|
1
|
Bharathidevi SR, Babu KA, Jain N, Muthukumaran S, Umashankar V, Biswas J, Angayarkanni N. Ocular distribution of antioxidant enzyme paraoxonase & its alteration in cataractous lens & diabetic retina. Indian J Med Res 2017; 145:513-520. [PMID: 28862184 PMCID: PMC5663166 DOI: 10.4103/ijmr.ijmr_1284_14] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Background & objectives: The enzyme paraoxonase (PON), an antioxidant enzyme that has both arylesterase and thiolactonase activity, is well studied in cardiovascular diseases. Although a few studies have shown altered PON activity in ocular diseases such as age-related macular degeneration and diabetic retinopathy, but the tissue-wise expression of PON in its three gene forms has not been studied. This study was conducted to see the ocular distribution of PON for any altered expression in ocular pathologies such as in cataract and diabetes mellitus. Methods: Immunohistochemistry (IHC) of the ocular tissues was done for localizing all three forms of the PON in the human donor eyeballs. The PON arylesterase (PON-AREase) and thiolactonase (PON-HCTLase) activities were determined by spectrophotometry in kinetic mode, and the mRNA expression of the PON genes (PON1-3) was determined by reverse transcription-polymerase chain reaction. Results: IHC showed the presence of both PON1 and 2 in all the ocular tissues and PON3 was seen only in retina. The mRNA expression analysis showed that PON2 and PON3 were present in all the tissues, whereas PON1 was seen only in ciliary and retina. Both the PON-AREase and PON-HCTLase activities were detected in all ocular tissues and was in the order of lens>retina>choroid>ciliary body>iris. The expression and activity were studied in cataractous lens and in diabetic retina of the donor eyes. A significant decrease in PON-AREase activity was seen in cataractous lens (P<0.05) but not in diabetic retina, and there was an increase in PON- HCTLase activity (P<0.05) only in diabetic retina. Bioinformatic studies and in vitro experiments indicated that advanced glycation end products (AGE) such as carboxymethyl -lysine might decrease the PON- AREase activity of the PON. Interpretation & conclusions: Distribution of PON enzyme and its activity in ocular tissues is reported here. The study revealed maximal PON activity in lens and retina, which are prone to higher oxidative stress. Differential activities of PON were observed in the lens and retinal tissues from cataractous and diabetic patients, respectively.
Collapse
Affiliation(s)
| | - Kannadasan Anand Babu
- RS Mehta Jain Department of Biochemistry & Cell Biology, KBIRVO Block, Vision Research Foundation, Chennai, India
| | - Nishit Jain
- Department of Pharmacy, Birla Institute of Technology and Science, Pilani, India
| | | | - Vetrivel Umashankar
- Centre for Bioinformatics, KBIRVO Block, Vision Research Foundation, Chennai, India
| | - J Biswas
- Uveitis Services, Sankara Nethralaya, Chennai, India
| | - Narayanasamy Angayarkanni
- RS Mehta Jain Department of Biochemistry & Cell Biology, KBIRVO Block, Vision Research Foundation, Chennai, India
| |
Collapse
|
|
2
|
Lao X, Wang X, Liu Y, Lu Y, Yang D, Liu M, Zhang X, Rong C, Qin X, Li S. Association of Paraoxonase 1 Gene Polymorphisms With the Risk of Hepatitis B Virus-related Liver Diseases in a Guangxi Population: A Case-control Study. Medicine (Baltimore) 2015; 94:e2179. [PMID: 26632904 PMCID: PMC4674207 DOI: 10.1097/md.0000000000002179] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Paraoxonase 1 (PON1), a liver-induced glycoprotein enzyme responsible for antioxidant defense against reactive oxygen species and anti-inflammatory, has been linked to various cancers. The objective of this study was to explore the association of PON1 rs662 and rs705382 with the risk of chronic hepatitis B (CHB), hepatitis B virus-related liver cirrhosis (LC), and hepatocellular carcinoma (HCC) in patients living in the Guangxi region of southern China. The PON1 rs662 and rs705382 single-nucleotide polymorphisms (SNPs) were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 99 CHB patients, 84 LC patients, 258 HCC patients, and 221 healthy controls.Significant associations with CHB risk were observed for the rs705382 SNP after adjusting for sex, age, ethnicity, smoking, alcohol consumption, and body mass index. When stratified by sex and age, this positive association was significantly strengthened among men and individuals over 40 years old. Moreover, a decreased risk of LC was associated with the rs705382 CG and the combined GG + CG genotypes among women, with borderline statistical significance. In haplotype analyses, the haplotype GA was associated with a 1.68-fold increase in the risk of HCC.Our results showed that the PON1 rs705382 SNP might be a risk factor for CHB in Guangxi populations.
