1

LACTATE DEHYDROGENASE: APPLICATIONS IN DIAGNOSTIC

ENZYMOLOGY AND CLINICAL MEDICINE.

Ibrahim Hassan Garba* and Ubom Gregory Abraham**

*
Chemistry Programme, School of Science, Abubakar Tafawa Balewa University,

PMB 0248, Bauchi, Nigeria. Email: ihgarba2002@yahoo.com (Correspondin

author).

**
Department of Biochemistry, Faculty of Medical Sciences, University of Jos, PMB

2048, Plateau, Nigeria.

TABLE OF CONTENTS

1 LACTATE DEHYDROGNASE (LDH): AN INTRODUCTION

Lactate dehydrogenase (LDH) (LD; L – lactate: NAD oxidoreductase, EC 1.1.1.

27) is a pyridine-linked enzyme found in virtually all animal and human tissues

where it functions primarily in the metabolism of glucose, by catalyzing the

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reduction of free pyruvate to lactate in the last step of glycolysis as well as

participating in the conversion of lactate to pyruvate during gluconeogenesis

(Emad et al. 2003). At high concentration of pyruvate the enzyme exhibits

feedback inhibition and the rate of conversion of pyruvate to lactate is decreased.

It uses either nicotinamide adenine dinucleotide (NAD) or reduced nicotinamide

adenine dinucleotide (NADH) as co-factors. Within the L-LDH family, allosteric

and non-allosteric forms are known to exist. However it should be noted that there

is another variant of the LDH known as d-lactate dehydrogenase (d-LDH) found

particularly in bacteria where it exist as a peripheral membrane respiratory

enzyme involved in electron transfer, located on the cytoplasmic side of the inner

membrane. Functionally, d-LDH catalyzes the oxidation of d-lactate to pyruvate,

which is coupled to the transmembrane transport of amino acids and sugars (Orly

et al. 2000). Apart from its specificity for d-lactate, d-LDH uses flavin adenine

dinucleotide (FAD) as a cofactor. L-LDH and d-LDH belong to evolutionarily

unrelated enzyme families (Melania et al. 2008). Except otherwise specified, the

reference to LDH in this work refers to l-lactate dehydrogenase (l-LDH). LDH is

a tetramer composed of two immunologically distinct subunits (M and H). The

five theoretically possible forms of LDH (H4, HM, H2M2, H1M3 and M4) are found

in human tissues, with their proportions differing in various organs. An extra

LDH isoenzyme designated LDH-1ex has also been reported to exist. LDH is

widely distributed in the tissues of the body. High concentrations are found in the

heart and liver. Significant amounts are present in erythrocytes. Skeletal muscles

and kidney also contain considerable concentrations of LDH (Calbreath, 1992).

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Overall concentrations of LDH are some 500-fold greater than the serum levels

under normal circumstances. Therefore even small amounts of tissue damage are

reflected in measurable elevations of total lactate dehydrogenase activity

(Podlasek and McPherson, 1989).

2 ROLE OF ENZYMES IN DIAGNOSIS / CLINICAL MEDICINE

Some plasma / serum enzymes and proenzymes and their substrates are known to

be present at all times in the circulation of normal individuals where they perform

physiologic roles on the blood (Murray et al. 1990). Examples of functional

plasma enzymes are pseudocholinesterase (Bert and Lockridge, 1986) and

proenzymes of blood circulation. They are generally synthesized in the liver but

are present in blood in equivalent or higher concentration than in tissues

(Schumacher et al. 1986). Non-functional plasma / serum enzymes on the other

hand perform no known physiological role in blood and are present in the blood

of normal individuals at levels up to a million-fold lower than in tissues.

