Simplified and economic measurement of glyoxalase I activity using 2,4-dinitrophenylhydrazine: A valuable tool for researchers

Abstract

Glyoxalase I (Glo I) is an enzyme essential for detoxifying methylglyoxal, a toxic compound associated with advanced glycation end products. Given Glo I’s multifaceted roles in various physiological and pathological processes, accurately measuring its activity is crucial for understanding its implications in metabolic disorders. The current assay utilizes 2,4-dinitrophenylhydrazine (2,4-DNPH) to measure Glo I activity. This reagent has previously been employed to evaluate a group of enzyme protocols. The procedure involves incubating Glo I enzyme samples in a controlled phosphate buffer at pH 6.6, optimizing conditions for enzymatic activity. Glutathione and methylglyoxal serve as substrates, with Glo I catalyzing the conversion of the hemithioacetal adduct into S-D-lactoylglutathione. Unreacted methylglyoxal is quantified by forming a colored hydrazone complex with 2,4-DNPH. The 2,4-DNPH method is rigorously validated for linearity, stability, resistance to interference, and sensitivity from several chemicals. It strongly correlates with the existing ultraviolet method, offering enhanced simplicity and cost-effectiveness. The protocol allows precise quantification of Glo I activity, with potential in research and diagnostics. Intra- and inter-day analyses confirm accuracy as percentage relative error, ensuring reliable measurement activity. The DNPH-Glo I method exhibited excellent sensitivity, with low limits of detection and quantification at 0.006 U/L and 0.018 U/L, respectively. This research provides valuable insights into the quantification of Glo I, highlighting significant implications for future studies in metabolic disorders and related health fields. This study contributes to a deeper understanding of its role in health and disease management by advancing the methods available for measuring Glo I activity.

Introduction

Glyoxalase I (Glo I) stands as a crucial enzymatic defender against methylglyoxal (MG), a reactive oncometabolite generated during energy metabolism, particularly under conditions of elevated glycolytic flux [1]. This enzyme operates within the broader glyoxalase system, which orchestrates the conversion of MG to D-lactate through a two-step process. In the initial phase, Glo I catalyzes MG’s glutathione (GSH)-dependent transformation into S-D-lactoylglutathione (SLG). Subsequently, glyoxalase 2 facilitates the hydrolysis of this intermediate compound to D-lactate, simultaneously regenerating the GSH consumed in the first step [2]. As the rate-limiting enzyme in this detoxification pathway, Glo I is pivotal in preventing the formation of advanced glycation end products by efficiently metabolizing MG [3]. When Glo I function becomes impaired, it triggers carbonyl stress, leading to advanced glycation end-product accumulation in brain tissue [4, 5]. This positioning makes Glo I a central regulator in maintaining the delicate balance between cellular detoxification and toxification of reactive carbonyl species [6], with implications for various pathological conditions, including schizophrenia [7].

Metabolic disorders like obesity and diabetes show that increased MG formation correlates with glyceroneogenesis, hyperglycemia, and reduced Glo I expression. Enhancing Glo I expression can reduce dicarbonyl stress, slow obesity progression, cut insulin resistance, and prevent diabetic nephropathy. This led to Glo I inducers, such as trans-resveratrol and hesperetin, effectively improving glycemic control and reducing vascular inflammation in obese individuals [8]. Dicarbonyl stress, marked by excess dicarbonyl metabolites, leads to cellular dysfunction in aging and disease [9]. Typically, the glyoxalase system manages MG, but diabetes can overwhelm this system, causing MG toxicity [10, 11].

There is growing interest in the glyoxalase system for interventions like MG reduction through arginine or pyridoxamine and upregulating the glyoxalase system with isothiocyanate [12]. However, more studies are needed to determine how well Glo I detoxifies MG during human inflammation or hypoxia [13, 14]. Glo I protects against glycation damage in cancer biology, but elevated activity in cancer cells boosts growth and chemotherapy resistance. Yet, suppressing Glo I can induce apoptosis via MG, highlighting its potential as a cancer therapy target, especially for cancer stem cells. Additionally, research reveals a link between angiotensin II and MG formation, emphasizing the Glo I/GSH system’s role in vascular health. Evidence shows MG’s role in metabolic, psychiatric, and neurodegenerative disorders. Many findings from rodent studies suggest MG significantly contributes to Alzheimer’s, Parkinson’s, depression, anxiety, and age-related conditions [15].

