13.9 mmol/L. Blood sugar levels were measured immediately before starting experiments. The experimental rats were provided with standard rat chow (ToxiCoop, Budapest, Hungary). Food was withdrawn 12 h before glucose levels were measured. Small intestine perfusate and bile samples were obtained from the same rats in four different groups. After the experiments, the organs (liver, small intestine, kidneys) were removed, and the animals were over-anesthetized by urethane. The samples were stored in refrigerator (-70 °C) until the analysis was performed. Then they were measured in 2 weeks. For the measurement of oxidized and reduced GSH, the same experiment was performed with Krebs-Tris buffer perfusion without paracetamol in four different groups (control and STZ treated). Each group had five rats.

The protocol to determine paracetamol and its major metabolites in the intestinal perfusate was developed and validated in our previous study [27]. Before the reverse phased high performance liquid chromatography (RP-HPLC), UV/Vis analysis was performed. The collected perfusate samples were allowed to reach the ambient temperature and then vortex-mixed for 5 s. Then 75 µL of each sample was mixed with 25 µL 0.00159 M theophylline (dissolved in Krebs-Tris buffer). The samples were vortex-mixed for 5 s, and then 10 µL of 3.88 M HClO₄ solution was added, vortexed for 5 s, then centrifuged at 10,000 × g for 10 min to sediment the precipitated proteins. After centrifugation, 50 µL was transferred and mixed with 5 µL of 3.96 M NaOH. After vortexing and centrifugation processes, 20 µL sample was injected.

Analysis of the perfusates was performed using Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA, USA), which was equipped with a quaternary HPLC pump (G1311A), degasser (G1322A), an autosampler (G1313A), a thermostated column compartment (G1316A), and a UV-Vis detector (G1314A). Data were recorded and evaluated using Agilent ChemStation software (Rev.B.03.02-SR2). Reversed-phase PerfectSIL 120 ODS C18 (4.6 × 100 mm, 5 µm particle size) (MZ-Analysentechnik GmbH, Mainz, Germany) were used for analytical separation.

The mobile phase consisted of water, acetonitrile, triethylamine in a ratio of 92.95:7.00:0.05 v/v% with the pH of 2.25 (adjusted with formic acid). The flow rate of the eluent was 1 mL/min. The measurements were done at ambient temperature, and the wavelength of the detection was 245 nm.

Organ samples were placed into homogenization buffer (250 mM sucrose, 1 mM DL-dithiothreitol, 10 mM Hepes-Tris buffer, pH = 8). The ratio of buffer to the tissue was 10:1 (mL/g) (v/m). Organs were disrupted with homogenizer. Into 1 mL homogenate, 100 µL 0.388 M perchloric acid was added, and the mixture was vortex-mixed for 5 s, and then centrifuged at 14,000 × g for 5 min to sediment the precipitated proteins. After centrifugation 800 µL was transferred, and it was mixed with 80 µL 0.396 M sodium-hydroxide. After vortexing and centrifugation process, a 10-µL sample was injected.

Analysis of the homogenates was performed using Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA, USA), which was equipped with a quaternary HPLC pump (G1311A), degasser (G1322A), an autosampler (G1313A), a thermostated column compartment (G1316A), and a UV-Vis detector (G1314A). Data were recorded and evaluated using Agilent ChemStation software (Rev.B.03.02-SR2). Reversed-phase Kinetex Hypersil C18 (4.6 × 150 mm, 5 µm particle size) was used for analytical separation. The mobile phase consisted of 0.1% phosphoric acid with pH of 2, adjusted with NaOH and methanol. The ratio of the buffer and the methanol was 9:1. The flow rate of the eluent was 1 mL/min. The volume of the samples was 10 µL, and the measurements were done at 25 °C. Detection was performed at a wavelength of 215 nm.

Data were presented as the means ± standard error (SE) of five independent experiments. Data were analyzed by one-way analysis of variance (ANOVA). The difference among groups was determined by the Student’s t-test.

