Introduction

Lactate dehydrogenase (LDH) is an important enzyme of the anaerobic metabolic pathway. It belongs to the class of oxidoreductases, with an enzyme commission number EC 1.1.1.27. The function of the enzyme is to catalyze the reversible conversion of lactate to pyruvate with the reduction of NAD+ to NADH and vice versa.[1] The enzyme is present in a variety of organisms, that include plants and animals. It is ubiquitously present in all tissues and serves as an important checkpoint of gluconeogenesis and DNA metabolism. A species-wide analysis of LDH demonstrates its well-preserved structure with only a few changes in the amino acid sequence across species.[2] The structural similarity with slight amino acid changes provides a logical platform for designing functional molecules to modulate the catalytic potential and expression of the enzyme. This article will focus on the biochemical function, testing methods, and clinical relevance of the LDH enzyme. 

Issues of Concern

Lactate dehydrogenase is an enzyme that is present in almost all body tissues. Conditions that can cause increased LDH in the blood may include liver disease, anemia, heart attack, bone fractures, muscle trauma, cancers, and infections such as encephalitis, meningitis, encephalitis, and HIV. LDH is also a non-specific marker of tissue turnover, which is a normal metabolic process. Many cancers cause a general increase in LDH levels or an increase in one of its isozymes. Thus it can be a non-specific tumor marker not useful in identifying the type of cancer. Because LDH is non-specific and routine isozyme measurement is usually unavailable in clinical laboratories, LDH measurements provide incomplete information, and alternate assays such as CK for muscle, ALT for liver, troponin for heart diseases, etc. are needed.

Additionally, LDH activity is affected by hemolysis of the blood sample. Since red blood cells (RBCs) contain their own LDH protein, hemolysis causes an artifactual increase leading to false-positive high results. Besides, any cellular necrosis can result in increased serum concentration, and its ubiquitous distribution throughout tissues confers a severe handicap to its wider clinical utility as a biomarker.

Cellular Level

LDH is a cytoplasmic enzyme that is present in almost all tissues but at high concentrations in muscle, liver, and kidney. Red blood cells also contain moderate concentrations of this enzyme. LDH exhibits five isomeric forms assembled in tetramers of either of the two types of subunits, namely muscle (M) and heart (H). The isoforms called isozymes are named LDH-1 through LDH-5, each having differential expression in different tissues.[3] This differential expression of LDH is the basis of its importance as a clinical diagnostic marker. Isozyme LDH-1 has four heart subunits (4H) and is the major isozyme present in the heart tissue. Isozyme LDH-2 has three heart and one muscle subunit (3H1M) and is the major isozyme of the reticuloendothelial system and RBCs. The LDH-3 isozyme consists of two heart and two muscle subunits (2H2M) and is the major isozyme of the lungs. Isozyme LDH-4 has one heart and three muscle subunits (1H3M) and is the primary isozyme present in the kidneys. The LDH-5 isozyme has four muscle subunits (4M) and has significant expression in liver and skeletal muscle.[4][5] These five isoforms, although catalyzing the same overall reaction, differ in their affinity to the substrate, inhibition concentration, isoelectric point, and electrophoretic mobility. These five isoforms can be visualized in the active state using LDH zymography.

Though LDH is predominantly a cytoplasmic enzyme, its mitochondrial presence is also demonstrated by various studies. The presence of a mitochondrial L-lactate dehydrogenase (mL-LDH) was confirmed in yeast, plant, and animals.[6] L-lactate, which is the substrate for mL-LDH, is transported to the mitochondria through L-lactate/H symporter and the L-lactate/pyruvate and L-lactate/oxaloacetate antiporters. Subsequently, mL-LDH facilitates the oxidation of L-lactate to pyruvate in the mitochondrial matrix.[7] Many cancer cells reprogram the mitochondrial processes to fulfill their higher demands for energy. Glycolysis becomes elevated in cancer cells, and hence, mL-LDH may be a player in accelerating oxidative phosphorylation.[8]

