Exploring the untapped potential: a systematic review of novel enzymes as biomarkers over the past 12 years

Introduction

Background

Biomarkers are any substances or molecules that can be detected or quantified in human clinical samples for disease diagnosis, prognosis, and monitoring of treatment responses. Biomarkers are also used to monitor the physiology and changes of various health-related parameters (1-3). In a clinical sample, an ideal biomarker should have a concentration that is constant in healthy populations, has a strong correlation to a specific disease or medical condition, is significantly increased or decreased in various disease states, and is directly measurable in easy-to-collect samples such as blood and other bodily fluids (4,5). Currently, many biomarkers have enabled clinicians to treat patients more safely and effectively (6). An example of biomarkers are enzymes, which are biological catalysts in various physiological and pathological processes within the human body (7). With high levels of substrate specificity and expected products always seen in its catalyzed reaction, enzymes have been conveniently measured in biological fluids by determining their activity, which is then related to their concentration (8,9). This enabled the enzymes to serve as discriminatory biomarkers since their leak from cells and tissues and the duration of their activity in biological fluids (e.g., serum) can give valuable information about the source and severity of cell and tissue damage (7,10). At present, the use of enzymes to diagnose diseases has intensified further since pancreatitis was associated with increased serum amylase in 1908 (11). Other notable examples were the association of increased serum alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT) to hepatobiliary diseases, and increased serum creatine kinase (CK) and aspartate aminotransferase (AST) to heart diseases and myopathies (12-14). Given these characteristics of enzymes, they are now widely used as one of the biomarkers routinely measured in clinical chemistry laboratories.

Rationale and knowledge gap

Many enzymes have been explored in the past, but some were less specific and less sensitive and, therefore, were replaced with more reliable non-enzymatic biomarkers (8). Thus, to date, not so many enzymes and their isotypes have been added to the list of diagnostics biomarkers. In addition, some enzyme determinations may require invasive sampling and tedious sample preparation and analysis; hence, they may not be suitable for accessible and routine testing. Therefore, more enzyme biomarkers, particularly in easy-to-collect biological fluids, may need to be studied for their potential in assessing organ function, diagnosing and prognosing various diseases, and monitoring treatment.

Objective

This review aims to summarize the novel enzymes that were studied as potential biomarkers in the past 12 years (from January 2012 to December 2023). This review also aims to explore the current research landscape (trends, methodologies, gaps, and updates) surrounding these novel enzymes and the progress of their application in clinical settings. If their determination is optimized and validated, they might become reliable biomarkers for diagnosis, prognosis, and therapeutic monitoring of various diseases and medical conditions. A potential addition to the list of biomarkers that can be routinely analyzed in clinical chemistry laboratories. We present this article in accordance with the PRISMA reporting checklist (available at https://jlpm.amegroups.org/article/view/10.21037/jlpm-24-2/rc).

Methods

Search strategy

Studies related to diagnostic tests by quantifying enzyme activity or concentration, published from January 2012 to December 2023, were systematically searched in PubMed. The search term used to find the potentially relevant studies was “enzyme activity”, which studies potential enzymes whose activity and concentration can be measured in blood and other bodily fluids for the assessment of organ function, diagnosis and prognosis of diseases, and treatment monitoring. In PubMed, the authors ticked the article types “clinical trial” and “randomized control trial” only.

The eligibility criteria and study selections

The inclusion criteria for selection in the current review was that any included studies must be original research or short communications about measuring enzyme activity or concentration in biological fluids for diagnosis, prognosis, and therapeutic monitoring of various medical conditions. The study design should be either clinical trial or randomized controlled trial. The exclusion criteria were (I) studies without full text, (II) studies without data of interest, and (III) editorials, review articles, correspondence, book chapters and reviews, conference info and abstract, encyclopedia, news, and discussion. The authors (A.J.P., B.C., and M.R.P.C.) screened the studies based on eligibility criteria to identify potentially relevant studies.