The study was designed and conducted according to the European Legislation (Directive 2010/63/E.U.) and Hungarian Government Regulation (40/2013., II.14.) on the protection of animals that are used for scientific purposes. The project was approved by the Animal Welfare Committee of the University of Pecs and by the Government Office of Baranya County (license No. BAI35/51-61/2016 (license supplement No. BAI35/90-5/2019) and BA02/2000-53/2023).

The onset of hyperglycemia was monitored before the experiments, and their values were 6.56 ± 1.05 mmol/L in the control, and 6.84 ± 1.67 mmol/L at the paracetamol-perfused group. After STZ pretreatment, in the buffer perfused group, the average of the blood sugar levels was 20.16 ± 2.83 mmol/L, while in the paracetamol-perfused group, it was 21.56 ± 2.97 mmol/L.

Figure 1 shows the slow, mainly passive absorption of the paracetamol during the 90-min experiment. The tendency of the rate of the absorption is reduced at the group of the animals treated with STZ. During the 90-min period, the scale of difference is increasing with final values of 5,245.94 ± 1,022.29 nmol of paracetamol at the non-pretreated group, while the STZ pretreatment modify the remaining paracetamol quantity in the perfusion buffer to 6,406.17 ± 954.21 nmol.

Figures 2 and 3 show that the appearance of PG (42.12 ± 7.31 nmol) and PS (6.68 ± 1.07 nmol) metabolites. These derivatives change parallelly toward the increasing direction under the influence of the STZ (PG (88.07 ± 17.33 nmol) and PS (8.45 ± 1.95 nmol)). The alteration at the PG was found to be significant.

Click for large image

Figure 2. Alteration in the total excreted amount of paracetamol β-D-glucuronide (PG) into the lumen of the small intestine of rat during perfusion of the jejunal loop with isotonic medium containing 500 µM paracetamol. Each value represents the mean of five independent experiments ± standard error. **P < 0.01. STZ: streptozotocin.

Click for large image

Figure 3. Alteration in the total excreted amount of paracetamol sulfate (PS) into the lumen of the small intestine of rat during perfusion of the jejunal loop with isotonic medium containing 500 µM paracetamol. Each value represents the mean of five independent experiments ± standard error. No statistical significance was found within the tested parameters. STZ: streptozotocin.

Figures 4 and 5 show that the significant elevation in the PC (4.57 ± 0.68 nmol) and PM (0.74 ± 0.69 nmol) levels can be seen after STZ treatment (PC (8.91 ± 2.19 nmol), and PM (4.07 ± 1.27 nmol)) in the perfusion medium, and both of the alterations were found to be significant.

Click for large image

Figure 4. Alteration in the total excreted amount of paracetamol cysteine (PC) into the lumen of the small intestine of rat during perfusion of the jejunal loop with isotonic medium containing 500 µM paracetamol. Each value represents the mean of five independent experiments ± standard error. **P < 0.01. STZ: streptozotocin.

Click for large image

Figure 5. Alteration in the total excreted amount of paracetamol mercapturate (PM) into the lumen of the small intestine of rat during perfusion of the jejunal loop with isotonic medium containing 500 µM paracetamol. Each value represents the mean of five independent experiments ± standard error. **P < 0.01. STZ: streptozotocin.

The detection of the GSH and the GSSG was performed by a simple reversed phase HPLC method after the appropriate sample preparation. The chromatogram of the applied HPLC method can be seen in Figure 6.

Click for large image

Figure 6. HPLC chromatogram of the glutathione (GSH) and oxidized glutathione (GSSG) standards prepared with the blank perfusate. Peak 1 represents the reduced GSH (tR = 2.454 min); peak 2 represents the GSSG (tR = 2.776 min).

By the effect of the STZ-induced hyperglycemia and paracetamol perfusion, the control GSH concentration in the small intestinal tissue homogenate (78.84 ± 7.24 µM) is going to change toward a decreasing direction at all treatments (in case of STZ pretreatment (63.10 ± 9.20 µM), at 500 µM paracetamol perfusion (77.21 ± 9.27 µM) and with STZ pretreatment and 500 µM paracetamol perfusion (72.60 ± 14.53 µM) (Fig. 7).