Examples of such enzymes include those of exocrine secretions such as amylase

and prostatic acid phosphatase (Crerar et al. 1983; Suguira et al. 1980) which

diffuse into the plasma and true intracellular enzymes such as 5’- nucleotidase

(Sullivan and Alphers, 1971) which is normally absent in circulation. Accordingly

the low plasma circulating levels of such enzymes is due to normal cellular

turnover (Calbreath, 1992). Because disease states usually lead to moderate or

extensive tissue damage, depending on the time of onset, such disease conditions

is accompanied by the release of enzymes specific to the diseased organ / tissue

into the circulation. The resultant effect is an increase in activity of these enzymes
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in body fluids. However not all tissue-specific damage leads to an increase in the

activity of the organ / tissue-specific enzymes concerned. Some disease

conditions have been reported to involve a decrease in organ / tissue-specific

enzyme activity in body fluids (Lum et al. 1993b). In this context, the

measurement of enzymatic activity in serum /plasma has been employed

diagnostically for over thirty years (Wacker and Coombs, 1969). Changes in

enzyme activity have proven to be among the most sensitive diagnostic methods,

a reflection of the implication inherent in the catalytic property of enzymes

(Ferdinand, 1976). Thus minimal changes in the concentration of an enzyme are

reflected in easily measurable alterations in enzymatic rates. Enzymatic methods

are now crucial to the diagnosis of acute myocardial infarction (Adams et. al.

1994), liver diseases (Castaldo et. al. 1994), acute pancreatitis (Patenghini and

Pagani, 1989) and various types of anaemia (Fujita et. al. 1994). While alterations

in the activity of enzymes in serum and other body fluids are usually general

diagnostic indicators, recently developed methods to identify various isoenzymes

have added specificity to the approach (Wu et al. 1994; Van Hoof et al. 1995).

Efforts have also been made to develop assays for enzymes which are limited in

their organ distribution, in a further attempt to achieve diagnostic specificity

(Collazos et al. 1994).

3 LACTATE DEHYDROGENASE ISOENZYMES

Normal human serum / plasma contain five different isoenzymes of LDH

(Giannoulaki et al. 1989). These isoenzymes are formed by the random

combination of two different subunits encoded by two structurally distinct genes,

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LDHA and LDHB (Markert 1963). Furthermore, the expression of mammalian

LDHA and LDHB has been shown to be regulated during development and is

tissue-specific, indicating that alterations in serum LDH isoenzyme pattern can

serve as indicators of pathologic involvement and the development of cancer

(Markert et al. 1975; Maekawa, 1988). In accordance with international

nomenclature the one which migrates farthest from the origin after electrophoresis

is called LDH-1, while LDH-5 has the slowest migration from the origin. There is

some considerable degree of tissue specificity associated with the various

isoenzymes. LDH-1 and LDH-5 are homotetramers of heart predominant H and

muscle-specific M subunits and form random heterotrameric combinations giving

rise to LDH-2, LDH-3 and LDH-4 respectively (Riaz et al. 2009). Skeletal muscle

and liver contain high amounts of LDH-5 (Castaldo et al. 1994). Kidney tissue

seems to have a significant amount of LDH-4 and LDH-5 (Kalpaxis and

Giannoulaki, 1989) whereas the major component of the lung is LDH-3. In

addition to the regular isoenzyme bands, many workers have reported the

presence of additional bands in electrophoretograms of human serum. Some of

these bands have been shown to be genetic variants, but more commonly they

have been established to be immunoglobulin complexes, tumour products, or

recombination products between H, M, and C monomers made during storage of

samples at -200C (Giannoulaki et al. 1989). The primary approach to the

fractionation and assay of LDH isoenzymes is electrophoretic separation on

agarose or cellulose acetate (Cohen et al. 1993). Other notable analytical

approaches are chromatography and immunological methods (Lambeth and

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Muhonen, 1994; Gosling, 1990). The serum activities of individual isoenzyme

bands are determined by the physiological function and metabolic or clinical state

of affected tissue. Tissue-specific normal isoenzyme profiles of humans are

therefore found disturbed in a number of clinical conditions and thus carry

diagnostic value (Riaz et al. 2009). Studies on LDH isoenzymes have shown that

serum / plasma LDH-1 increases in myocardial infarction and haemolysis

(Calbreath, 1992). LDH-3 is elevated in patients with pulmonary infarction, while

an increased serum / plasma LDH-5 is associated with hepatic damage. Elevations

in serum LDH-1, LDH-3 and LDH-5 have also been reported in patients with a

variety of cancers (Kopetzki et al. 1994). Specifically, in cancer patients LDH

isoenzymes originate primarily from tumour tissues and partly from healthy

tissues damaged by tumour expansion and invasion (Masato et al. 2003). The

increase in LDH-1 in cancer has been shown to correlate with the total copy

number of the short arm of chromosome 12 in tumour cells, a finding which

correlates with the high proportion of LDH-1 found in a patient with

retinoblastoma (Von Eyben et al. 1992; Maekawa et al. 2002). Gastric cancer was

also recently found to be associated with increased concentrations of

electrophoretically slow-moving LDH isoenzymes, which was attributed to

transcriptional silencing of LDHB expression by aberrant promoter methylation

(Ishikawa et al. 2004). Furthermore, serum LDH-1 concentrations are also found

to increase in patients with brain tumour, various types of germinal cell tumours