Molecular Level

The genes that encode LDH are LDHA, LDHB, LDHC, and LDHD. LDHA, LDHB, and LDHC encode for L-isomers of the enzyme, whereas LDHD encodes the  D-isomer. The L-isomers use and produce L-lactate, which is the major enantiomeric form of lactate present in the vertebrates. The gene that encodes the LDHA form of the enzyme is located on chromosome 11p15.4 and is transcribed into a 332 amino acid protein. The LDHB gene is located on chromosome 12p12.1 and encodes a protein of 334 amino acids. The isozyme forms of Lactate dehydrogenase enzymes, LDH-1 through LDH-5 are translational products of two genes LDHA and LDHB gene [9]. The two genes provide instructions for making the lactate dehydrogenase-A and lactate dehydrogenase-B subunits of the lactate dehydrogenase enzyme. There are five different forms of LDH, each made up of four subunits. Various combinations of the protein products of lactate dehydrogenase-A subunits and lactate dehydrogenase-B subunits produced from a different gene) make up the different forms of the enzyme.[4]

In the mammalian system, two more subunits, LDHC and LDHBx, are also included to form LDH tetramer. The LDHC gene encodes the LDHC protein that is specific to the testes, while the LDHBx gene encodes the LDHBx protein specific to the peroxisome.[10] LDHBx is the readthrough form of the LDHB gene. LDHBx is generated by translation of the LDHB mRNA, where the stop codon is read as encoding an amino acid. Consequently, translation proceeds to the next stop codon, which adds seven amino acid residues encoding the peroxisomal targeting signal to the normal LDH-H protein so that LDHBx is imported into the peroxisome.[11] The secondary structure of LDH comprises 40% alpha helices and 23% beta-sheets; this makes LDH as mixed beta-alpha-beta, with parallel beta sheets as the main component of the protein structure.[12]

The active site of the enzyme is located in its substrate-binding pocket and contains catalytically important His-193 as well as Asp-168, Arg-171, Thr-246, and Arg-106. His-193 is the active amino acid present in the active site of humans as well as other species of the animal kingdom. All the LDH isozymes are structurally very similar; however, each one has distinct kinetic properties resulting from the differences in the charged amino acids flanking the active site [2][13].

The two different subunits of LDH (the M subunit and H subunit of LDH) both maintain the same active site structure and amino acids that participate in the reaction. In the tertiary structure, the alanine of the M-chain is replaced with glutamine in the H-chain. Alanine is a nonpolar and small molecular weight amino acid, while glutamine is a positively charged amino acid. This chemistry provides different biochemical properties to the two subunits. Hence, the H subunit can bind faster but has fivefold reduced catalytic activity as compared to the M-subunit. LDHA subunit carries a net charge of -6 and exhibits a higher affinity towards pyruvate, thus converting pyruvate to lactate and  NADH to NAD+. On the other hand, LDHB has a net charge of +1 and demonstrates a higher affinity towards lactate, resulting in a preferential conversion of lactate to pyruvate and NAD+ to NADH.[4]

LDH is inherent in maintaining homeostasis when there is a lack of oxygen. Oxygen levels in the muscle tissues drop quickly upon heavy exercise. Since oxygen is typically the final electron acceptor of the electron transport chain (ETC), the chain halts along with ATP synthase. Nonetheless, muscle cells continue to function by creating ATP through NAD+. LDH produces lactic acid as an end product through a fermentation reaction. In the process, LDH removes electrons from NADH and makes NAD+, which is channelized in the glycolysis pathway to create ATP.[1] Though this process creates less ATP as compared to the ETC, it allows the cell to carry out its physiological and biochemical functions in the absence of oxygen.