Click for large image

Figure 7. Concentration of reduced glutathione (GSH) (the ratio of buffer to the wet tissue was 10:1 (mL/g)) in the small intestine, liver and kidney in control animals. Each value represents the mean of five independent experiments of five rats ± standard error. STZ: streptozotocin.

The formation of the GSSG cannot be detected in the small intestinal homogenate with our method of those animals, which were not treated previously with STZ. But the STZ pretreatment contributed to the appearance of the oxidative derivative (0.1539 ± 0.0592 µM), and its level has increased after the perfusion of 500 µM paracetamol (0.6686 ± 0.1183 µM) (Fig. 8).

Click for large image

Figure 8. Concentration of oxidized glutathione (GSSG, the ratio of buffer to the tissue was 10:1 (mL/g)) in the small intestine without and with paracetamol perfusion in case of diabetic rats. Each value represents the mean of five independent experiments ± standard error. **P < 0.01. STZ: streptozotocin.

The determination of the metabolites from the small intestinal perfusate was performed with an isocratic, validated RP-HPLC UV-Vis method, which was developed for the quantification of paracetamol and its major metabolites (PG, PS, PC and PM) [27]. The concentration of the paracetamol was aimed to model an elevated dose between the regular and the maximal doses on one occasion. Some of the experimental animals were treated with STZ to provoke a diabetic condition. We found that the experimental diabetes caused changes in intestinal absorption and metabolism, resulting in an increase of all of the metabolic processes [27].

In Figure 1, the predominantly slow and passive absorption of the paracetamol can be observed, and the absorption tendency is reduced in the STZ-pretreated animals. Although the STZ-induced diabetes can increase the permeability of the membrane of the gastrointestinal mucosa, at higher concentrations of the administered paracetamol molecule, the saturation of the transporters, and the slower change of the concentration gradient can be emphasized. The number of the transporters can limit the rate of the absorption, although their function can be important as well. The STZ pretreatment during the modelling of a long-term diabetes increases the membrane permeability, but inhibits the function and the expression of P-gp transporter in the small intestine [41, 42]. In short-term diabetes (9 days), the decrease of the P-gp expression is reported [42-44]. Besides affecting the small intestine, the experimental diabetes reduces the activity of efflux transporters in the liver and the kidney, such as MDR1B (multidrug resistance transporter, P-gp), multidrug resistance-associated protein 2, and breast cancer resistance protein [29].

In the small intestine, the excreted quantity of the PG increased significantly. Although the PS showed an increasing tendency, the change was not significant after STZ pretreatment. These alterations correspond with the changes that were found at the tests for activity of the UDP-glucuronyltransferase and the sulfotransferase enzymes in the control and STZ-pretreated rats [45], and these results were directly proportional with the applied concentrations [27].

The changes of the PC and PM levels can be explained with the reactive metabolites formed after metabolic transformation by spontaneous and enzyme-catalyzed pathways [36]. The oxidation itself can be facilitated by the CYP3A4 and prostaglandin H-synthase in the small intestine [46]. The numbers are similar to those obtained with treatments at lower substrate concentrations [27]. This phenomenon also can be explained by the limited capacity of the GSH conjugate degradation process or the interorgan shift of the cysteine and mercapturate derivatives [47]. It can be noted that during the elimination process of paracetamol metabolites, especially paracetamol GSH derivatives, these can enter the bloodstream from the kidney and liver. Moreover, the applied method is available not only for the investigation of the small intestinal absorption but also for detection of the excreted intestinal metabolites. Because the intestinal vascularization is intact, the metabolic roles of the livers and kidneys can also be important [48, 49]. Besides the covalent connection to the reactive metabolites, the formation of the reactive intermediates (phenoxy- or N-acetyl benzosemiquinone radicals) theoretically allows for the direct oxidation of the GSH to GSSG, although this transformation can also occur due to the elevated oxidative stress provoked by the experimental diabetes.

In the small intestine, the most metabolically active layer is composed of the enterocytes of the mucosa, which contains the GSH in the highest concentration [4]. When the GSH content of the small intestine is compared with that in the hepatic and renal tissues, it tends to be the lowest. However, diurnal variations in GSH levels, which can be measured in the liver tissue homogenate, cannot be detected. It partly can be explained by the hepatic resupplementation of the GSH [50, 51].