Collapse
Affiliation(s)
- Xianjun Lao
- From the Department of Clinical Laboratory, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
3
|
Abstract
Oxidative stress and inflammation underpin most diseases; their mechanisms are inextricably linked. Chronic inflammation is associated with oxidation, anti-inflammatory cascades are linked to decreased oxidation, increased oxidative stress triggers inflammation, and redox balance inhibits the inflammatory cellular response. Whether or not oxidative stress and inflammation represent the cause or consequence of cellular pathology, they contribute significantly to the pathogenesis of noncommunicable diseases (NCD). The incidence of obesity and other related metabolic disturbances are increasing, as are age-related diseases due to a progressively aging population. Relationships between oxidative stress, inflammatory signaling, and metabolism are, in the broad sense of energy transformation, being increasingly recognized as part of the problem in NCD. In this chapter, we summarize the pathologic consequences of an imbalance between circulating and cellular paraoxonases, the system for scavenging excessive reactive oxygen species and circulating chemokines. They act as inducers of migration and infiltration of immune cells in target tissues as well as in the pathogenesis of disease that perturbs normal metabolic function. This disruption involves pathways controlling lipid and glucose homeostasis as well as metabolically driven chronic inflammatory states that encompass several response pathways. Dysfunction in the endoplasmic reticulum and/or mitochondria represents an important feature of chronic disease linked to oxidation and inflammation seen as self-reinforcing in NCD. Therefore, correct management requires a thorough understanding of these relationships and precise interpretation of laboratory test results.
Collapse
|
|
4
|
Interrelationships between paraoxonase-1 and monocyte chemoattractant protein-1 in the regulation of hepatic inflammation. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2011; 660:5-18. [PMID: 20221866 DOI: 10.1007/978-1-60761-350-3_2] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Oxidative stress and inflammation play a central role in the onset and development of liver diseases irrespective of the agent causing the hepatic impairment. The monocyte chemoattractant protein-1 is intimately involved in the inflammatory reaction and is directly correlated with the degree of hepatic inflammation in patients with chronic liver disease. Recent studies showed that hepatic paraoxonase-1 may counteract the production of the monocyte chemoattractant protein-1, thus playing an anti-inflammatory role. The current review summarises experiments suggesting how paraoxonase-1 activity and expression are altered in liver diseases, and their relationships with the monocyte chemoattractant protein-1 and inflammation.
Collapse
|
|
5
|
Kedage V, Muttigi MS, Shetty MS, Suvarna R, Rao SS, Joshi C, Prakash M. Serum paraoxonase 1 activity status in patients with liver disorders. Saudi J Gastroenterol 2010; 16:79-83. [PMID: 20339175 PMCID: PMC3016510 DOI: 10.4103/1319-3767.61232] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
BACKGROUND/AIM Paraoxonase 1 (PON1) is an esterase, exclusively synthesized by liver. The present study has two objectives: to determine the PON1 activity status in various disorders associated with hepatocellular damage and to correlate the changes of PON1 activity with the standard liver function and fasting lipid profile tests in these disorders. PATIENTS AND METHODS The study groups consisted of 95 patients with liver diseases including acute viral hepatitis (14), cirrhosis with portal hypertension (33), leptospirosis (14), sepsis and multi organ failure (15), left ventricular failure (9), and falciparum malaria (10); and 53 healthy controls. Serum PON1 activity was measured manually using spectrophotometer. Liver function test parameters and fasting lipid profile were performed in clinical chemistry auto analyzer (Hitachi 912). RESULTS The serum PON1 activity in patients with acute viral hepatitis and sepsis decreased significantly ( P < 0.001) and moderately in falciparum malaria ( P < 0.05). However, in patients with cirrhosis, leptospirosis and left ventricular patients, its activity did not change significantly. On applying Pearson correlation, serum PON1 activity correlated positively with high-density lipoprotein-cholesterol (HDL-C) in patients with sepsis (r=0.633, P < 0.05), left ventricular failure patients (r=0.814, P < 0.05) and negatively with acute viral hepatitis patients (r=-0.528, P <0.05). CONCLUSION PON1 activity has decreased significantly in acute viral hepatitis, sepsis with multi organ failure and falciparum malaria patients. Determination of PON1 activity may serve as a useful additional test in assessing these conditions.