Function

Lactate Dehydrogenase is one of the H transfer (oxidoreductase) enzymes, which catalyzes the reversible conversion of pyruvate to lactate using NADH.  Basically, the enzyme is involved in the anaerobic metabolism of glucose when oxygen is absent or in limited supply.[14]

• Pyruvate + NADH + H+ ­--> Lactate + NAD+

When cells become exposed to anaerobic or hypoxic conditions, the production of ATP by oxidative phosphorylation becomes disrupted. This process demands cells to produce energy by alternate metabolism. Consequently, LDH is upregulated in such conditions to cater to the need for energy production. However, lactate produced during the anaerobic conversion of glucose meets a dead end in metabolism. It cannot undergo further metabolism in any tissue except the liver. Hence, lactate is released in the blood and transported to the liver, where LDH performs the reverse reaction of converting lactate to pyruvate through the Cori cycle.[6]

During exercise, when muscles exhaust the oxygen, pyruvate gets catalyzed into lactic acid by the lactate dehydrogenase enzyme. In erythrocytes, pyruvate is not further metabolized due to the absence of mitochondria but remains within the cytoplasm, finally converting to lactate. In this reaction, NADH oxidizes to NAD+. The availability of high intracellular concentrations of NAD is necessary to carry out the preparatory phase of glycolysis. The net ATP production of anaerobic glycolysis is only 2 ATP per glucose molecule as compared to oxidative phosphorylation, which produces 36 ATP per glucose molecule. LDH can also catalyze the dehydrogenation of 2-hydroxybutyrate, but it is the less preferred substrate for LDH than lactate.[14]

The subunit composition of the LDH enzyme (H and M subunits) varies among tissues (mentioned previously in the 'cellular' section). This variation is due to the difference in the metabolic rates, energy needs, and function of the tissues, which reflects in their LDHA: LDHB ratio. Almost 40% of lactate in the circulation is released from the skeletal muscle. This lactate is further absorbed mostly by the liver and kidney, where it undergoes oxidation for the synthesis of glucose. In the brain, about 10% of the lactate oxidizes to fuel 8% of cerebral energy needs during resting conditions, and the remaining lactate is released in circulation.[14][15] However, hyperlactatemia and physical exertion can lead to the uptake of lactate, which supports 60% of brain metabolism, with the contribution of cerebral lactate oxidation only up to 33%.

In cancer cells, the function of LDH, specifically LDHA, is modified as compared to the normal cells. Cancer cells employ LDH to increase their aerobic metabolism (glycolysis and ATP production, and lactate production) even in the presence of oxygen. This process is known as the Warburg effect. The abnormal cancer cells benefit from switching to anaerobic metabolic phenotype by avoiding the generation of oxidative stress by the ETC. Additionally, the cancer cells also gain access to the metabolic intermediates of the tricarboxylic acid cycle, generated through glucose and pyruvate, to synthesize lipids and nucleic acid for rapid cell proliferation.[16][17]

Mechanism

LDH catalyzes the synchronized inter-conversion of pyruvate to lactate and NADH to NAD+ and increases the speed of reaction by 14 times. The chemical reaction proceeds by transferring a hydride ion from NADH to pyruvate at its C2 carbon. The molecular mechanism involves the binding of NADH to the enzymes as a first step. Many residues at the active site are involved in this binding. Once NADH is bound, it facilitates the binding of lactate, through an interaction between the NADH ring and the LDH residues. Transfer of a hydride quickly occurs in both directions forming two tertiary complexes, namely,  LDH-NAD+-lactate and LDH-NADH-pyruvate.[18] Subsequently, pyruvate Is dissociated from the enzyme first, and then NAD+ is released. The rate of dissociation of NADH  and NAD+ proves to be the rate-limiting step in this reaction, and the final conversion of pyruvate to lactate leading to the regeneration of NAD+ is thermodynamically favored in the reaction.[19]