Due to the STZ-induced hyperglycemia, the GSH concentrations are reduced at all treatments (or show a decreasing tendency). As shown in Figure 7, the reduction of the GSH level can be observed. The obtained results, at least partly, were comparable to those obtained with other methods [27]. Surprisingly, at the recently perfused paracetamol concentration (500 µM), an increase can be observed. This phenomenon can be explained with the observation that the presence of the paracetamol can inhibit the activity of the glutathione-S-transferase enzyme at higher concentrations in the liver homogenate of the mice and in the gills of the eel fish [52, 53]. Moreover, the oxidative stress might induce a protective compensatory increase in the synthesis of the endogenous antioxidant GSH [54]. The formation of the GSSG (Fig. 8) is only observable in the STZ-pretreated animals and is even increased by the effect of the paracetamol.

After the STZ administration, there could be an increased oxidative stress in the metabolizing cells. These results suggest that the quantity of the reactive species formed during the metabolism can transform to O-, or N-centered derivatives or reactive fragment molecules, which can directly interact with GSH [31]. The obtained results can also be the result of the activity change of the enzymes involved in the GSH biosynthesis, e.g., GSH synthetase and γ-glutamylcysteine synthetase [55]. The influence of the GSH resupplementation can be further investigated by experiments on intact tissues [56].

Following the administration of higher (500 µM) dose of paracetamol, all the tested metabolites (PG, PS, PC and PM) appear in elevated quantities after STZ pretreatment. The increase in PC and PM indicates the formation of the oxidative metabolite. With the introduced HPLC method, the GSH and GSSG levels were measured. There are no significant decreases in GSH levels by the effect of the paracetamol and the STZ; but GSSG only appears after STZ pretreatment, in the small intestine. At higher doses of the paracetamol administration, the physico-chemical character of the gastrointestinal mucosa might change, and educed tendency of the absorption can be observed in experimental diabetes. The main metabolic pathways (glucuronidation and sulfation) are intact, although the GSH levels show a downhill tendency both after paracetamol perfusion and STZ pretreatment. After STZ pretreatment the elevation of the GSSG is an indicator of oxidative stress. The exact role of the liver and kidney in the formation of the PC and PM metabolites is to be clarified. In diabetes, the absorption of paracetamol and the protective GSH level can be lowered, and the level of the oxidative stress is higher.

Acknowledgments

None to declare.

Financial Disclosure

This study was supported by the Faculty of Medicine/Pharmacy Research Fund (GYTK KA-2023-01), University of Pecs.

Conflict of Interest

Authors declare no conflict of interest.

Informed Consent

The study is not a human research, no informed consent was obtained.

Author Contributions

A. Almasi: conceptualization, methodology, visualization, and writing. S. Kovacs: formal analysis. P. Meszaros: investigation, data curation, writing, and statistics. All authors have read and agreed to the published version of the manuscript.

Data Availability

Any inquiries regarding supporting data availability of this study should be directed to the corresponding author.

