Malondialdehyde: A Toxic Stress Marker for Periodontitis
Correspondence Address :
Arthy Arrvind,
Room No. 8, Fazal Resiplex, Al-Shafi Compound, Medical Road, Aligarh-202001, Uttar Pradesh, India.
E-mail: art10.dent@gmail.com
Recently, it has been discovered that one of the primary causes of a variety of inflammatory illnesses, including periodontitis, is an excess of Reactive Oxygen Species (ROS). It is recognised that the host’s response to pathogens plays an equal or even bigger part in determining how connective tissue breaks down. The process of Polymorphonuclear (PMN) phagocytosis greatly increases ROS formation through the metabolic pathway known as the “respiratory burst.” Because the antioxidant defense system is unable to neutralise these high levels or activities of ROS, Oxidative Stress (OS) and tissue damage ensue. ROS can directly affect tissue through Lipid Peroxidation (LPO), Deoxyribonucleic Acid (DNA) damage, protein breakdown, and the oxidation of essential enzymes. Numerous aldehydes can be produced as byproducts of LPO, including Malondialdehyde (MDA), propanal, hexanal, and 4-Hydroxynonenal (4-HNE). They can be detected in bodily fluids and suggest a pro-oxidant state. MDA is a by-product created by the enzymatic or non enzymatic breakdown of Arachidonic Acid (AA) and larger Polyunsaturated Fatty Acids (PUFAs). MDA is the most studied OS tissue damage indicator. After it is formed, MDA can either be broken down by enzymes or combine with DNA or proteins in cells and tissues to form adducts that can cause harm to biological molecules.
Lipid peroxidation, Oxidative stress, Reactive oxygen species
Chronic illnesses are becoming more common worldwide and are affecting people from all socio-economic backgrounds. Since periodontal disease and other chronic illnesses share many risk factors, they contribute to the increasing worldwide burden of disease. Approximately 320 million adults in India, or roughly one in two, suffer from periodontal disease. The prevalence of periodontal disease among American adults (47.2%) is comparable to this estimate (1).
Periodontitis is a chronic, multifactorial inflammatory disease characterised by host-mediated inflammation that results in the progressive breakdown of periodontal attachment. According to a growing body of recent research, ROS have been implicated in the establishment of an oxidatively stressed environment that underpins the pathogenesis of many long-term chronic inflammatory diseases, such as type 2 diabetes, atherosclerosis, Rheumatoid Arthritis (RA), cancer, inflammatory lung disease, and periodontitis (2). While pathogenic bacteria are an important cause in the development of periodontitis, periodontal breakdown is ultimately caused by an overreaction of the immune system. The host’s defense mechanism against spreading periodontal pathogenic bacteria is mostly mediated by PMN leukocytes. The antioxidant defense system is unable to combat the excessive production of Reactive Oxygen or Nitrogen Species (ROS/RNS) like Hydrogen Peroxide (H2O2) and superoxide (O2–) that are produced by hyperactivated neutrophils or from the direct release of microbes. This leads to oxidative stress and subsequently apoptosis of connective periodontal tissue (3). The pathogenesis of periodontitis has drawn more attention to the importance of LPO products and antioxidant biomarkers so far.
The OS is characterised by elevated LPO metabolites or end products, protein damage, and DNA damage. When PUFAs in cell membranes are peroxidised by ROS, carbon-centered radicals (PUFA radicals) or lipid peroxide radicals are produced, which can lead to a loss of membrane function. Compared to free radicals, the byproducts of LPO are more stable. MDA, 4-hydroxy-2-nonenal (4-HNE), and isoprostanes are examples of such LPO breakdown products. Additionally, DNA can be affected by OS, which generates 8-hydroxy-2’-deoxyguanosine (8-OHdG) (4). MDA has been the subject of the most research since it is a dependable indication of OS tissue injury in a variety of biological samples. The present narrative review aimed to summarise the key facts about MDA synthesis and metabolism and to distill the various approaches to estimating MDA levels in individuals with periodontitis.