Data extraction

The data from each study were extracted as follows: author name, year of publication, enzyme studied, specimen used, method of measurement, organ or tissues assessed, associated disease or medical condition, and purposes (e.g., diagnoses, prognosis, or monitoring of therapeutic efficiency). After data extraction, the data were arranged into three broad categories according to (I) enzymes used as tumor markers (see Table 1); (II) enzymes used for assessing and monitoring of tissues, organs, and organ systems (see Table 2); and (III) enzymes used to assess other diseases or conditions not confined to a specific organ (see Table 3).

Results

Search results

The initial search yielded 3,255 records from PubMed (Figure 1). Out of those records, no duplicates were found. After screening the titles and abstracts of the 3,255 records, 3,110 records were excluded. All the articles from the remaining 145 records were retrieved. Following the evaluation of articles for eligibility, an additional 103 articles were excluded for reasons such as the studies were about therapeutic enzymes, genetic studies (e.g., polymorphism, mutations) of enzymes, animal (non-human) enzymes, or the studied enzyme has no significant association or correlation to the disease or medical condition of interest. Other records that were excluded were studies that used tissue sampling and genotyping methods only.

Figure 1 Study selection process shown by PRISMA 2020 flow diagram.

Characteristics of the included studies

A total of 42 articles were utilized in this review. The characteristics of the included studies are shown in Tables 1-3. Table 1 summarizes the 8 studies about enzymes as potential tumor or cancer markers (15-22). Among the 8 studies, 7 analyzed blood samples such as whole blood, serum, or plasma (16-22), and 1 analyzed a pleural fluid (15). Table 2 summarizes the 28 studies of enzymes as potential biomarkers for organ system function. Among the 28 studies, 20 analyzed blood samples, of which 10, 8, and 2 were serum (23,25,29,34-36,40,45,46,48), plasma (24,27,28,31,37-39,50), and whole blood (44,47) samples, respectively. In addition, eight studies analyzed other bodily fluids. Two of which were tear (32,33), and sputum samples (41,42), and one each was saliva (26), cerebrospinal fluid (CSF) (30), gingival crevicular transudate (43), and synovial fluid samples (49). Table 3 summarizes the six studies of enzymes as potential biomarkers of other medically related conditions not specified to an organ system (51-56). Among the 6 studies, 5 analyzed blood samples (51,52,54-56), while 1 analyzed a wound fluid (53). In the reports, most enzymes were measured in blood samples (35 out of 42 studies) since leaked enzymes from several damaged tissues and organs can readily reach the bloodstream in significant quantities (57). Regarding the methods of enzyme measurements in biological samples, enzyme activities or concentration were quantified in 23 studies using some basic techniques in analytic chemistry such as spectrometry, luminescence, and chromatography. Specifically, 12, 7, and 4 studies used spectrophotometry (17,20,22,23,26,37,39,41,46,49,52,55), fluorimetry (16,27,28,30,33,35,56), and column liquid or lateral flow chromatography (44,47,51,53), respectively. In addition, 16 studies used other methods such as immunoassays, of which 14 and 2 studies used enzyme (15,19,21,24,25,29,31,34,38,40,42,45,48,54) and luminescent labels (36,43), respectively. Finally, three more studies used other methods that incorporate immunoassay principles, such as flow cytometry (18), immunochromatography (32), and radioimmunoassay (50). It is also notable that based on the chemical reactions they catalyze, enzymes in these studies belong in the nomenclature of mostly hydrolases (16,18,20,23-25,27-32,35,37,38,40,42,43,45,46,48-50,53,55), followed by transferases (17,19,21,39,41,44,47,52), oxidoreductases (22,26,33,34,51,54), and lyases (15,36), with 25, 8, 6, and 2 studies, respectively. None of the included reports studied enzyme biomarkers that were classified as isomerases and ligases. Nonetheless, glucose-6-phosphate isomerase (GPI) deficiency has been associated with nonspherocytic hemolytic anemia (58). Moreover, although ligases are not commonly used as biomarkers, DNA ligases have applications in molecular biology and diagnostics, particularly in techniques like DNA amplification via ligase chain reaction (59).