1. Wu G, Fang YZ, Yang S, Lupton JR, Turner ND. Glutathione metabolism and its implications for health. J Nutr. 2004;134(3):489-492.
   doi pubmed
2. Forman HJ, Zhang H, Rinna A. Glutathione: overview of its protective roles, measurement, and biosynthesis. Mol Aspects Med. 2009;30(1-2):1-12.
   doi pubmed pmc
3. Lu SC. Regulation of glutathione synthesis. Mol Aspects Med. 2009;30(1-2):42-59.
   doi pubmed pmc
4. Samiec PS, Dahm LJ, Jones DP. Glutathione S-transferase in mucus of rat small intestine. Toxicol Sci. 2000;54(1):52-59.
   doi pubmed
5. Jefferies H, Bot J, Coster J, Khalil A, Hall JC, McCauley RD. The role of glutathione in intestinal dysfunction. J Invest Surg. 2003;16(6):315-323.
   doi pubmed
6. Lauterburg BH. Analgesics and glutathione. Am J Ther. 2002;9(3):225-233.
   doi pubmed
7. Tang W, Stearns RA, Bandiera SM, Zhang Y, Raab C, Braun MP, Dean DC, et al. Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: identification of glutathione conjugated metabolites. Drug Metab Dispos. 1999;27(3):365-372.
   pubmed
8. Teffera Y, Waldon DJ, Colletti AE, Albrecht BK, Zhao Z. Identification of a novel glutathione conjugate of diclofenac by LTQ-Orbitrap. Drug Metab Lett. 2008;2(1):35-40.
   doi pubmed
9. Suzuki H, Sugiyama Y. Role of metabolic enzymes and efflux transporters in the absorption of drugs from the small intestine. Eur J Pharm Sci. 2000;12(1):3-12.
   doi pubmed
10. Kaminsky LS, Zhang QY. The small intestine as a xenobiotic-metabolizing organ. Drug Metab Dispos. 2003;31(12):1520-1525.
    doi pubmed
11. Raffa RB, Pergolizzi JV, Jr., Taylor R, Jr., Decker JF, Patrick JT. Acetaminophen (paracetamol) oral absorption and clinical influences. Pain Pract. 2014;14(7):668-677.
    doi pubmed
12. Watkins JB, Klaassen CD. Absorption, enterohepatic circulation, and fecal excretion of toxicants. Comprehensive Toxicology (Second Edition), Elsevier. 2010; p. 77-91.
13. Mazaleuskaya LL, Sangkuhl K, Thorn CF, FitzGerald GA, Altman RB, Klein TE. PharmGKB summary: pathways of acetaminophen metabolism at the therapeutic versus toxic doses. Pharmacogenet Genomics. 2015;25(8):416-426.
    doi pubmed pmc
14. Hayward KL, Powell EE, Irvine KM, Martin JH. Can paracetamol (acetaminophen) be administered to patients with liver impairment? Br J Clin Pharmacol. 2016;81(2):210-222.
    doi pubmed pmc
15. Chen W, Koenigs LL, Thompson SJ, Peter RM, Rettie AE, Trager WF, Nelson SD. Oxidation of acetaminophen to its toxic quinone imine and nontoxic catechol metabolites by baculovirus-expressed and purified human cytochromes P450 2E1 and 2A6. Chem Res Toxicol. 1998;11(4):295-301.
    doi pubmed
16. Graham GG, Davies MJ, Day RO, Mohamudally A, Scott KF. The modern pharmacology of paracetamol: therapeutic actions, mechanism of action, metabolism, toxicity and recent pharmacological findings. Inflammopharmacology. 2013;21(3):201-232.
    doi pubmed
17. McGill MR, Jaeschke H. Metabolism and disposition of acetaminophen: recent advances in relation to hepatotoxicity and diagnosis. Pharm Res. 2013;30(9):2174-2187.
    doi pubmed pmc
18. Powis G, Svingen BA, Dahlin DC, Nelson SD. Enzymatic and non-enzymatic reduction of N-acetyl-p-benzoquinone imine and some properties of the N-acetyl-p-benzosemiquinone imine radical. Biochem Pharmacol. 1984;33(15):2367-2370.
    doi pubmed
19. Fischer V, West PR, Nelson SD, Harvison PJ, Mason RP. Formation of 4-aminophenoxyl free radical from the acetaminophen metabolite N-acetyl-p-benzoquinone imine. J Biol Chem. 1985;260(21):11446-11450.
    pubmed