The evolving understanding of Glo I has catalyzed the development of targeted therapeutic strategies, including inhibitors for cancer treatment and activators for managing diabetic complications. Glo I levels are promising biomarkers for disease progression and treatment response across various conditions. Moreover, its protective role against oxidative stress positions Glo I as a key target in developing interventions for age-related diseases and promoting cellular health [9].

The pivotal role of Glo I in cellular detoxification necessitates precise and reproducible methods for measuring its enzymatic activity, both for research purposes and potential clinical applications [16]. Currently, researchers employ several well-established analytical protocols to quantify Glo I activity. The primary methodological protocols include spectrophotometric analysis, fluorometric assays, and enzyme-linked immunosorbent assays. Each method offers distinct advantages regarding sensitivity, specificity, and practical application, allowing researchers to select the most appropriate method based on their specific experimental requirements and available resources.

Many protocols have been established to measure Glo1 activity in different samples, including enzyme preparations and biological specimens. The most commonly used method is a spectrophotometric assay that quantifies the production of SLG from MG and reduced GSH [17]. The assay detects an increase in absorbance at 240 nm, which reflects the formation of SLG. The process typically involves incubating MG and GSH to produce the hemithioacetal and adding a cell extract or sample containing Glo I. The change in absorbance at 240 nm is then monitored over a set period. A previous study measured Glo I by incubating the Glo I enzyme with GSH and MG, forming SLG [18]. This compound is then converted to lactic acid by adding sulfuric acid, which is oxidized to acetaldehyde using copper. Finally, acetaldehyde reacts with p-phenylphenol in sulfuric acid, creating a colored complex that absorbs light at 570 nm.

An alternative approach to measuring Glo I activity is monitoring changes in fluorescence. This technique tracks shifts in fluorescence intensity to assess Glo I activity [19]. The protocol for evaluating Glo I activity utilizes a novel near-infrared (NIR) fluorescent probe known as MEBTD, specifically designed to detect MG, a substrate of Glo I. This probe demonstrates exceptional selectivity for MG, with a detection limit of 18 nM and a remarkable 131-fold increase in fluorescence upon binding to MG.

The present study introduces a novel application to dinitrophenylhydrazine (DNPH) chemistry for assessing Glo I activity. Given Glo I’s multifaceted roles in various physiological and pathological processes, accurately measuring its activity is crucial for understanding its implications in metabolic disorders. This study presents a novel method for assessing Glo I activity using 2,4-dinitrophenylhydrazine (2,4-DNPH). The assay involves incubating Glo I enzyme samples in a phosphate buffer (pH of 6.6), which is optimal for enzymatic activity. The substrates for the reaction are GSH and MG, which Glo I converts into SLG. After the reaction, we quantify the unreacted MG by forming a colored hydrazone adduct with 2,4-DNPH. Specifically, the assay uses 2,4-DNPH to determine the activity of Glo I, with enzyme samples incubated in a controlled phosphate buffer at pH 6.6, which is ideal for the enzyme’s function. The GSH and MG substrates react with Glo I to produce SLG. In contrast, any unreacted MG subsequently forms a colored hydrazone complex with 2,4-DNPH, allowing us to quantify it effectively. In 1926, Oscar L. Brady introduced the DNPH reagent, demonstrating its ability to identify carbonyl compounds by forming colored products called dinitrophenylhydrazones [20]. When carbonyl groups from aldehydes and ketones react with DNPH, they produce a yellow, orange, or red adduct, depending on the carbonyl compound’s structure [21]. This reagent was also employed to evaluate enzymes such as monoamine oxidase, aspartate aminotransferase (formerly glutamic oxaloacetic transaminase), and alanine aminotransferase (formerly serum glutamic pyruvic transaminase) [22, 23]. DNPH is considered cost-effective as it is a relatively affordable reagent for detecting carbonyl groups in aldehydes and ketones [24]. The process is straightforward, allowing easy application in a laboratory environment. When DNPH reacts with carbonyl compounds, hydrazone derivatives form, which can be readily quantified using a spectrophotometer [21]. This technique requires only inexpensive equipment, such as a single or double-beam spectrophotometer or a plate reader, ensuring it is accessible for regular laboratory use [23].