Novel enzymes as tumor or cancer markers

Enzymes have been used as surrogate biomarkers for cancer diagnoses, monitoring of metastases, and evaluation of patient’s prognoses during cancer therapy (60). This review enumerated new enzymes that were studied for their potential use as tumor or cancer biomarkers using biological fluids as samples, specifically for tumors or cancers of the lung, head and neck, kidney, breast, liver, and white blood cells (see Table 1). First, some novel enzymes have shown diagnostic potential for certain malignancies (15,19,21,22). For example, Liu et al. showed that the concentration of carbonic anhydrase XII (CAXII) in pleural effusions was significantly higher amongst small cell lung cancer (SCLC) patients than in non-malignant (tuberculosis) patients. Thus, the enzyme can be used as a surrogate biomarker for cytological examinations, especially if the diagnosis is inconclusive (15). In addition, Wu et al. showed that the thioredoxin reductase (TrxR) activity in plasma/serum was significantly higher among primary liver cancer patients than healthy controls. It was even found to be more discriminating than known tumor markers such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and cancer antigen 19-9 (CA19-9) (22). Second, some novel enzymes have shown prognostic potential, as they can predict patient outcomes such as survival rates for certain cancers (16-19,21). For example, John et al. showed that the activity of caspase-3/7 in the serum of patients with squamous cell cancer of the head and neck (SCCHN) was significantly higher for those with short progression-free survival (PFS) than those with longer PFS (16). In addition, Takemura et al. showed that the activity of GGT in the serum of patients with advanced urothelial carcinoma (aUC) was significantly higher for those with shorter overall survival (OS) than those with longer OS (17). Finally, El-Sisi et al. showed that the concentration of focal adhesion kinase (FAK), protooncogene tyrosine kinase SRC (Src), and protein kinase C (PKC) were significantly higher among acute myeloid leukemia (AML) patients with shorter OS (19). In short, the concentration or activity of caspase-3, GGT, FAK, Src, and PKC in blood and/or pleural fluid were all shown to be inversely correlated to patient survival. Lastly, some novel enzymes have also shown potential for monitoring therapeutic efficiency for certain cancers (16,18,20,22). For example, Spiess et al. showed that the activity of the enzyme separase in white blood cells (WBC) of tyrosine kinase inhibitor (TKI) treated chronic myelogenous leukemia (CML) patients was found to positively correlate with the enhanced proliferation of hematopoietic cells (18). Moreover, Gheler et al. showed that the activity of CD73 (ecto-5'-nucleotidase) can be used for treatment monitoring of patients with breast cancer as its activity in plasma is reduced with chemotherapy, radiotherapy, hormone therapy, or surgery (20). Therefore, enzyme activities and concentration in biological fluids, in conjunction with other methods, can help enhance the accuracy of diagnosis, prognosis, and monitoring of therapeutic efficiency for tumors and cancers.

Novel enzyme biomarkers for the assessment of organ system function

Enzymes have been used as biomarkers for assessing various tissues, organs, and organ systems. This review enumerated novel enzymes that were studied for their potential use as biomarkers and some well-established enzyme biomarkers with their newfound clinical association (see Table 2). This includes enzymes for the evaluation of diseases of the endocrine system, such as diabetes mellitus (24); nervous system, such as intracerebral hemorrhage, hypoxic-ischemic encephalopathy, cerebral infarction, embolic stroke, and Alzheimer’s disease (AD) (25-31); visual system such as the dry eye disease and keratoconus (32,33); circulatory system, such as acute coronary syndrome (ACS), acute myocardial infarction (AMI), and paroxysmal atrial fibrillation (34-40); respiratory system, such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) (41,42); digestive system, such as inflammatory bowel disease (IBD) and hemorrhoidal disease (43-45); integumentary system such as recurrent aphthous stomatitis and psoriasis (46,47); musculoskeletal system such as rheumatoid arthritis, and osteoarthritis (48,49); and the genitourinary system such as renal impairment (50).