Collapse
Affiliation(s)
| | | | - Mahesh S. Shetty
- Department of Biochemistry, Kasturba Medical College, Manipal, India
| | - Renuka Suvarna
- Department of Biochemistry, Kasturba Medical College, Manipal, India
| | - Soumya S. Rao
- Department of Biochemistry, Kasturba Medical College, Manipal, India
| | - Chitralekha Joshi
- Department of Biochemistry, Kasturba Medical College, Manipal, India
| | - Mungli Prakash
- Department of Biochemistry, Kasturba Medical College, Manipal, India,Address for correspondence: Dr. Mungli Prakash, Department of Biochemistry, Kasturba Medical College, Manipal, India. E-mail:
| |
Collapse
|
|
6
|
Camps J, Marsillach J, Joven J. Measurement of serum paraoxonase-1 activity in the evaluation of liver function. World J Gastroenterol 2009; 15:1929-33. [PMID: 19399923 PMCID: PMC2675081 DOI: 10.3748/wjg.15.1929] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Paraoxonase-1 (PON1) is an esterase and lactonase synthesized by the liver and found in the circulation associated with high-density lipoproteins. The physiological function of PON1 seems to be to degrade specific oxidized cholesteryl esters and oxidized phospholipids in lipoproteins and cell membranes. PON1 is, therefore, an antioxidant enzyme. Alterations in circulating PON1 levels have been reported in a variety of diseases involving oxidative stress including chronic liver diseases. Measurement of serum PON1 activity has been proposed as a potential test for the evaluation of liver function. However, this measurement is still restricted to research and has not been extensively applied in routine clinical chemistry laboratories. The reason for this restriction is due to the problem that the substrate commonly used for PON1 measurement, paraoxon, is toxic and unstable. The recent development of new assays with non-toxic substrates makes this proposal closer to a practical development. The present editorial summarizes PON1 biochemistry and function, its involvement with chronic liver impairment, and some aspects related to the measurement of PON1 activity in circulation.
Collapse
|
|
7
|
Camps J, Marsillach J, Joven J. The paraoxonases: role in human diseases and methodological difficulties in measurement. Crit Rev Clin Lab Sci 2009; 46:83-106. [PMID: 19255916 DOI: 10.1080/10408360802610878] [Citation(s) in RCA: 166] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Research into the paraoxonase (PON) gene family has flourished over the past few years. In the 1970s and 1980s, only PON1 was known, and the investigations were conducted, essentially, by toxicologists focusing on protection against organophosphate poisoning. Since then, two new members of the family, PON2 and PON3, have been identified, both being shown to play antioxidant and anti-inflammatory roles. Evidence exists indicating that the PON family is central to a wide variety of human illnesses such as cardiovascular disease, diabetes mellitus, metabolic syndrome, obesity, non-alcoholic steatohepatitis, and several mental disorders. However, research is hampered considerably by the methods currently available to measure the activity of these enzymes. In this review, we summarize the state of knowledge on PON biochemistry and function, the influence of genetic variations, and the involvement of PON in several diseases. The problems associated with PON measurement, such as sample acquisition, lack of reference methods, and variety of substrates, will be presented. Also, we cover some of the present lines of research and propose some others for future progress in this field.
Collapse
Affiliation(s)
- Jordi Camps
- Centre de Recerca Biomedica, Hospital Universitari de Sant Joan, Institut d'Investigació Sanitària Pere Virgili, Universitat Rovira i Virgili, Reus, Spain.