Enzyme regulation: LDH activity is dependant on the metabolic switch to anaerobic respiration. LDH is modulated by three types of regulations, namely, allosteric modulation, substrate-level regulation, and transcriptional regulation. The relative availability and concentration of substrates regulate the activity of LDH. The enzyme becomes more active during extreme muscular activity when there is an increase in substrates. The demand for ATP compared to aerobic ATP supply causes the accumulation of ADP, AMP, and Pi. Glycolytic flux leads to the production of pyruvate that exceeds the metabolic capacity of pyruvate dehydrogenase and other shuttle enzymes that metabolize pyruvate. This process channelizes the flux of pyruvate and NAD+ through LDH, subsequently generating lactate and NADH.[20]

In conditions of increased NADH/NAD+ ratio, as usually happens in individuals who drink alcoholic beverages, high concentrations of ethanol lead to the production of high concentrations of lactate and NADH, and thus the depletion of NAD+. This reaction subsequently leads to pyruvate conversion to lactate linked to the regeneration of NAD+. Thus, the high NADH/NAD+ ratio shifts the LDH equilibrium towards lactate.[20]

Testing

LDH assays can measure the amount of LDH present in the serum that leaks from the tissues when damaged. The catalytic property of LDH leading to reversible oxidation of L-lactate to pyruvate, mediated by the hydrogen acceptor, NAD+, is harnessed as a basis of the measurement of LDH activity. Clinical diagnostic laboratories assess the rate of production of NADH that changes the optical density of the sample measured spectrophotometrically at 340 nm. The conversion of pyruvate to lactate or reverse reaction of oxidation of L-lactate to pyruvate can be monitored spectrophotometrically. LDH activity is measurable in various samples such as plasma, serum, tissue, cells, and in the culture medium for research purposes. Care is required when handling serum and plasma samples because hemolysis can cause an artefactual increase in the enzyme levels due to its release from ruptured erythrocytes. 

Typically, the normal range of LDH is between 140 to 280 U/L. However, the clinical interpretation depends upon the signs and symptoms of the patient. The serum usually has a higher level of LDH as compared with plasma because of LDH release during clotting. The LDH activity also increases during strenuous exercise to generate lactic acid under normal physiological conditions.[21] LDH test is affected by drugs and medications, which could interfere with accurate testing for LDH. The presence of high concentrations of vitamin C may lead to a falsely low LDH result. On the other hand, the presence of anesthetics, aspirin, alcohols, and certain narcotics, and procainamide may falsely increase the LDH result. The LDH test may only show an elevated concentration of one or more types of isozymes. Liver diseases, kidney diseases, muscle injury, trauma, heart attack, certain infectious diseases, pancreatitis, cancer, and anemia are some of the health conditions that can lead to a rise in serum LDH levels. Furthermore, the concentration of LDH varies with age, infants and young children usually have much higher normal levels of LDH levels as compared to older children and adults. Newborns have a normal range of 135 to 750 U/L (units/L), children up to 12 months have 180 to 435 U/L, and above 18 years of age have a normal range of 122 to 222 U/L (as indicated by Mayo Clinic Labs).

Besides testing the concentration of LDH in samples, LDH isozyme testing also helps to assess the type, location, and severity of tissue damage.  LDH is a tetrameric enzyme made of H and M subunits. The assembly of the enzymes occurs in a defined ratio through a tissue-specific synthesis of subunits, hence providing tissue specificity, i.e., heart-specific LDH (LDH-1) preferentially synthesizes all four H subunits, while liver LDH (LDH-5)  is exclusively made of all M-subunits. Similarly, other tissues synthesize the subunits in a specified ratio. LDH isozyme testing identifies the isozymes as LDH 1 to 5 based on the electrophoretic mobility shift. The different subunit composition imparts a difference in the net charges and hence a different migration in an electric field. A good separation pattern of LDH isozymes is obtained in a buffer of pH of 8.6. In a typical LDH isozyme electrophoretic pattern, LDH-1 moves as a fast band, followed by LDH-2, LDH-3, LDH-4, and LDH-5 being the slowest band. The normal serum percentage of LDH-1 (4H) is 30.4 to 36.4%, LDH-2 (3H1M) is 30.4 to 36.4%, LDH-3 (2H2M) is 19.2 to 24.8%, LDH-4 (1H3M) is 9.6 to 15.6%, and LDH-5 (4M) is 5.5 to 12.7%.