of the ovary, testis, and mediastinum (Maekawa, 1988). In addition, in terms of

diagnostic efficiency in patients with metastatic liver disease, a combined

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measurement of total LDH of 225U/L (upper limit of normal) and increased

LDH-4 and 5 was shown by Rotenberg et al (1989) to be much better than

measuring total LDH alone. They further went on to conclude that serum LDH

and LDH isoenzymes should be determined in every patient with suspected liver

metastatic disease since the isomorphic pattern of LDH isoenzymes is apparently

associated with higher values of total LDH and was found to be common among

the patients with multiple metastatic sites. An unusual extra electrophoretic band

has occasionally been detected in sera from cancer patients with brain tumours,

esophageal cancer and neuroblastomas and in erythrocytes, first trimester placenta

trophoblast, and a choriocarcinoma cell line. The electrophoretic mobility of this

extra band (designated LDH-2ex) is often close to that of LDH-2 and between the

mobilities of the LDH-2 and LDH-3 isoenzyme bands (Masato et al. 2002). This

is in addition to the LDH variant that was found to migrate between albumin and

LDH-1 isoenzyme during electrophoresis and designated LDH-1ex. The heat

stability of the LDH variant appears to be similar to that of LDH-1 but greater

than that of the other LDH isoenzymes. Statistical data on LDH-1ex has shown that

there is significant correlation between malignancy and the appearance of this

isoenzyme (Giannoulaki et al. 1989).

4 DISEASE CONDITONS ASSOCIATED WITH ALTERED SERUM /

PLASMA LACTATE DEHYDROGENASE ACTIVITY

LDH total activity is elevated in a wide variety of disease states. Increases in

serum lactate dehydrogenase activity have been reported to be associated with

renal infarction, myocardial damage, ineffective erythropoiesis and haemolysis.

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High serum LDH has also been reported in a variety of cancers, i.e. small cell

carcinoma of the lungs, nephroblastoma, neuroblastoma and metastatic

neuroendocrine tumours (David and Alan, 1994; Podlasek and McPherson, 1989).

LDH activity is also increased in the serum of patients with measles, cervical

lymphadenitis and malaria (Sugaya et. al., 1990: Garba and Ubom, 2005). A

variety of liver diseases and skeletal as well as cardiac muscle dysfunction also

cause elevated serum LDH activity (Riaz et al. 2009). The measurement of LDH

in fluid aspirated from pleural effusions or pericardial effusion has been used to

aid distinguishing between exudates (actively secreted fluid, e.g. due to

inflammation) and transudates (passively secreted fluid, due to high hydrostatic

pressure or a low oncotic pressure). In particular if the LDH measurement is done

simultaneously with pleural cholesterol, it permits the separation of pleural

exudates from transudates with an accuracy similar to the original report of Light

et al (1972), with the added advantage of requiring only two laboratory

investigation and no simultaneous blood sample (Costa et al. 1995). The enzyme

is elevated in an exudate and low in a transudate. LDH is also found in

cerebrospinal fluid (CSF) where high levels of the enzyme in the CSF is often

associated with bacterial meningitis, generally serving as an indicator of the

presence of encephalitis and poor prognosis. LDH is also measured in HIV

patients as a non-specific marker of Pneumocyctis jiroveci pneumonia (PJP).

Elevated LDH in the setting of the upper respiratory symptoms in an HIV patient

suggests, but is not diagnostic of PJP.