| | | | | |
Collapse
|
|
8
|
Aslan M, Horoz M, Nazligul Y, Bolukbas C, Bolukbas FF, Selek S, Aksoy N, Erel O. Serum paraoxonase and arylesterase activities for the evaluation of patients with chronic hepatitis. Int J Clin Pract 2008; 62:1050-5. [PMID: 17887991 DOI: 10.1111/j.1742-1241.2006.01206..x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/01/2022] Open
Abstract
The sensitivity of standard biochemical tests for liver function is low and insufficient for a reliable determination of the presence or absence of liver disease. The aim of the present study was to investigate serum paraoxonase and arylesterase activities and lipid hydroperoxide (LOOH) levels, and to find out that whether the measurement of serum paraoxonase and arylesterase activities would be useful as an index of liver function status in chronic hepatitis (CH). Fourty-four patients with CH (24 CHB and 20 CHC) and 38 controls were enrolled. Serum paraoxonase and arylesterase activities were detected spectrophotometrically. LOOH levels were measured by the FOX-2 assay. Serum paraoxonase and arylesterase activities were significantly lower in patients with CH than controls (p < 0.001 for both), while LOOH levels were significantly higher (p < 0.001). Paraoxonase and arylesterase activities were inversely correlated with LOOH levels (r = -0.394, p < 0.05; r =-0.362, p < 0.05, respectively). Fibrosis scores of CH patients were significantly correlated with paraoxonase and arylesterase activities and LOOH levels (r =-0.276, p < 0.05; r = -0.583, p < 0.001 and r = 0.562, p < 0.001, respectively). Our results indicated that decrease in the activities paraoxonase and arylesterase may play a role in the pathogenesis of CH. In addition, serum paraoxonase and arylesterase activities measurement may add a significant contribution to the liver function tests.
Collapse
Affiliation(s)
- M Aslan
- Department of Internal Medicine, School of Medicine, Harran University, Sanliurfa, Turkey.
| | | | | | | | | | | | | | | |
Collapse
|
|
9
|
Schulpis KH, Barzeliotou A, Papadakis M, Rodolakis A, Antsaklis A, Papassotiriou I, Vlachos GD. Maternal chronic hepatitis B virus is implicated with low neonatal paraoxonase/arylesterase activities. Clin Biochem 2007; 41:282-7. [PMID: 18035058 DOI: 10.1016/j.clinbiochem.2007.10.013] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2007] [Revised: 10/04/2007] [Accepted: 10/24/2007] [Indexed: 01/29/2023]
Abstract
BACKGROUND Paraoxonase/arylesterase activities are closely implicated with liver function and antiatherogenetic process. AIM To evaluate whether maternal chronic hepatitis B virus, disease (HBV) affect serum neonatal paraoxonase/arylesterase activities. PATIENTS AND METHODS 28 pregnant women with HBV and 28 healthy pregnant women (controls) in the delivery room and their newborns (cord blood) underwent laboratory examinations. Serological virus tests and liver function tests and paraoxonase (PON 1) activities were measured with the Siemens Advia 1650 Clinical Chemistry System, while total antioxidant capacity (TAC) levels and paraoxonase-arylesterase (PON-aryl) activities were measured spectrophotometrically. RESULTS Serological HBV tests and HBV DNA showed chronic HBV (precore mutant G1896A) in the diseased mothers whereas anti-HBc and anti-HBe were detected in their neonates. Liver function parameters were found normal in controls and both groups of newborns. Moderately increased transaminase levels were measured in HBV mothers, whereas TAC levels were decreased in hepatic mothers and their newborns. Interestingly albumin levels did not differ among the studied groups. PON 1 and PON-aryl activities in the diseased mothers (148+/-14 U/mL/min, 130+/-16 KU/mL/min) and their infants (32+/-6 U/mL/min, 24+/-5 KU/mL/min) were significantly lower as compared to those of control mothers (217+/-16 U/mL/min, 196+/-14 KU/mL/min p<0.001) and their newborns (57+/-6 U/mL/min, 48+/-8 UK mL/min p<0.001). Inverse significant correlations were found between the studied enzyme activities and liver enzymes in all the groups of study except in infants born from HBV mothers and positive with TAC in all the studied groups. CONCLUSIONS Decreased PON 1 and PON-aryl activities were measured in infants born from hepatic mothers probably as a consequence of their low TAC. Infants born from HBV mothers are at risk for developing LDL oxidation perinatally.