Pathophysiology

The quantification of LDH is of clinical interest as a serum concentration of LDH isozymes reflect tissue-specific pathological conditions. Hence, LDH can be used as a marker for diverse tissue injuries owing to its isozyme form, and its ubiquitous presence. Upon tissue damage, the cells release LDH in the bloodstream. Depending upon the type of tissue injury, the enzyme can remain elevated for up to 7 days in the bloodstream. The elevated LDH in serum as a result of organ destruction occurs due to significant cell death that results in loss of cytoplasm. Causes of tissue damage can be diseases such as acute myocardial infarction, anemia, pulmonary embolism, hepatitis, acute renal failure, etc.[22] LDH can be used as a satisfactory marker for the staging of a disease (S-classification), monitor prognosis or response to treatment, and evaluate body fluids other than blood. The decrease in LDH levels during treatment is indicative of a better prognosis and/or good response to treatment in conditions such as acute myocardial infarction or liver injury. In acute myocardial infarction, LDH-1 isozyme remains elevated from the second day up to the 4th day. Similarly, in liver injury, LDH-5 is elevated. A significant increase in LDH-5 higher than LDH-4 is a marker of hepatocellular injuries such as hepatitis or cirrhosis.

LDH increases during effusion in serous body fluids such as pericardial and peritoneal fluids. Hence, it serves to characterize effusion. In cerebrospinal fluid, LDH increases in bacterial meningitis, while it is observed to be normal in viral meningitis. The ratio of fluid LDH compared to the upper limit of normal serum LDH (> 0.6) indicates an inflammatory process, and hence exudate.[23]

There is a marked increase in LDH during intracranial hemorrhage. More than 40 U/L increase above the normal levels is observed in the central nervous system lymphoma, leukemia, and metastatic carcinoma. Elevated levels of more than one isoenzyme may be indicative of more than one cause of tissue damage, e.g., in conditions where pneumonia may also be associated with a heart attack. Very levels of LDH appear to correlate with severe disease or multiple organ failure.

LDH is the only serum biomarker useful for assessing metastatic melanomas.[24] In malignancy, the growth of tumor cells consumes oxygen more than the supply; thus, hypoxia is quite common. The growing tumors undergo LDH mediated energy production to fulfill the demand for fast cellular growth.[25] Hence, LDH is an established marker of metastases, especially in the liver. It is also a crucial single prognostic factor since patients with high LDH have reduced survival rates. LDH levels serve to predict incidences of metastasis in uveal melanoma.[26] LDH has a good correlation with tyrosine kinase expression in tumors.[27]

This enzyme also constitutes a potential therapeutic target for diseases such as malaria and cancer. The LDH isoform expressed by Plasmodium falciparum, the malarial parasite, is a crucial enzyme for the generation of energy in the parasite. Since these malarial parasites lack a tricarboxylic acid cycle for ATP formation, anaerobic glycolysis serves as a source of energy.[28] The inhibitors of Plasmodium falciparum LDH would only be directed towards the parasite and would selectively kill the parasite.

Most invasive tumors undergo a metabolic switching (Warburg effect) from oxidative phosphorylation to higher anaerobic glycolysis. This switch occurs through upregulation of the LDH-5 (also called LDH-A), the isoform normally present in muscles and the liver.[22] Hence, inhibition of LDH-5 can specifically target the site of tumor progression and invasiveness. An analog, N-hydroyxindole class LDH inhibitors are also tested effectively as anticancer agents.[29][30] Many clinical trials and translational data demonstrated that targeting LDHA genes or its protein product LDH-5 may be harnessed as a metabolic treatment of cancer.[31][28]