Procedure

Chemicals

MG was purchased from Shanghai Macklin Biochemical Co., Ltd. Sodium phosphate dibasic [(Na₂HPO₄)], Potassium phosphate monobasic[(KH₂PO₄)], 2,4-DNPH, hydrochloric acid (HCl), sodium hydroxide (NaOH), and ethanol were obtained from Thomas Baker (Chemicals) Pvt. Ltd.

Reagents

To prepare a 50 mM phosphate buffer with a pH of 6.6, 20.214 g of sodium hydrogen phosphate heptahydrate (Na₂HPO₄.7H₂O) and 3.394 g of sodium dihydrogen phosphate monohydrate (NaH₂PO₄·H₂O) are dissolved in approximately 800 mL of distilled water. Once the salts are fully dissolved, the pH of the solution is adjusted to 6.6 by slowly adding 1 M HCl or 1 M NaOH while continuously monitoring the pH with a calibrated pH meter. The acid or base is added dropwise near the target pH to avoid overshooting. After the desired pH is achieved, the total volume of the solution is brought to 1 liter by adding distilled water.

Next, a 2 mM MG solution is prepared by carefully pipetting 17 µL of MG. This compound is dissolved in 100 mL of distilled water, and complete dissolution is ensured by gently stirring or shaking the solution. A GSH substrate solution was prepared by dissolving 61.46 mg in 100 mL of the previously prepared phosphate buffer. The compound is ensured to dissolve entirely before use.

To prepare the 2,4-DNPH color reagent, a stock solution was first prepared by dissolving 100 mg of 2,4-DNPH in 100 mL of a solvent mixture containing 4 mL of HCl, 46 mL of distilled water, and 50 mL of ethanol. The solution was mixed thoroughly to ensure homogeneity. A working solution was then prepared by diluting 10 mL of the stock solution with 90 mL of the same solvent mixture. After mixing, the solution was stored in a brown bottle to protect it from light and maintain stability.

Finally, a 1 M NaOH solution is prepared by carefully dissolving 4 grams of NaOH pellets in 100 mL of distilled water.

Instrument

A Shimadzu UV-visible spectrophotometer model 1800 measured UV-visible spectra using matched 1 cm quartz cells, while a Spectramax 340 Plus UV-Vis plate reader performed the 96-well plate measurements.

Source of Glo I

Male albino rats sourced from the University of Babylon’s animal facility were utilized to prepare a tissue homogenate solution. After the rats were humanely euthanized, a surgical procedure was performed to harvest the organs carefully. The excised organs were then immersed in a 0.9% (w/v) sodium chloride solution to preserve their structural integrity. Animal tissue samples were preserved at −80°C for later analysis. The tissue homogenization protocol was adapted from Skapare et al. [25]. The process involved homogenizing the tissue in cold sodium phosphate buffer (50 mM, pH 7.4) at a 1:10 weight-to-volume ratio using a 20 kHz ultrasonic processor for 30 seconds. After centrifugation at 20,000 g for 10 minutes at +4°C, the supernatant was extracted for Glo I activity measurements. Protein content was quantified using the Lowry method.

Ethics approval

College of Science Ethics Committee, University of Babylon, Iraq; Ref. No. 2217C, dated October 27, 2023.

The method for measuring the activity of the Glo I enzyme is detailed in Table 1.

Table 1.

Detailed steps for the Glo I enzyme activity protocol. The table clearly outlines the steps involved in the Glo I enzyme activity protocol. Each step ensures accurate measurements and optimal enzyme performance for successful experimental outcomes.

Reagents	Test (µL)	Control (µL)	STD (µL)	Blank (µL)
Sodium phosphate buffer (pH 6.6)	50	550	540	590
GSH solution	10	100	100	100
The test tube contents were mixed by vortexing, followed by a 10-minute incubation at 37°C. Then, add:
Sample	40	40
The test tube contents were mixed by vortexing, followed by a 10-minute incubation at 37°C.
MG	50		50

Notes: The mixed test tube was incubated for 10 minutes at 37°C, and the enzymatic reaction was stopped by adding 1 mL of DNPH solution. Following a 20-minute incubation at 37°C, the solutions were further treated by adding 1 ml of 1 N NaOH to each tube. The tubes were then allowed to sit at room temperature for a minimum of 30 minutes before measuring the absorbance at 480 nm.