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5 OTHER APPLICATIONS OF LACTATE DEHYDROGENASE IN

CLINICAL MEDICINE

Dental caries is an infectious disease in which Streptococcus mutans (S. Mutans)

play a major role. In this disease LDH has been shown to serve as an important

virulence factor for acid production on these bacteria on which the cariogenic

potential of S. Mutans depends (Yang and Liu, 2004). Increases in serum LDH

activity, particularly in the region of 600 U/L or greater has also been postulated

to serve as an adjunct laboratory marker in the diagnosis of histoplasmosis, with

the added suggestion that physicians should look for a diagnosis other than that of

Pneumocyctis jiroveci pneumonia (PJP) once the serum LDH level is up to 600

U/L (Adeel et al. 2002). Kotoh et al. (2008) have recently reported the combined

use of serum LDH and alanine aminotransferase levels as a potentially valuable

parameter for predicting the prognosis of patients at an early stage of acute liver

injury. They were able to show that the ALT-LDH index is useful in predicting

the prognosis of patients with acute liver injury. LDH has also been used as a

component of the biochemical markers of cardioversion or defibrillation of

arrythmias in acutely ill cardiac patients (Kjell et al. 2000). In relation to burns,

patient serum LDH activity has been reported to remain significantly elevated up

to 10 days during the recovery of post burn patients. Riaz et al. (2009) have

therefore concluded that this finding strongly recommends the use of LDH-5

assessment in particular, during post burn wound-healing as a post-burn marker

relative to serum creatine kinase due to its better reproducibility and stability up

to 10 days during post-burn recovery. Serum LDH measurement has also been
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postulated to be useful in identifying response to chemotherapy, and hence

underscoring its potential significance as a prognostic marker (Emad et al. 2003).

LDH isoenzymes are also well recognized as indicators of up- or down-regulation

of anaerobic glycolytic mechanism under diverse physiological conditions,

including hypoxia. Specifically, observations on Channa punctatus suggest that it

differs from members of the genus Fundus in its response to signals of respiratory

distress by inducing either LHD-A or LDH-B or by inducing both the genes

depending on respiration mode or in a tissue / organ-specific manner (Riaz and

Hanain, 2005).

6 LACTATE DEHYDROGENASE: FUTURE PROSPECTS

From the foregone discussion it can be deduced that the role of lactate

dehydrogenase in clinical medicine goes way beyond its application in enzymatic

diagnosis. Based on the difference in the amino acid composition of non-

conserved motifs of this enzyme and its isoforms, there is the potential of using

this enzyme as an entigenic determinant for developing antibodies against a

number of pathogens and the application LDH antibodies to diagnose various

infectious disease conditions. Structural studies of this enzyme in various

organisms can also give lead insights into the evolutionary diversity / relationship

existing between these organisms.

7 REFERENCES

Adams JE, Schectman KB, Landt Y, Ladenson JH and Jaffe AS. (1994). Comparative

detection of acute myocardial infarction by creatine kinase MB isoenzyme and

cardiac troponin. Clin. Chem. 40(7): 1191-1295

 11

Adeel AB, Michaels S, Donald G, Clark R, Kissinger P and Martin HD. (2002). Serum

LDH level as clue to the diagnosis of histoplasmosis. The AIDS Reader 12 (7):

317-321

Bert NLD and Lockridge O. (1986). Molecular biology of human serum cholinesterase.

Fed. Proc. 45 (13): 2965-2969

Calbreath DF. (1992). Clinical Chemistry. Philadelphia: W. B. Saunders Company, 468p

Castaldo G, Oriani G, Cimino L, et al. (1994). Total discrimination of peritoneal

malignant ascites from cirrhosis and hepatocarcinoma-associated ascites by assays

of ascitic cholesterol and lactate dehydrogenase. Clin. Chem. 40(3): 478-483

Cohen AS, Smisek DL, and Keohavong P. (1993). Capillary gel electrophoresis of

biopolymers. Trends Anal. Chem. 12(5): 195-202

Collazos J, Esteben C, and Ferdinand A. (1994). Neuron-specific enolase concentrations

in serum in non-neoplastic patients with pneumonia. Clin. Chem. 40(2): 266-267

Costa M, Quiroga T and Cruz E. (1995). Measurement of pleural fluid cholesterol and

lactate dehydrogenase. A simple and accurate set of indicators for separating

exudates from transudates. Chest 108, 1260-1263

Crerar MM, Sullivan WM, Pictet RL, Nokovits W and Rutter WJ. (1983). Isolation and

characterization of rat amylase gene family. J. Biol. Chem. 258 (2): 1311-1317

David K and Alan C. (1994). Increased lactate dehydrogenase isoenzyme-1 in a case of