The Defective Host Response in Periodontitis
The tissue destruction in the periodontium is primarily caused by host-derived enzymes called matrix Metalloproteinases (MMPs) and changes in osteoclast activity triggered by cytokines and prostanoids. As subgingival plaque bacteria accumulate, various microbial components, including chemotactic elements like Lipopolysaccharide (LPS), microbial peptides, and other bacterial antigens, penetrate the gingival connective tissues over the junctional epithelium. An inflammatory response occurs in the tissues due to the stimulation of epithelial and connective tissue cells to produce inflammatory mediators. The gingival vasculature expands (also known as vasodilation) and opens up to more fluid and cells. The tissues fill with fluid, and defense cells migrate from the circulation to the gingival crevice, where the bacteria and their byproducts serve as the source of chemotactic stimulation. In the early stages of gingival inflammation, neutrophils, a type of PMN leukocyte, predominate to phagocytose and eliminate plaque microorganisms. PMNs utilise both intracellular and extracellular mechanisms to destroy bacteria. Committed lymphocytes return to the infection area as bacterial products circulate, and B lymphocytes transform into plasma cells that produce antibodies to specific bacterial antigens. Antibodies generated in gingival tissues aid and enhance PMN phagocytosis and bacterial killing in the presence of complement (5). During the process of PMN phagocytosis, ROS formation is significantly increased through the metabolic pathway known as the “respiratory burst” (6). Due to the inadequate antioxidant defense system to counteract high levels or activities of ROS, oxidative stress and tissue damage occur. While ROS function as signalling molecules or inflammatory mediators, they can also directly damage tissue through processes such as lipid peroxidation, DNA damage, protein degradation, and the oxidation of essential enzymes when they accumulate to toxic levels (7).
Lipid peroxide radicals or carbon-centered radicals (PUFA radicals) are formed when PUFAs undergo peroxidation in cell membranes, leading to membrane dysfunction (Table/Fig 1). During the LPO of PUFAs, Malondialdehyde (MDA), also known as MDA, can be generated through the actions of polyamine oxidase and amine oxidase on spermine, as well as by human platelet thromboxane synthetase on prostaglandins PGH2, PGH3, and PGG2. In biological fluids, breakdown products of LPO such as MDA, 4-hydroxy-2-nonenal (4-HNE), and isoprostanes are detected, indicating a pro-oxidant state (4). It is noteworthy that individuals with this “hyperactivated” PMN neutrophil response, characterised by an excess of ROS and proteases, are more susceptible to periodontitis (8).
The Production of MDA
Numerous oxidation products are generated during LPO. Lipid Hydroperoxides (LOOH) are the primary byproducts of LPO. Several aldehydes, including MDA, propanal, hexanal, and 4-HNE, can be formed as secondary products during LPO. Esterbauer H et al., conducted significant research on these compounds in the 1980s (9),(10).
The MDA is a by-product created through the enzymatic or non enzymatic breakdown of Arachidonic Acid (AA) and larger Polyunsaturated Fatty Acids (PUFAs). Arachidonic acid is acted upon by cyclooxygenases to produce PGH2. The enzyme thromboxane A2 synthase converts prostaglandin endoperoxide or Prostaglandin H2 (PGH2) into Thromboxane A2 (TXA2), a physiologically active metabolite of AA (11). MDA can originate in-vivo as a by-product of enzymatic events during the formation of thromboxane A2 (12).
The process of LPO leads to the creation of a mixture of LOOHs. Hydroperoxides with a homoallylic cis-double bond to the peroxyl group can cyclically generate new radicals by introducing an intramolecular radical to the double bond. Cycle-generated intermediate free radicals can form bicyclic endoperoxides, which structurally resemble prostaglandins and can further break down to produce MDA. Arachidonic acid is the primary precursor of bicyclic endoperoxide through a non enzymatic oxygen radical-dependent process. To produce MDA, this compound subsequently undergoes additional reactions, either independently or in conjunction with other compounds. However, other eicosanoids that can also be formed through non enzymatic oxygen radical-dependent reactions may serve as precursors of bicyclic endoperoxide and MDA (11),(13).
MDA Metabolism
After its production, MDA can either be broken down by enzymes or combine with proteins or DNA in cells and tissues to form adducts that can harm biological molecules. Early research suggested that MDA is oxidised by mitochondrial aldehyde dehydrogenase, followed by decarboxylation to produce acetaldehyde. Acetaldehyde is then oxidised by aldehyde dehydrogenase to acetate, which is further converted to CO2 and H2O (11),(14). On the other hand, cytoplasmic MDA is likely converted by phosphoglucose isomerase to Methylglyoxal (MG), which is subsequently transformed into D-lactate by glyoxalase system enzymes with the help of Glutathione (GSH) as a cofactor (15). Certain enaminals (RNH-CH-CH-CHO), such as N-epsilon-(2-propenal)lysine or N-2-(propenal) serine, are among the distinct fractions of MDA that are eliminated in the urine (11).