Calculation

The activity of the Glo I enzyme was quantified using the current assay and expressed in International Units (IU). One IU is defined as the quantity of Glo I that catalyzes the consumption of one micromole of MG per minute per liter of sample at a temperature of 37°C. To determine the activity in each sample, the following formula was employed:

The residual MG concentration in each test tube was calculated using the following equation:

$$A\mathrm{ }\mathit{corrected}\mathrm{\hspace{0.17em}}=\mathrm{\hspace{0.17em}}\mathit{ATest}\mathrm{\hspace{0.17em}}-\mathrm{\hspace{0.17em}}\mathit{AControL}$$ (1)

 

$$\mathit{Residual}\mathrm{ }MG\mathrm{ }\left(\frac{\mathrm{\mu }\mathit{mole}}{L}\right)=\frac{A\mathrm{ }\mathit{Corrected}}{A\mathrm{ }\mathit{Standard}}\mathrm{\hspace{0.17em}}\mathrm{*}\mathrm{\hspace{0.17em}}\mathit{Concentration}\mathrm{ }of\mathrm{ }\mathit{Standard}$$ (2)

Glo I activity was calculated using the following equation:

$$\mathit{Glo}\mathrm{ }I\mathrm{ }\mathit{activity}\mathrm{ }\left(\frac{U}{L}\right)=\left[\frac{\mathit{Concentration}\mathrm{ }of\mathrm{ }MG\mathrm{ }in\mathrm{ }\mathit{STD}-\mathrm{ }\mathit{Concentration}\mathrm{ }of\mathrm{ }MG\mathrm{ }in\mathrm{ }\mathit{test}\mathrm{ }\mathit{tube}\mathrm{ }}{\mathit{Incubation}\mathrm{ }\mathit{time}}\right]\mathrm{*}\left[\frac{\mathit{Total}\mathrm{ }\mathit{Volum}\mathrm{e}\mathrm{ }}{\mathit{Volume}\mathrm{ }of\mathrm{ }\mathit{Sample}}\right]\mathrm{\hspace{0.17em}}\mathrm{*}\mathrm{\hspace{0.17em}}D.F.\mathrm{ }$$ (3)

D.F.: The dilution factor.

Glo I activity could be determined from a standard curve (Fig. 1) generated using a MG stock solution and the DNPH-Glo I method. This standard curve plotted absorbance at 480 nm against known lactate concentrations, allowing for interpolating unknown sample concentrations from their absorbance readings. The procedure for generating this curve followed the same assay protocol as the sample measurements. The standard curve visually represented the relationship between absorbance and lactate concentration, facilitating the determination of Glo I activity from the test sample’s absorbance.

Figure 1.

The MG standard curve for the Glo I method

Reference method

A reaction mixture for Glo I activity measurement was prepared in a 1 mL cuvette, combining 500 μL of sodium phosphate buffer (100 mM, pH 6.6, pre-warmed to 37°C), 100 μL of reduced GSH solution (20 mM GSH), and 100 microliters of MG solution (20 mM MG) [17]. The mixture was then diluted with 280 μL of deionized water. After a 10-minute incubation period at 37°C, 20 μL of the sample was added to the cuvette, and the absorbance at 240 nm (A₂₄₀) was continuously monitored for 5 minutes. The initial rate of change in A₂₄₀ (dA₂₄₀/dt)₀ was calculated using a linear regression of the data points obtained during the first minute of the reaction. The Glo I activity (units) was then determined using the equation: Glo I activity = (dA₂₄₀/dt)₀/2.86.

Method validation

Linearity and sensitivity

The DNPH-Glo I method was thoroughly evaluated for its sensitivity and linearity by testing its performance over a wide range of Glo I enzymatic activities, covering the spectrum from 0.05 to 450 units per liter (U/L). A detailed comparative analysis was performed against the established ultraviolet assay to assess the method’s linearity. This comparison utilized linear regression analysis, conducted with specialized statistical software, to quantitatively determine the degree of agreement between the two methods. Additionally, the data were processed and visualized using a commercially available software package designed for advanced data analysis and graphical representation. The method’s sensitivity was further characterized by calculating both the limits of detection (LOD) and the limits of quantitation (LOQ), which provide precise thresholds for the smallest measurable and reliably quantifiable Glo I activity, respectively [26]. This thorough evaluation confirmed that the DNPH-Glo I method offers reliable measurements of Glo I activity across a wide concentration range, demonstrating its robustness and applicability for various experimental conditions.