20. Bertolini A, Ferrari A, Ottani A, Guerzoni S, Tacchi R, Leone S. Paracetamol: new vistas of an old drug. CNS Drug Rev. 2006;12(3-4):250-275.
    doi pubmed pmc
21. Sharma CV, Long JH, Shah S, Rahman J, Perrett D, Ayoub SS, Mehta V. First evidence of the conversion of paracetamol to AM404 in human cerebrospinal fluid. J Pain Res. 2017;10:2703-2709.
    doi pubmed pmc
22. Ayoub SS. Paracetamol (acetaminophen): A familiar drug with an unexplained mechanism of action. Temperature (Austin). 2021;8(4):351-371.
    doi pubmed pmc
23. Eyanagi R, Hisanari Y, Shigematsu H. Studies of paracetamol/phenacetin toxicity: isolation and characterization of p-aminophenol-glutathione conjugate. Xenobiotica. 1991;21(6):793-803.
    doi pubmed
24. Fowler LM, Moore RB, Foster JR, Lock EA. Nephrotoxicity of 4-aminophenol glutathione conjugate. Hum Exp Toxicol. 1991;10(6):451-459.
    doi pubmed
25. Schwab M, Oetzel C, Morike K, Jagle C, Gleiter CH, Eichelbaum M. Using trade names: a risk factor for accidental drug overdose. Arch Intern Med. 2002;162(9):1065-1066.
    doi pubmed
26. Saeedi P, Petersohn I, Salpea P, Malanda B, Karuranga S, Unwin N, Colagiuri S, et al. Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas, 9(th) edition. Diabetes Res Clin Pract. 2019;157:107843.
    doi pubmed
27. Meszaros P, Kovacs S, Kulcsar G, Paskuj M, Almasi A. Investigation of intestinal absorption and excretion of paracetamol in streptozotocin-induced hyperglycemia. Int J Mol Sci. 2022;23(19):11913.
    doi pubmed pmc
28. Fischer E, Almasi A, Bojcsev S, Fischer T, Kovacs NP, Perjesi P. Effect of experimental diabetes and insulin replacement on intestinal metabolism and excretion of 4-nitrophenol in rats. Can J Physiol Pharmacol. 2015;93(6):459-464.
    doi pubmed
29. Kovacs NP, Almasi A, Garai K, Kuzma M, Vancea S, Fischer E, Perjesi P. Investigation of intestinal elimination and biliary excretion of ibuprofen in hyperglycemic rats. Can J Physiol Pharmacol. 2019;97(11):1080-1089.
    doi pubmed
30. Mohammed HO, Almasi A, Molnar S, Perjesi P. The intestinal and biliary metabolites of ibuprofen in the rat with experimental hyperglycemia. Molecules. 2022;27(13):4000.
    doi pubmed pmc
31. Maritim AC, Sanders RA, Watkins JB, 3rd. Diabetes, oxidative stress, and antioxidants: a review. J Biochem Mol Toxicol. 2003;17(1):24-38.
    doi pubmed
32. Asmat U, Abad K, Ismail K. Diabetes mellitus and oxidative stress-A concise review. Saudi Pharm J. 2016;24(5):547-553.
    doi pubmed pmc
33. Callaghan MJ, Ceradini DJ, Gurtner GC. Hyperglycemia-induced reactive oxygen species and impaired endothelial progenitor cell function. Antioxid Redox Signal. 2005;7(11-12):1476-1482.
    doi pubmed
34. Ceriello A. Oxidative stress and diabetes-associated complications. Endocr Pract. 2006;12(Suppl 1):60-62.
    doi pubmed
35. Lazo de la Vega Monroy ML, Fernndez-Mej C. Oxidative stress in diabetes mellitus and the role of vitamins with antioxidant actions. Environment Sci. 2013.
    doi
36. Coles B, Wilson I, Wardman P, Hinson JA, Nelson SD, Ketterer B. The spontaneous and enzymatic reaction of N-acetyl-p-benzoquinonimine with glutathione: a stopped-flow kinetic study. Arch Biochem Biophys. 1988;264(1):253-260.
    doi pubmed
37. Tesauro M, Nistico S, Noce A, Tarantino A, Marrone G, Costa A, Rovella V, et al. The possible role of glutathione-S-transferase activity in diabetic nephropathy. Int J Immunopathol Pharmacol. 2015;28(1):129-133.
    doi pubmed
38. Mason RP, Fischer V. Free radicals of acetaminophen: their subsequent reactions and toxicological significance. Fed Proc. 1986;45(10):2493-2499.