The MDA is a bifunctional aldehyde electrophile whose reactivity varies with pH. The enolate ion, a conjugate base with a negative charge on oxygen and an adjacent C-C double bond, is present at healthy pH levels and exhibits low reactivity. Beta-hydroxyacrolein, the form of MDA that arises when pH drops, is more reactive (11). MDA’s electrophilicity, which makes it highly reactive towards nucleophiles such as basic amino acid residues (lysine, histidine, or arginine), is the main reason for its high reactivity. Advanced LPO end-products (ALE), also known as Schiff-base adducts, result from early interactions between MDA and free amino acids or proteins (16). Malondialdehyde Acetaldehyde (MAA) adducts are formed by acetaldehyde (a by-product of MDA metabolism) in the presence of MDA under oxidative stress conditions (17). Evidence suggests that MAA adducts are highly immunogenic (17),(18),(19),(20). Research reveals a list of up to 33 proteins, including enzymatic proteins, carrier proteins, cytoskeletal proteins, mitochondrial proteins, and antioxidant proteins, that are known to be modified by MDA (21).
Adducts of deoxyguanosine and deoxyadenosine have been proposed as the result of several nucleosides, such as cytidine and deoxyguanosine, reacting physiologically with MDA. The primary by-product of this reaction is pyrimido (1,2-a) purin-10 (3H-) one, also referred to as M1G or M1dG. MDA plays a significant role in DNA damage and mutation (22). The Nucleotide Excision Repair (NER) pathway is believed to be the primary route for repairing M1dG residues in genomic DNA. MDA-DNA adducts have the potential to cause strand breakage, apoptosis induction, point and frameshift mutations, and cell cycle arrest if left unrepaired. Research also indicates that long-lasting M1dG adducts in mitochondrial DNA inhibit the transcription of mitochondrial genes (23). Lower levels of DNA oxidation markers (M1dG and 8-oxodG) in peripheral white blood cells of healthy individuals have been associated with the consumption of specific antioxidants, such as vitamins (24).
Detection of MDA in Body Fluids
The MDA is utilised as a versatile biomarker in various disease indicators to differentiate between health and illness. The main technique used to measure plasma MDA levels is the colorimetric reaction with Thiobarbituric Acid (TBA). Standard Enzyme-linked Immunosorbent Assay (ELISA) kits, validated against High-Performance Liquid Chromatography (HPLC), can be used to conduct novel antibody-based tests and have produced reliable and precise results (25). Recently, a self-assembled organic-inorganic nanohybrid technology was employed to develop a non invasive method for measuring the MDA biomarker in human exhaled breath condensate. This technique has shown promise as a suitable, dependable, and cost-effective diagnostic tool for lung diseases (26). Long-term clinical studies on circulating MDA as a biomarker of LPO have determined that gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) is a useful approach (27). In 2017, a study by Hanff E et al., revealed that measuring nitrite and MDA in human urine using GC-Electron-Capture Negative Ion Chemical Ionisation (ECNICI)-MS is crucial as a surrogate internal standard for MDA (28). Takamura T aki et al., also demonstrated that MDA-modified Low-density Lipoprotein (MDA-LDL) was a favourable option for predicting the outcomes of endovascular therapy in patients with peripheral artery disease (29).
MDA Assay Methods and Studies that Evaluated the Association of MDA with Periodontitis
In 2023, Mohideen K et al., conducted a comprehensive systematic review and meta-analysis of the literature on the OS-mediated LPO end product MDA in periodontitis. The meta-analysis utilised the standardised mean differences approach and a random-effects model with a 95% confidence interval. The trials examined showed that the periodontitis group had signalling higher MDA levels in gingival crevicular fluid, saliva, and blood samples compared to the healthy control group. A total of 16 studies provided consistent data suitable for quantitative synthesis (30). Veljovic T et al., also found a statistically significant positive correlation between periodontitis and MDA levels in blood and saliva. They observed that scaling and root planing resulted in reduced levels of probing depth and MDA in the blood and saliva of periodontitis patients (31). Another study by Altngöz SM et al., assessed the relationship between OS and periodontitis in patients with and without diabetes (4). The study revealed a positive association between the Clinical Attachment Level (CAL) and MDA levels in saliva. The results of the research using MDA evaluation methods on patients with periodontitis clearly indicate that MDA levels in patients with chronic periodontitis are signalling higher than those in periodontally healthy individuals. The reaction with Thiobarbituric Acid (TBA) is used in the majority of MDA assay procedures to produce a chromogen that can be quantified spectrophotometrically (Table/Fig 2) (31),(32),(33),(34),(35),(36),(37),(38),(39),(40),(41),(42),(43),(44),(45),(46),(47),(48),(49),(50).