The LOD and LOQ for the Glo I colorimetric method were established using the calibration curve’s intercept and the standard deviation of blank measurements. The equations used for these calculations are:

LOQ = yB + 10SB and LOD = yB + 3SB, where yB is the calibration curve’s intercept, and SB is the standard deviation of the blank. These equations provide a statistically reliable means to define the method’s sensitivity. LOD indicates the lowest detectable concentration, and LOQ represents the lowest concentration that can be accurately and precisely quantified [26].

Precision, or the method’s repeatability, was assessed by analyzing samples obtained from animal tissues under intraday and inter-day conditions. The relative standard deviation (RSD%) was calculated as the standard deviation divided by the mean multiplied by 100. Low RSD% values indicate that the method is precise and reproducible. Accuracy, or the method’s trueness, was evaluated by calculating the percentage relative error (RE%) using the formula: %RE = [(measured activity—known activity)/known activity] × 100. This metric quantifies the deviation of the measured Glo I activity from the known spiked concentrations. Accuracy was evaluated at both inter-day and intraday levels for the studied animal tissues.

Signal stability

The stability of the DNPH-Glo I assay’s final product, a colored adduct, was investigated to determine its suitability for long-term use. A MG solution (1 mM) was prepared, and its absorbance was measured at 480 nm at various time intervals ranging from 15 minutes to 120 hours. The sample was maintained at 25°C throughout the study to minimize potential degradation effects caused by fluctuations in storage conditions. The stability of the colored adduct was assessed by monitoring changes in absorbance values at each time point, providing insight into the temporal stability of the assay’s end point product and allowing for an estimation of its shelf life and usability over extended periods.

Matrix effect

The matrix effect, which belongs to the impact of non-target components in a sample on the precision of an analytical measurement, is a critical consideration in the DNPH-Glo I assay. In this context, dicarbonyl-containing compounds in biological samples can interfere with accurately assessing Glo I activity. These compounds can react with the assay reagents, resulting in skewed outcomes.

A control test tube was included in the experimental design to counteract the matrix effect. This control consists of all assay components, excluding the Glo I substrate, enabling absorbance to be measured solely from the interfering compounds in the sample matrix. By comparing the absorbance of both the sample and the control, researchers can quantify the interference caused by these additional compounds.

The methodology involves subtracting the absorbance of the control from that of the sample, thereby eliminating the contribution of the interfering compounds. This subtraction more accurately determines the unreacted substrates, a reliable indicator of Glo I activity. By controlling for these influences, researchers can enhance the precision of the results and ensure the accuracy of the modified DNPH-Glo I assay, thereby minimizing potential biases introduced by the matrix effect. Ultimately, this method fosters more reliable and reproducible findings in biological studies.

Validation

The relationship between the new DNPH-Glo I assay and the standard UV-Glo I method for Glo I activity measurement was evaluated through Bland-Altman statistical analysis [27]. Using spinal cord-derived Glo I enzyme [25], samples with activities ranging from 0.5 to 100 U/L were prepared. These samples were analyzed in parallel using both methods to obtain corresponding measurements at each activity level.

*The Bland-Altman analysis generated a plot visualizing the agreement between the two methods. The x-axis of the plot represented the average of the Glo I activity measurements obtained from both methods for each paired data point: ([DNPH-Glo I activity] + [UV-Glo I activity])/2. The y-axis represented the difference between the Glo I activity measurements obtained from the two methods for each paired data point: [DNPH-Glo I activity]—[UV-Glo I activity]. This difference is often referred to as the bias. The limits of agreement were determined by calculating the mean difference between the two methods and their standard deviation. Precisely, 95% limits of agreement were calculated as the mean difference ± 1.96 standard deviation of the difference. These limits define the range within which 95% of the differences between the two methods are expected to fall, assuming a normal distribution of the differences. According to Bland and Altman’s criteria [22], an acceptable agreement between the two methods is indicated if 95% of the data points on the Bland-Altman plot fall within these calculated limits of agreement.

Statistical analysis

Each experiment was conducted in triplicate, with results shown as means ± standard deviation (SD). Data were analyzed with Microsoft Excel 2020 and statistically evaluated using GraphPad Prism 8, confirming the DNPH-Glo I method's reproducibility and accuracy.