First, some novel enzymes have shown diagnostic potential for certain life-threatening cardiovascular conditions. For example, Qu et al. showed that the activity of ADAMTS13 (a disintegrin and a metalloproteinase with a thrombospondin type I motif, member 13) is reduced in patients with thrombosis and cerebral infarction (27). In addition, Yamac et al. showed that the activity of aspartic lysosomal endopeptidase cathepsin (CatD) was significantly higher in patients with AMI than their age-matched healthy controls (35). Second, in addition to diagnostic potential, some novel enzymes have shown a prognostic potential for specific cardiovascular conditions. Here are some examples: Sajeev et al. showed that the elevated activity of angiotensin-converting enzyme 2 (ACE2) is associated with an increased risk for embolic stroke of undetermined source (ESUS) (28); Stammet et al. showed that the elevated concentration of neuron-specific enolase (NSE) is associated with an increased risk for poor neurological outcome and death after a patient’s out-of-hospital cardiac arrest (36); and, Wallentin et al. showed that the elevated activity of lipoprotein-associated phospholipase A2 (Lp-PLA2) is associated with the risk of cardiovascular death, myocardial infarction, or stroke (37). Furthermore, some enzyme assays have shown diagnostic potential for the diagnosis of neurodegenerative diseases such as AD. For example, Watabe-Rudolph et al. showed that chitinase activity was significantly higher in AD patients’ CSF than in control patients (30). In addition, Wu et al. 2021 showed that the activity of β-Secretase was also significantly higher in AD patients than in controls (31). Lastly, some novel enzymes have shown potential for predicting responses to therapeutic interventions. For example, Corti et al. showed that the resupply of glutathione by inhalation to alleviate lung inflammation may further aggravate airway damage in CF patients with increased GGT in sputum. Thus, measuring GGT may help discriminate CF patients who would more likely benefit from inhaled glutathione (41). In addition, Coenen et al. showed that screening for IBD patients who carry a lower thiopurine S-methyltransferase (TPMT) activity in their whole blood and received a lesser (≤50%) thiopurine dose (e.g., azathioprine or 6-mercaptopurine) led to a 10-fold reduction in hematologic adverse reactions such as leukopenia, compared with the same group who were treated according to standard IBD guidelines (44). In other words, exploring the ability of the enzymes to predict pharmacologic responses to a therapeutic intervention may reduce adverse drug reactions, thus improving favorable therapeutic outcomes.

Novel enzyme biomarkers for the assessment of other diseases

Novel enzyme biomarkers have also been used to assess other diseases or medical conditions not specified to an organ (see Table 3). This includes enzymes for the evaluation of conditions related to infectious diseases such as tuberculosis and dengue (51-53); sleep-wake disorders such as sleep-disordered breathing (SDB) (54), and symptoms, signs, or clinical findings, not elsewhere classified, such as systemic inflammation, poor physical function, and frailty (55,56). First, some novel enzymes in blood samples have shown diagnostic potential for age-related syndromes. For example, Sanz et al. showed that higher ACE2 activity was associated with increased frailty (susceptibility to minor stressors, which may increase the risk of hospitalization and dependence) among older people living in nursing homes. Second, some novel enzymes have shown a prognostic potential for infectious diseases. For example, Senaratne et al. showed that established enzyme biomarkers for liver diseases such as AST, ALT, and AST/ALT ratio were also significantly higher in dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS) patients relative to dengue fever (DF) patients (52). Therefore, it has prognostic potential for predicting dengue severity and its outcomes in addition to definitive molecular and serological markers (52,61,62). Third, some novel enzymes have shown prognostic potential for conditions relating to surgical injury. Loffredo et al. have shown that the elevated activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is associated with endothelial dysfunction in children with SDB, such as primary snoring and obstructive sleep apnea, relative to healthy controls (54). In addition, Zivkovic et al. have shown that butyrylcholinesterase (BChE) activity changes (decreases) earlier in patients with systemic inflammation during major surgery when compared to changes (increases) in the concentration of an established acute phase reactant, C-reactive protein (CRP) (55). This shows that BChE levels can potentially facilitate the earlier detection of systemic inflammation. Lastly, some novel enzymes can also be used for determining therapeutic intervention responses. For example, Tanner et al. showed that indoleamine 2,3-dioxygenase (IDO) can be used to monitor the immunogenicity of a vaccine for Mycobacterium tuberculosis (51). In other words, exploring the unusual uses of enzyme biomarkers, such as assessing frailty, generalized inflammation, and vaccine immunogenicity, could be a potential direction for discovering novel enzyme biomarkers.