Association of MDA with other Oral Diseases
Numerous studies have demonstrated that OS contributes to the pathophysiology of Oral Lichen Planus (OLP) (51). Salivary ROS, LPO, nitric oxide, and nitrite levels were signalling higher in OLP patients compared to control subjects (52),(53). In OLP patients with higher salivary MDA levels than the healthy control group, total antioxidant activity was considerably lower, suggesting a potential role for oxidants in driving the disease through an LPO-mediated pathway (54),(55). Additionally, patients with oral leukoplakia and oral squamous cell carcinoma exhibited signalling higher levels of LPO, as evidenced by salivary MDA levels (56),(57). OS biomarkers have been used in studies as markers for various oral pathologies, including odontogenic cysts, inflammatory disorders of the oral mucosa, and salivary gland pathologies (58).
Various pathological issues, such as pericoronitis, swelling, odontogenic cysts or tumours, bone loss, or root resorption of adjacent teeth, are commonly associated with impacted mandibular third molars. These diseases can disrupt normal oral functions and reduce a patient’s quality of life, even if they remain unnoticed for the rest of their lives. Tekin U et al., compared MDA levels in 40 dental follicle samples from asymptomatic impacted mandibular third molars with 40 samples from healthy gingival tissue. They found higher MDA levels in the dental follicles of impacted mandibular third molars, with a statistically significant difference (59). In a long-term study, blood serum samples were used to investigate the decrease in inflammation and MDA levels after impacted third molar extraction (60). Fabio Camacho-Alonso F et al., reported that myeloperoxidase and MDA salivary concentrations were higher in patients with impacted mandibular third molars compared to participants without impacted third molars (61).
Association of MDA with Systemic Diseases Linked to Periodontitis
According to a study by Isola G et al., patients with Coronary Heart Disease (CHD) who also have periodontitis exhibit higher levels of serum and salivary MAA and C-reactive protein. The observed decrease in endothelial function due to elevated MAA levels in CHD patients may be attributed to the co-existence of periodontitis. In instance, periodontal therapy dramatically lowered serum MAA levels in periodontitis patients (62).
It has been suggested that the transition from subclinical autoimmunity to clinically evident arthritis may be influenced by the production of malondialdehyde acetaldehyde (MAA) and immune responses against MAA. In Rheumatoid Arthritis (RA) inflamed synovial tissues, MAA adducts localise independently and, more signalling, colocalise with citrullinated proteins. Notably, MAA-modified proteins have been identified in inflamed periodontal tissue due to oxidative damage induced by inflammation. Further research is needed; however, the available data suggest a potential role for anti-MAA autoantibodies in the development of RA (63). RA (autoimmune) and periodontal disease (dysbiotic microbial biofilm) have distinct aetiologies, but they share parallel biological processes, such as citrullination, autoantibody response (64), and the crucial role of bacterial dysbiosis, which may serve as direct links between these two conditions (65). Monea A et al., discovered that diabetics had higher tissue MDA levels than controls in their study. Elevated tissue MDA levels could support the hypothesis that oxidative stress contributes to the development of periodontal disease in diabetics (66).
A substantial body of research has demonstrated that OS is the main cause of the development of various inflammatory diseases, including periodontitis. Given this, it is no surprise that Malondialdehyde (MDA) is considered one of the most valuable biomarkers in numerous diseases. In conclusion, MDA serves as a crucial biomarker for assessing OS and potential cellular damage. Its measurement offers valuable insights into the extent of LPO, reflecting the body’s response to different stressors, toxins, or pathological conditions. As a reliable indicator, MDA helps in understanding the complex mechanisms of OS-related diseases. Ongoing research on MDA and its associations with various stressors holds promise for enhancing our understanding of cellular damage and developing targeted treatments to mitigate its adverse effects. The advancements in detecting OS in patients with periodontitis will be intriguing to follow, and further research in this area is recommended.
DOI: 10.7860/JCDR/2024/68440.19195
Date of Submission: Nov 04, 2023
Date of Peer Review: Dec 23, 2023
Date of Acceptance: Feb 05, 2024
Date of Publishing: Mar 01, 2024
AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was informed consent obtained from the subjects involved in the study? No
• For any images presented appropriate consent has been obtained from the subjects. No
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ETYMOLOGY: Author Origin
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