    pubmed
39. Field KJ, White WJ, Lang CM. Anaesthetic effects of chloral hydrate, pentobarbitone and urethane in adult male rats. Lab Anim. 1993;27(3):258-269.
    doi pubmed
40. Furman BL. Streptozotocin-induced diabetic models in mice and rats. Curr Protoc. 2021;1(4):e78.
    doi pubmed
41. Zhang LL, Lu L, Jin S, Jing XY, Yao D, Hu N, Liu L, et al. Tissue-specific alterations in expression and function of P-glycoprotein in streptozotocin-induced diabetic rats. Acta Pharmacol Sin. 2011;32(7):956-966.
    doi pubmed pmc
42. Nawa A, Fujita Hamabe W, Tokuyama S. Inducible nitric oxide synthase-mediated decrease of intestinal P-glycoprotein expression under streptozotocin-induced diabetic conditions. Life Sci. 2010;86(11-12):402-409.
    doi pubmed
43. Bojcsev S, Rafiei A, Fischer E. Changes in the biliary excretion of exogenous organic anions by streptozotocin-induced diabetes. Acta Physiol Hung. 1996;84(3):263-264.
    pubmed
44. Anger GJ, Magomedova L, Piquette-Miller M. Impact of acute streptozotocin-induced diabetes on ABC transporter expression in rats. Chem Biodivers. 2009;6(11):1943-1959.
    doi pubmed
45. Almasi A, Bojcsev S, Fischer T, Simon H, Perjesi P, Fischer E. Metabolic enzyme activities and drug excretion in the small intestine and in the liver in the rat. Acta Physiol Hung. 2013;100(4):478-488.
    doi pubmed
46. Manyike PT, Kharasch ED, Kalhorn TF, Slattery JT. Contribution of CYP2E1 and CYP3A to acetaminophen reactive metabolite formation. Clin Pharmacol Ther. 2000;67(3):275-282.
    doi pubmed
47. Bessems JG, Vermeulen NP. Paracetamol (acetaminophen)-induced toxicity: molecular and biochemical mechanisms, analogues and protective approaches. Crit Rev Toxicol. 2001;31(1):55-138.
    doi pubmed
48. Inoue M, Okajima K, Nagase S, Morino Y. Inter-organ metabolism and transport of a cysteine-S-conjugate of xenobiotics in normal and mutant analbuminemic rats. Biochem Pharmacol. 1987;36(13):2145-2150.
    doi pubmed
49. Hinchman CA, Ballatori N. Glutathione conjugation and conversion to mercapturic acids can occur as an intrahepatic process. J Toxicol Environ Health. 1994;41(4):387-409.
    doi pubmed
50. Kelly FJ. Glutathione content of the small intestine: regulation and function. Br J Nutr. 1993;69(2):589-596.
    doi pubmed
51. Hazelton GA, Lang CA. Glutathione contents of tissues in the aging mouse. Biochem J. 1980;188(1):25-30.
    doi pubmed pmc
52. Nunes B, Verde MF, Soares AM. Biochemical effects of the pharmaceutical drug paracetamol on Anguilla anguilla. Environ Sci Pollut Res Int. 2015;22(15):11574-11584.
    doi pubmed
53. Wang H, Peng RX. [Effects of paracetamol on glutathione S-transferase activity in mice]. Zhongguo Yao Li Xue Bao. 1993;14(Suppl):S41-44.
    pubmed
54. Bea F, Hudson FN, Chait A, Kavanagh TJ, Rosenfeld ME. Induction of glutathione synthesis in macrophages by oxidized low-density lipoproteins is mediated by consensus antioxidant response elements. Circ Res. 2003;92(4):386-393.
    doi pubmed
55. Anderson ME. Assay of the enzymes of glutathione biosynthesis. Anal Biochem. 2022;644:114218.
    doi pubmed
56. de Graaf IA, Olinga P, de Jager MH, Merema MT, de Kanter R, van de Kerkhof EG, Groothuis GM. Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies. Nat Protoc. 2010;5(9):1540-1551.
    doi pubmed

This article is distributed under the terms of the Creative Commons Attribution Non-Commercial 4.0 International License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Journal of Endocrinology and Metabolism is published by Elmer Press Inc.