Other novel enzymes biomarkers and their applications

In this systematic review, some excluded studies were also examined as they have essential information for suggesting needed actions in the later discussion. Some of the novel enzymes used as biomarkers for tumors/cancer and tissues/organs require biopsies, surgically resected tissues, and tissue scrapings. Most of the specimens were also analyzed using immunohistochemistry (IHC), fluorescence in-situ hybridization (FISH), real-time polymerase chain reaction (Q-PCR), western blots, and complementary DNA (cDNA) microarray, which are usually done in histopathology, molecular diagnostics, or cytogenetics sections of pathology laboratories (63-75). See Tables S1,S2.

Discussion

Key findings

As pointed out in the results, some remarkable findings about select enzyme biomarkers are as follows. First, some enzymes have better discriminating potential than some established non-enzyme biomarkers for certain tumors (21,22). Second, some enzymes can potentially facilitate earlier detection of certain conditions (e.g., inflammation) than some well-established acute phase biomarkers (55). Although these stated first two potentials were also the grounds why many enzymes were superseded or replaced by non-enzyme biomarkers (8,76,77). Third, some enzymes have the potential to diagnose signs and symptoms that do not fit into specific categories of diseases or conditions, such as poor physical function, higher dependence, and frailty (56). Fourth, some enzymes (amongst non-drug-metabolizing enzymes) can potentially predict pharmacologic responses to therapeutic intervention to reduce adverse drug reactions and identify patients who would more likely benefit from therapeutic intervention (41,44,78). Fifth, some enzymes have the potential to determine the severity of an infectious disease and the effectiveness of vaccination against certain infectious diseases (51,79). Lastly, among the selected articles, the concentration of most novel enzymes was measured by methods that incorporate immunoassay principles, and they belong mostly to the nomenclature of hydrolases.

Strengths and limitations

To our knowledge, this study was the first review of potential enzyme biomarkers that have been studied through randomized controlled trials or clinical trials in the past 12 years. This type of information is essential since the number of clinically significant enzymes in the menus of clinical chemistry laboratories has remained stagnant for years (8). Therefore, more novel enzyme biomarkers must be uncovered and validated so that they can be introduced in the future menus of clinical chemistry laboratories. In this review, the enzyme biomarkers were also grouped and sorted according to the medical classification of diseases and medical conditions they aimed to assess (80). However, there are limitations. First, the extracted data was derived from a single abstract database, which is PubMed. This was the only abstract database selected, as it also contains medical biochemistry journals indexed in Scopus and Web of Science but with an emphasis on research articles about biomarkers that have undergone randomized control trials or clinical trials. The relevant literature obtained from this search may not be exhaustive; therefore, a search in other biomedical and life science-focused databases that do not overlap with PubMed is encouraged to gather more information. Second, relevant enzymes analyzed only with techniques associated more with molecular biology, immunogenetics, and immunohistology were excluded (63-75). PCR and western blotting often require a specialized and higher level of technical skills and are time-consuming, which could limit their accessibility and feasibility in routine clinical practice. Regardless, Tables S1,S2 were shown to be discussed for the later part of “implications and actions needed”. Third, this systematic review highlighted only novel enzymes; thus, non-enzyme biomarkers studied along or in adjunct with the enzyme were excluded. Lastly, this review reported only the enzymes with a statistically significant correlation or association with a disease or medical condition.

Comparison with similar researches

Several review articles focused only on a specific group of diagnostic enzymes. For example, Lioudaki et al. reviewed only the routinely analyzed liver enzymes (ALT, AST and GGT activity) and their potential use as cardiovascular risk markers (81); Cecerska-Heryć et al., Marrocco et al., Yang et al. reviewed only the antioxidant enzymes (e.g., glutathione reductase, glutathione peroxidase, etc.) for the diagnosis and monitoring of oxidative stress, cancer and other diseases (82-84); Brancaccio et al. focused only on enzyme markers of muscular damage and stress (e.g., creatine kinase, lactate dehydrogenase) that affects both metabolic and mechanical factors (85); and Kimura et al. focused only on new biomarkers for cardiovascular diseases (86). On the other hand, this current review is different as it seeks to include various enzyme biomarkers for diverse sets of diseases and medical conditions using patient samples (e.g., blood and bodily fluids) and methods (e.g., spectrophotometry, immunoassays, etc.) that can be routinely done in a clinical chemistry laboratory.