Results

The current study has developed a new methodology for measuring Glo I activity. The new method employs 2,4-DNPH as a chromogenic reagent, facilitating the quantification of Glo I activity. The process involves incubating Glo I enzyme samples in an accurately controlled 50 mM phosphate buffer, maintaining the pH at 6.6 to ensure optimal enzymatic function. GSH and MG serve as essential substrates, with Glo I catalyzing the conversion of the hemithioacetal adduct into SLG. After the enzymatic reaction, the unreacted MG reacts with 2,4-DNPH to form a colored hydrazone complex (as shown in Scheme 1). This reaction occurs through a nucleophilic addition mechanism where the nitrogen atom in the hydrazine (2,4-DNPH) attacks the carbonyl carbon of the aldehyde or ketone (MG). This nucleophilic attack forms a tetrahedral intermediate, which subsequently undergoes dehydration, creating a stable hydrazone linkage characterized by a C = N bond. This complex exhibits a distinct color, measurable by spectrophotometry. The intensity of this color is directly proportional to the concentration of the unreacted MG, which, in turn, allows for the quantification of Glo I activity. This new hydrazone-based detection method for measuring Glo I activity is critical for understanding the glyoxalase system and its role in cellular metabolism. By measuring the concentration of unreacted carbonyl compound (MG) through the intensity of the distinct colored hydrazone complex, researchers can effectively quantify the activity of the Glo I enzyme. As illustrated in Fig. 2, there is a direct relationship between color intensity and absorbance, reflecting Glo I’s consumption of MG. One unit of enzyme activity is the quantity that catalyzes the conversion of one micromole of MG per unit of time. NaOH converts the adduct into a bright brown end product, which displays strong absorbance at 480 nm.

Scheme 1.

The Glo I enzymatic reaction involves the unreacted MG from the Glo I-catalyzed process, which subsequently reacts with the 2,4-DNPH reagent. This reaction results in the formation of a brown-colored hydrazone product in the presence of NaOH

Figure 2.

The brown-colored hydrazone product’s spectral characteristics were associated with the remaining MG in the Glo I enzymatic reaction. The product was quantified using spectrophotometry at a wavelength of 480 nm. The values (a–i) represent the MG concentrations of 300, 270, 240, 210, 180, 150, 120, 90, and 0 µmole/L, respectively

Signal stability

The results of the current study indicate that the brown-colored hydrazone product exhibits remarkable stability when stored at 25°C, as demonstrated by consistent absorbance readings at 480 nm over three days. While a slight decrease in absorbance was recorded on the second day (9%) and the third day (13%), this indicates an impressively low rate of compound degradation, even after prolonged exposure to ambient temperatures. These results unequivocally demonstrate that the colored compound exhibits remarkable stability under standard temperature conditions, rendering it an ideal candidate for prolonged analysis and storage.

Linearity and sensitivity

The DNPH-Glo I method linearly responds to MG (MG) concentrations between 0.05 and 300 μM, with a very high Pearson's coefficient of 0.998. This strong agreement confirms the method's ability to quantify MG levels accurately, as demonstrated in Fig. 1. Additionally, the DNPH-Glo I method was evaluated against the UV-Glo I method across a wide range of Glo I enzyme activity levels, from 0.05 to 100 U/L, further validating the reliability of the DNPH-Glo I method (Fig. 3).

Figure 3.

Evaluation of the linearity of the DNPH-Glo I method for quantifying Glo I enzyme activity: comparison to the UV-Glo I assay across a range of activity levels (0.01–100 U/L), with results representing the average of three independent experiments, following 30-minute incubation at 37°C