glucagonoma. Clin. Chem. 40(1): 158-159

Emad AA, Lamia MA, and Janan KH (2003). Serum lactate dehydrogenase (LDH)

activity in children with malignant disease. Bahrain Med Bul 25 (2) 71-73

Ferdinand W. (1976). The enzyme molecule. London: John Wiley and Sons, 289p
 12

Fujita T, Hamasaki H, Furukata T and Nanobe M. (1994). New enzymatic assay of iron

in serum. Clin. Chem. 40(5): 763-767

Garba IH and Ubom GA (2005) Total serum lactate dehydrogenase activity in acute

plasmodium falciparum malaria infection. Singapore Med. J. 46(11): 632- 634

Giannoulaki EE, Kalpaxis DL, Tentas C and Fessas P. (1989). Lactate dehydrogenase

isoenzyme pattern in sera of patients with malignant diseases. Clin. Chem. 35 (3):

396-399

Gosling JP. (1990). A decade of development in immunoassay methodology. Review.

Clin. Chem. 36(8): 1402-1427

Hiroyuki U, Shinya F, Wakagi T, Konno M, Taguchi H and Matsuzawa H. (2001).

Crystal structure of non-allosteric L-lactate dehydrogenase from Lactobacillus

pentosus at 2.4A resolution: Specific interactions at subunit interfaces. Proteins:

Structure, Functions and Bioinformatics, 46 (2): 206-214

Ishikawa J, Terumi T, Higashi H, Muira K, Suzuki K, Akihiro T and Masato M. (2004).

High lactate dehydrogenase isoenzyme 1 in a patient with malignant germ cell

tumour is attributable to aberrant methylation of the LDHA gene. Clin. Chem. 50

(10): 1826-1828

Kalpaxis DL and Giannoulaki EE. (1989). Partial characterization of an abnormal lactate

dehydrogenase isoenzyme, LDH-1ex in serum from a patient with hepatocellular

carcinoma. Clin. Chem. 35(5): 844-848

Kjell V, Omvik P, Farstad M and Jan Erik N. (2000). Cardiac biochemical markers after

cardioversion of atrial fibrillation or atrial flutter. Am. Heart J. 140, 690-694

 13

Kopetzski E, Lehnert K and Buckel P. (1994). Enzymes in diagnostics. Achievements

and possibilities of recombinant DNA technology. Clin. Chem. 40(5): 688-704

Kotoh K, Enjoji M, Kato M, Nakamuta M and Takayanagi R. (2008). A new parameter

using serum alanine aminotransferase level is useful for predicting the prognosis

of patients at an early stage of acute liver injury: A retrospective study. Comp.

Hepatol., 7:6. doi:10.1186/1476-5926-7-6

Lambeth DO and Muhonen WE. (1994). High performance liquid chromatography-based

assays of enzyme activities. Review. J. Chromatog-B. 651(1): 143-157

Light RW, MacGregor MI, Luchsinger PC et al. (1972). Pleural effusions: the diagnostic

separation of exudates and transudates. Ann. Intern. Med. 77: 507-513

Lum G, Marquard C and Khuri SF. (1989). Hypomagnesaemia and low alkaline

phosphatase activity in patient’s serum after cardiac surgery. Clin. Chem. 35(4):

164-167

Maekawa M, Inomata M, Sasaki MS et al. (2002). Electrophoretic variant of lactate

dehydrogenase isoenzyme and selective promoter methylation of the LDHA gene

in a human retinoblastoma cell line. Clin. Chem. 48: 1938-1945

Maekawa M. (1988). Lactate dehydrogenase isoenzymes. J. Chromatog. 429: 373-398

Markert CL, Shakelee JB and Whitt GS. (1975). Evolution of a gene: multiple genes for

LDH isoenzymes provide a model for the evolution of gene structure, function

and regulation. Science 189: 102-114

Markert CL. (1963). Lactate dehydrogenase isoenzymes: dissociation and recombination

of subunits. Science 140: 1329-1330

 14

Masato M, Inomato M, Sasaki MS, Kaneko A, Mineko U, Sugano K, Takayama J and

Takashi K. (2002). Electrophoretic variant of a lactate dehydrogenase isoenzyme

and selective promoter methylation of the LDHA gene in a human retinoblastoma

cell line. Clin. Chem. 48 (11); 1938-1945

Masato M, Taniguchi C, Ishikawa J, Sugimura H, Sugano K and Takashi K. (2003).