Researchers have conducted clinical trials in melanoma patients with low LDH, and treatment with Ipilimumab showed higher efficacy when treatment started with low baseline LDH.[32][33] Another trial using a combination of Bcl2 antisense oligonucleotide plus dacarbazine also showed efficacy in patients with low baseline LDH.[34] Thus, the overall importance of LDH as a tumor marker becomes masked because of its low sensitivity and specificity.[35] High LDL levels are usually associated with advanced stages of cancer.[36]

The deficiency of the enzyme LDH is very rare, and not much data is available for its prevalence. This deficiency can result from either the mutations in the LDHA gene or the LDHB gene leading to a deficiency in LDH-A (M- subunit protein) and LDH-B (H-subunit protein) proteins, respectively.[37] LDHA gene mutations result in the formation of an abnormal M subunit protein. This protein subunit cannot bind to other subunits to form the LDH enzyme.

LDHA gene mutation mostly affects skeletal muscles, because skeletal LDH has all M-subunits. However, a lack of a functional subunit reduces the amount of enzyme formed in all other tissues as well. This chemistry results in an ineffective breakdown of glycogen. Hence, LDHA gene deficiency is also called glycogen storage disease XI. The unavailability of sufficient energy, especially to the muscle cells, causes muscle weakness and breakdown of muscle tissue (rhabdomyolysis).[38] The effect is more pronounced during strenuous activities, and muscle tissue destruction releases protein myoglobin. This protein is processed in the kidneys and released in the urine, causing myoglobinuria. The high accumulation of myoglobin protein can damage the kidneys, which can also lead to kidney failure. In some patients, LDHA deficiency causes skin rashes of varying severity.[39]

On the other hand, LDHB gene mutations affect the heart muscle primarily because the heart LDH is made of all four H-subunits. In cardiac muscle, the involuntary muscle movement is fueled by the conversion of lactate to pyruvate through the LDH enzyme. Such conditions lead to a reduced LDH activity in cardiac muscle of patients with LDHB deficiency. Interestingly, no visible phenotype, signs, or symptoms are observed in such patients. Both LDHA and LDHB gene mutations have shown relevance in tumorigenesis.[40]

Clinical Significance

LDH serves as a general indicator of acute and chronic diseases. Elevation in serum LDH activity follows isoenzyme patterns that are characteristic of various diseases.

• LDH increase can serve as a prognostic marker of cancer progression for different types of cancer. LDH also serves as one of the important diagnostic markers of cutaneous lymphoma. The concentration of LDH-5 is demonstrated as a predictor of radiotherapy and chemotherapy response in cancer patients. LDH is useful in evaluating metastatic cancer patients. Contrarily, LDH-5 serves as a marker for radio-sensitization.
• The enzymes can function to monitor progressive conditions such as muscular dystrophy or HIV infection.
• In sports medicine, LDH potentially indicates muscle response to training, with an increase in skeletal and cardiac muscles after 3-5 hours of training.
• If LDH-1 is found to be greater than LDH-2, it indicates myocardial infarction, with a ‘flipped’ ratio of LDH-1/LDH-2 greater than 1. The increase in LDH persists for approximately ten days. It increases at 12 hours and peaks at 24-48 hours. A very high level thus indicates acute myocardial infarction.
• Greater than a 50 times increase in LDH-1 and LDH-2  indicates megaloblastic anemia.
• Increased LDH-5 in serum is a marker for muscular dystrophy.
• A ten times increase in serum LDH indicates toxic hepatitis with jaundice.
• An increase in LDH-3 is associated with the massive destruction of platelets as in pulmonary embolism.
• LDH is used to assess the nature, or pathological accumulation of pleural, peritoneal, or pericardial fluids. Serum LDH, compared to serous fluid LDH, helps in distinguishing exudate from transudate effusions.
• In patients with non-seminomatous testicular cancer LDH is used as a staging (S classification) marker.
• Abnormally low levels of LDH are very rare and usually not considered harmful.