Selectivity of the DNPH-Glo I method

Table 2 presents a comprehensive analysis of the effects of various biochemical substances on the DNPH-Glo I method, evaluating its ability to accurately measure Glo I activity in the presence of potential interfering agents. A systematic and meticulous interference study was conducted to assess the robustness of the assay against eight specific combinations of biomolecules, according to a previous study [28]. In the current study, 1 mL of a Glo I solution with an initial activity of 50 U/L was mixed with 1 mL of different biochemical solutions, and the Glo I activity was subsequently adjusted to 25 U/L using a validated ultraviolet method. The experimental design included eight flasks, each containing a specific combination of potentially interfering biomolecules: Flask 1 served as a control, containing phosphate-buffered saline (PBS) at pH 7.4; Flask 2 investigated the effects of three monosaccharides (1 mM each of mannose, sucrose, and fructose); Flask 3 analyzed four amino acids (1 mM each of isoleucine, aspartic acid, histidine, and valine); Flask 4 assessed the impact of 1% (w/v) bovine serum albumin (BSA); Flask 5 explored the effects of two small molecules 100 µM sodium linoleate and 100 µM uric acid; Flask 6 examined the influence of 1 mM ethylenediaminetetraacetic acid (EDTA); Flask 7 evaluated the effect of a protease inhibitor cocktail (10 µL/mL); and Flask 8 analyzed the impact of 0.1% (w/v) sodium dodecyl sulfate (SDS). The results, summarized in Table 2, present a detailed analysis of the relative percentage of errors associated with each biochemical combination tested, providing valuable insights into the performance and reliability of the DNPH-Glo I method in the presence of various interfering substances and enhancing our understanding of its applicability in biochemical research.

Table 2.

Impact of interfering biomolecules on the Glo I activity assessment accuracy using the DNPH-Glo I method.

Flask number	Contents	Added Glo I U/L	Found Glo I U/L	Relative errora (%)
1	PBS at pH 7.4	25	25	0
2	(1 mM each of mannose, sucrose, and fructose)	25	25.5	2
3	(1 mM each of isoleucine, cysteine, aspartic acid, histidine, and valine)	25	25.8	3.2
4	1% (w/v) BSA	25	26	4
5	100 µM sodium linoleate and 100 µM uric acid	25	26	4
6	1 mM EDTA	25	25.5	2
7	Protease inhibitor cocktail (10 µL/mL)	25	24.7	1.2
8	0.1% (w/v) SDS	25	25.7	2.8

^aRelative error is the ratio of absolute error to the actual measurement value, making it essential for accurately assessing measurement precision.

Method comparison

The precision of the DNPH-Glo I method was systematically evaluated using Bland-Altman plot analysis, a well-established statistical approach for assessing agreement between two quantitative measurement techniques. This analysis played a pivotal role in our study, allowing us to define the limits of agreement between the DNPH-Glo I and conventional UV-Glo I methods. To perform this comparative assessment, paired enzymatic samples were prepared to measure Glo I activity directly across a wide dynamic range. Specifically, enzyme dilutions were designed to produce Glo I activities ranging from 0.01 to 100 IU/L, enabling a systematic evaluation of the accuracy and reliability of the two methods. Figure 4 illustrates the Bland-Altman plot, clearly representing the measurement differences and the mean relative bias between the DNPH-Glo I and UV-Glo I methods. This graphical analysis is a cornerstone of our evaluation, providing direct insight into how the new method compares to the established reference. The Bland-Altman plot facilitates a robust examination of the new method’s accuracy concerning the reference standard. Key metrics analyzed include the mean difference, ideally close to zero, and the limits of agreement, which reveal potential discrepancies across the range of measurements. A reliable method is characterized by minimal bias, narrow limits of agreement, and the absence of systematic error. These attributes were carefully evaluated to determine the suitability of the DNPH-Glo I method.

Figure 4.

A graphical depiction of the Bland-Altman plot illustrates the differences and mean relative bias between the UV-Glo I and DNPH-Glo I methods. This comparative analysis offers a detailed evaluation of the relative bias, providing valuable insights into the performance and agreement of the two measurement methods

Reproducibility and precision

The accuracy and reliability of the DNPH-Glo I method were rigorously evaluated by comparing its performance in measuring Glo I activity in animal tissue samples with the UV-Glo I method. Tissue samples were carefully prepared according to a well-established protocol [17]. A comprehensive statistical analysis indicated a strong alignment between the results from the DNPH-Glo I method and the reference method, as demonstrated in Table 3.

Table 3.

A comparative analysis of Glo I activity: Evaluating the DNPH-Glo I and UV-Glo I methods.