Promoter hypermethylation in cancer silences LDHB, eliminating lactate

dehydrogenase isoenzymes 1-4. Clin. Chem. 49 (9): 1518-1520

Melania EC, Innes DJ, Jonathan SH, and Crease TJ. (2008). D- and L-lactate

dehydrogenases during invertebrate evolution. BMC Evolutionary Biol. 8: 268,

doi.10.1186/1471-2148-8-268

Murray RK, Mayes PA, Granner DK and Rodwell VW. (1990). Harper’s Biochemistry.

New York: Appleton and Lange, 695p

Orly D, Pratt AE, Chien H and Eisenberg D. (2000). The crystal structure of d-lactate

dehydrogenase, a peripheral membrane respiratory enzyme. Proceedings of the

National Academy of Sciences (USA), 97 (17): 9413-9418

Patenghini M and Pagani F. (1989). Diagnostic value of measuring pancreatic lipase and

the P3 isoform of the pancreatic amylase isoenzyme in serum of hospitalized

hyperamlasemic patients. Clin. Chem. 35(3): 417-421

Podlasek SJ, McPherson RA. (1989). Streptokinase binds lactate dehydrogenase subunit-

M which shares an epitope with plasminogen. Clin. Chem. 35(1): 69-73

Riaz A, Saba Q, Hanain A, Anjum A, Arshad HK, Mumtaz A. (2009). Observation on the

changes in lactate dehydrogenase isoenzymes in post-burn patients: Significance

in relation to creatine kinase. J Med Biochem 28 :( 1), 16-21

 15

Riaz A. and Hasnain A (2005). Ontogenic changes and developmental adjustments in

lactate dehydrogenase isoenzymes of an obligate air-breathing fish Channa

punctatus during deprivation of air access. Comp. Biochem. Physiol, Part B. 140,

271-278

Rotenberg Z, Weinberger I, Davidson E, Fuchs J, Harell D and Agmon J. (1989). Lactate

dehydrogenase isoenzyme patterns in serum of patients with metastatic liver

disease. Clin. Chem., 35 (5): 871-873

Schmacher M, Camp S, Maulet Y, Newton M, MacPhee-Quigley K, et al. (1986).

Primary structure of acetylcholinesterase: Implications for regulation and

function. Fed. Proc. 45(13): 2976-2981

Sugaya N, Kanno N, Nirasawa M et al. (1990). Increased activities of aminopeptidase

and lactate dehydrogenase in serum originate from lympocytes in necrotizing

lymphadenitis. Clin. Chem. 36(2): 304-306

Suguira KY, Kawabe H and Tanaka H. (1980). New manganese (III)-containing acid

phosphatase. Evidence for an intense charge-transfer band and tyrosine phenolate

coordination. J. Am. Chem. Soc. 102: 6581-6582

Sullivan JM and Alphers JP. (1971). In vitro regulation of rat heart 5’- nucleotidase by

adenine nucleotides and magnesium. J. Biol. Chem. 246(9): 3057-3063

Van Hoof VO, Martin M, Blockx P et al. (1995). Immunoradiometric method and

electrophoretic system compared for quantifying bone alkaline phosphatase. Clin.

Chem. 41(6): 853-857

Von Eyben FE, De Graff WE, Marrink J. et al. (1992). Serum lactate dehydrogenase

isoenzyme 1 activity in patients with testicular germ cell tumors correlates with
 16

the total number of copies of the short arms of chromosome 12 in the tumor. Mol.

Gen. Genet. 235:140-146

Wacker WEC and Coombs TL. (1969). Clinical Biochemistry: Enzymatic methods:

Automotion and atomic absorption spectroscopy. Ann. Rev. Biochem. 38: 539-568

Wu HBA, Valdes R, Apple SF et al. (1994). Cardiac troponin-T immunoassay for the

diagnosis of acute myocardial infarction. Clin. Chem. 40(6): 900-907

Yang D and Liu T. (2004). Study on the genetic diversity within lactate dehydrogenase of

Streptococcus mutans. Proceedings of the preliminary program for the 5th annual

meeting of the IADR Chinese Division.