Explanations of findings

The first notable finding is that some enzymes were more discriminating for certain tumors and diseases. This is possible since some enzymes can be selectively overexpressed or under expressed in tumor microenvironments through disturbances in allosteric regulation, molecular interactions (e.g., substrate binding and catalysis), and metabolism (e.g., oxygen and nutrient availability) (22,66,87). In addition, some tumors have been associated to genetic mutations or variations, which also led to the production of variant enzymes with altered function and catalytic activity (66,67). The second notable finding is that some enzyme biomarkers can facilitate earlier disease detection (55). This is possible since some enzymes exhibits rapid and detectable changes (e.g., concentration, activity, etc.) in blood and body fluids following a minor tissue damage, inflammation, and other small changes in physical conditions (55,88,89). This could be an early sign of a developing disease, condition, or disease remission (even before a distinct clinical symptom appears), thus allowing early medical intervention. The third notable finding is that some enzymes associated to drug metabolism (other than cytochrome P450s) can help identify patient subpopulations who are more likely to respond favorably to a particular therapeutic drug, thus making them as predictive biomarkers that enables adjustment or discontinuation of drug therapy for prevention of adverse effects (41,44). Enzymes involved in the biochemical pathways are often the target of therapeutic drugs and may vary in concentration and activity; therefore, measuring the enzyme activity or levels can assess a patient’s likely response from a particular drug whether a drug is effectively engaging its target (90,91). This personalized approach to drug therapy can help optimize treatment outcomes and improve patient safety. The fourth notable finding is that most analytical methods applied for the enzymes incorporate immunoassay principles. This is likely since immunoassays can be designed to selectively determine the concentration of enzymes and their isoforms in biological samples. This has an advantage over the determination of enzyme activity, as the rate of product formation can be contributed by multiple enzymes with similar catalytic activities, thus potentially affecting specificity (92,93). Moreover, automated immunoassays in clinical settings can have a high throughput with short turnaround times for routine diagnostic testing. The last notable finding is that most enzymes measured were hydrolases. This could be due to hydrolases being ubiquitous in the cellular processes of tissues, such as in metabolism and the breakdown of macromolecules (94). In addition, assays for hydrolase activity can be simple in that they may not require cofactors (e.g., NADH, NADPH, etc.), auxiliary enzymes, and indicator enzymes as components of its reagents (95).