Collapse
|
|
10
|
Kilic SS, Aydin S, Kilic N, Erman F, Aydin S, Celik I. Serum arylesterase and paraoxonase activity in patients with chronic hepatitis. World J Gastroenterol 2006; 11:7351-4. [PMID: 16437641 PMCID: PMC4725136 DOI: 10.3748/wjg.v11.i46.7351] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the relationship between serum paraoxonase (PON1), AST, ALT, GGT, and arylesterase (AE) activity alterations and the degree of liver damage in patients with chronic hepatitis. METHODS We studied 34 chronic hepatitis patients and 32 control subjects, aged between 35 and 65 years, in the Department of Infection and Clinical Microbiology at the Firat University School of Medicine. Blood samples were collected from subjects between 8:00 and 10:00 a.m. following a 12-h fast. Baseline and salt-stimulated PON1 activities were measured by the hydrolysis of paraoxon. Phenyl acetate was used as the substrate and formed phenol was measured spectrophotometrically at 270 nm after the addition of a 10-fold diluted serum sample in AE activity measurements. RESULTS The results of this investigation revealed that the levels of AE activity decreased from 132+/-52 to 94+/-36 (29%), baseline PON1 activity from 452+/-112 to 164+/-67 (64%), salt-stimulated PON1 activity from 746+/-394 to 294+/-220 (61%), HDL from 58.4+/-5.1 to 47.2+/-5.6 (20%), triglyceride from 133+/-51.2 to 86+/-34.0 (35%), while a slight increase in the level of LDL (from 163+/-54.1 to 177.3+/-56.0; 9%) and significant increases in the levels of AST (from 29+/-9.3 to 98+/-44), ALP (from 57.2+/-13.1 to 91+/-38.1), ALT (from 27.9+/-3.32 to 89+/-19.1), GGT (from 24.3+/-2.10 to 94+/-48.2), total bilirubin (from 0.74+/-0.02 to 1.36+/-0.06; 84%) and direct bilirubin (from 0.18+/-0.01 to 0.42+/-0.04; 133%) were detected. However, the levels of albumin, total protein, cholesterol, and uric acid were almost the same in chronic hepatitis and the control subjects. CONCLUSION Low PON1 and AE activity may contribute to the increased liver dysfunction in chronic hepatitis patients by reducing the ability of HDL to retard LDL oxidation and might be clinically useful for monitoring the disease of chronic hepatitis.
Collapse
Affiliation(s)
- Suleyman Sirri Kilic
- Department of Biochemistry and Clinical Biochemistry, Medical School (Firat Medical Center), Firat University, Elazig 23119, Turkey
| | | | | | | | | | | |
Collapse
|
|
11
|
Lie-Injo LE, Solai A, Ganesan J, Ganesan S. Arylesterase isoenzymes and activity in normal healthy adults and in patients with cancer and with other diseases. Clin Biochem 1980; 13:113-5. [PMID: 7418194 DOI: 10.1016/s0009-9120(80)90759-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Serum arylesterase isozyme patterns were studied in 184 normal healthy individuals, 290 cancer patients and 466 patients with various diseases. No abnormal patterns were seen in the normal healthy subjects. Several abnormal patterns found in the group of cancer patients and patients with various diseases are described. In the majority of patients with cancer of the liver there is an abnormal additional cathodal band. The most cathodal band in normals or the two most cathodal bands in the patients with hepatoma with double cathodal bands stained for cholinesterase as well as for arylesterase. We also studied serum arylesterase activity on the basis of the kinetic release of beta-naphthol in these groups. The mean activity in normal healthy individuals agrees with that reported earlier. In patients with cancer and with miscellaneous other diseases, the mean activity is lower but the range of values in the two groups is very wide.
Collapse
|
|
12
|
Burlina A. First J. Henry Wilkinson Memorial Lecture: The clinical relevance of isoenzyme assays. Clin Biochem 1979; 12:71-6. [PMID: 37000 DOI: 10.1016/s0009-9120(79)80069-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
|
|
13
|
Bøg-Hansen TC, Krog HH, Back U. Plasma lipoprotein-associated arylesterase is induced by bacterial lipopolysaccharide. FEBS Lett 1978; 93:86-90. [PMID: 212313 DOI: 10.1016/0014-5793(78)80811-4] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
|
|
14
|
Abstract
Human arylesterase is localized in liver microsomes where the presence of different electrophoretic bands corresponding to the serum bands can be recognized. Serum arylesterase is mainly a result of liver activity and its high level might be explained by a low rate of elimination in urine. The behaviour of arylesterase towards inhibitors shows certain similarities to that of some of the proteases, such as trypsin. The clinical value of serum arylesterase determination in assessing liver function is confirmed by its isoenzyme behaviour in cirrhosis and porto-caval shunt.
Collapse
|
|
15
|
Smith DA, Cole WJ. Identification of an arylesterase as the enzyme hydrolysing diacetylmorphine (heroin) in human plasma. Biochem Pharmacol 1976; 25:367-70. [PMID: 938568 DOI: 10.1016/0006-2952(76)90333-6] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
|