Implications and actions needed

As previously discussed, the enumerated enzyme biomarkers have diagnostic potential. Therefore, novel enzyme biomarkers for other disease classifications (not seen in this review) are potential prospects for further exploration. This includes nutritional or metabolic diseases; diseases of the blood-forming organs (e.g., spleen), the immune system (e.g., thymus), and connective tissue (e.g., ligaments, cartilage, etc.); and diseases related to sexual health, pregnancy, childbirth, and developmental anomalies (80). In addition, other potential prospects for enzyme biomarkers are for monitoring of pregnancy, puberty, and other physiological changes (96); diagnosis of senile weakness, mental frailty, and other age-related physical debilities (56); determination of vaccine immunogenicity and efficiency (51); determination of risks for future adverse medical conditions (97); monitoring of recovery from various infections or non-infectious diseases (93); diagnosis of potentially treatable genetic disease (98); and, discovery of enzyme biomarkers and their isoenzymes that are less affected by artifactual causes such as posture and physiologic activity (99). Furthermore, correlating these novel enzymes to other non-enzyme biomarkers and other laboratory tests, such as medical imaging, needs reinforcing studies (100). For the established enzyme biomarkers, their usefulness in diagnostics may need to be further elucidated. For example, ALT and alkaline phosphatase (ALP), a biomarker for liver diseases, were assessed as potential biomarkers for cardiovascular diseases (101-103). Therefore, the same course of studies could be applied to other conventional enzyme biomarkers. With a solid direction for exploring enzyme biomarkers, we are now moving on to specimens used in discovering enzyme biomarkers. As stated earlier, some excluded studies for this review explored specimens such as biopsies and surgically resected tissues along with analytical methods (e.g., PCR, IHC, etc.) not usually done in clinical chemistry laboratories (63-75). See Tables S1,S2. Therefore, this review focused on studies that sampled blood and other bodily fluids due to certain advantages. Biological fluids can be non-invasive (e.g., midstream clean catch urine, saliva, etc.) or minimally invasive (e.g., venipuncture) to collect, with fewer complications (e.g., less damage to surrounding tissues); may not necessitate hospitalization; require fewer resources; and, can be tolerated more by patients for repeated sampling over time (104). This is especially important for conditions requiring ongoing management or improving access to screening of at-risk populations. However, it is essential to note that biopsies and surgically resected tissues are necessary, especially when specific tissue characteristics or localized information are required (105). Moving on to the methodologies, this review also focused more on studies that measured enzyme activity or concentration via spectrophotometry and immunoassays in biological fluids. Spectrophotometric methods, in particular, aside from being simpler and faster, were already incorporated into discrete analyzers for an efficient, high-throughput, and simultaneous analysis of large number of samples in a relatively short time (9). This is advantageous when a broad range of other biomarkers in the blood (e.g., proteins, lipids, carbohydrates, non-protein nitrogenous compounds, etc.) is requested for the patient. Furthermore, in many modern clinical laboratories, spectrophotometric methods require only micro volumes of fluid samples, thus further allowing subsequent analyses from the remaining samples (106,107). In contrast, FISH, Q-PCR, and cDNA microarray comes with more technological and organizational limitations that restrict their widespread implementation in resource-limited healthcare settings. First, they require specialized equipment, reagents, and consumables. FISH requires a fluorescence microscope and probe design, Q-PCR requires a thermal cycler and amplification protocols, and cDNA microarray requires microarray platforms and complex data analysis (108). Second, their operation requires laboratories with controlled environments for sample preparation, instrument calibration, and data analysis. Establishing and maintaining such facilities requires considerable investment and continuing resources. Third, they require longer turnaround times compared to spectrophotometric tests. Sample processing, amplification, hybridization, and data analysis can take hours to days to complete, regardless of the workload in the laboratory. Lastly, recruitment, training, and retention of experienced clinical laboratory scientists and technologists are necessary for the reliable execution of assays and interpretation of results (108,109). All these technological and organizational factors can contribute to their high cost per test, thus affecting their accessibility and utility in everyday diagnostic laboratory practice. Regardless, it is essential to note that molecular techniques like PCR and western blotting is used in specialized laboratories for genotyping and determination of protein expression for a more comprehensive understanding of the underlying pathology associated with the enzyme (63-75). Therefore, with all things considered, determining enzyme activity or concentration via spectrophotometry or immunoassays using biological fluids as samples are potential prospects for those enzymes that were initially studied using biopsies and surgically resected tissues.

Conclusions

This review paper has shown the many opportunities of clinical enzymology and how “novel” enzymes can serve as potential markers for diagnosis, prognosis, and therapy monitoring. As the recent advances in the multi-omics landscape and the ongoing collaboration among researchers, clinicians, and industry stakeholders (e.g., pharmaceutical companies, clinical laboratories, etc.) continually reveal the complex roles enzymes play in diverse diseases, the integration and recalibration of this “novel” enzymes into routine clinical practice (e.g., diagnostic and therapeutic algorithms) holds promise for more accurate, timely, and personalized treatment strategies (110,111). This will significantly improve the accuracy of diagnosing diseases and pave the way for clinicians to treat patients more effectively. The challenge now is to perform rigorous and large-scale validation of these “novel” enzyme biomarkers, considering the changes in their concentration, cutoff values, and diagnostic interpretation in diverse patient populations, sexes, and age groups before they are introduced into routine clinical care (112-114).

Acknowledgments

We would like to thank our advisers from the Department of Medical Technology, Institute of Health Sciences and Nursing, Far Eastern University for their guidance and support to complete this review.

Funding: None.

Footnote

Reporting Checklist: The authors have completed the PRISMA reporting checklist. Available at https://jlpm.amegroups.org/article/view/10.21037/jlpm-24-2/rc

Peer Review File: Available at https://jlpm.amegroups.org/article/view/10.21037/jlpm-24-2/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jlpm.amegroups.org/article/view/10.21037/jlpm-24-2/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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