Sample type	Glo I activity (U/g protein)
	The DNPH-Glo I method		The UV-Glo I method
	Intraday analysis	Inter-day analysis	Intraday analysis	Inter-day analysis
	Mean ± SD (RSD%)	Mean ± SD (RSD%)	Mean ± SD (RSD%)	Mean ± SD (RSD%)
Spinal cord	483 ± 09 (1.86)	492 ± 17 (3.46)	491 ± 11 (2.24)	496 ± 15 (3.02)
Cortex	407 ± 07 (1.72)	423 ± 11 (2.6)	413 ± 09 (2.18)	427 ± 17 (3.98)
Liver	433 ± 12 (2.77)	441 ± 15 (3.4)	443 ± 14 (3.16)	449 ± 16 (3.56)
Kidney	405 ± 05 (1.23)	411 ± 09 (2.19)	413 ± 09 (2.18)	417 ± 14 (3.36)

Discussion

The present study introduces a novel and innovative enzymatic assay specifically designed to measure the activity of the Glo I enzyme with high precision, efficiency, and reliability. Glo-I plays a crucial role as the primary enzyme in the glyoxalase system, responsible for detoxifying MG, a highly reactive 2-oxoaldehyde that exhibits cytostatic and cytotoxic effects at low and high concentrations, respectively. The 2,4-DNPH method for measuring Glo I activity offers several significant advantages that enhance its application in biochemical research. Firstly, the protocol is straightforward and cost-effective, making it accessible for many laboratories. The method allows for a quantitative assessment of enzyme activity by measuring the concentration of unreacted MG rather than directly quantifying the formation of SLG. This indirect measurement is particularly beneficial when direct substrate analysis is challenging or impractical. Additionally, the 2,4-DNPH method is compatible with standard spectrophotometers, facilitating its integration into existing laboratory setups. These features make the 2,4-DNPH method a versatile and valuable tool for studying Glo I activity in various research contexts.

Validation results and methods comparison

The DNPH-Glo I method demonstrates exceptional linearity in response to MG concentrations from 0.05 to 300 µM, with a Pearson's correlation coefficient of 0.998 (Fig. 1). The obtained result indicates a strong and reliable alignment between MG levels and the intensity of the resulting-colored complex, which confirms the method’s precision in accurately quantifying MG levels.

The method was thoroughly assessed and compared with the ultraviolet-Glo I method across a wide range of Glo I enzyme activities, from 0.05 to 100 U/L. The results revealed a robust compatibility between the two methods, with a Pearson’s correlation coefficient of 0.998 (Fig. 3). This finding highlights the DNPH-Glo I method’s ability to produce results consistently closely aligned with the ultraviolet-Glo I method. Notably, the DNPH-Glo I method exhibits high sensitivity, featuring low LOD and LOQ at 0.006 U/L and 0.018 U/L, respectively. These attributes facilitate accurate measurements of even minimal Glo I enzyme activity.

Intra- and inter-day analyses validated the precision of the DNPH-Glo I assay, revealing that the relative errors remained within acceptable ranges. The method showed minimal interference from biological molecules commonly found in tissue homogenates, ensuring accurate results even when analyzing complex biological matrices.

The excellent agreement with the established UV-based method, shown by a Pearson’s correlation coefficient of 0.998, highlights the reliability of the DNPH-Glo I method. This correlation indicates that the DNPH-Glo I method is a solid alternative for assessing Glo I activity, providing notable benefits such as lower costs and ease of use. Confirming the DNPH-Glo I method emphasizes its promise as a dependable instrument in biochemical research and clinical applications.

Fluorometric and spectrophotometric methods for measuring MG have distinct advantages for different research contexts. The fluorometric method uses a NIR fluorescent probe, MEBTD, which is highly sensitive and selective with a detection limit of 18 nM, allowing the detection of low concentrations of MG [19]. This method is helpful for studies requiring trace amounts. In contrast, the spectrophotometric method measures absorbance changes at 480 nm, reflecting colored complex formation. It is more accessible and cost-effective but may be less sensitive in complex biological samples due to potential interferences. The choice between methods depends on research sensitivity requirements, sample nature, and available resources and equipment.

Method limitations and matrix effect

The current method employs a dual-pronged strategy to address potential interferents and matrix effects that may affect Glo I enzyme measurements. Biological samples with high Glo I activity are diluted before the assay to accommodate the DNPH-Glo I assay’s sensitivity. After dilution, a DNPH reagent is added to evaluate MG (MG) levels and Glo I activity. The correction factor from a control test counters interference from biomolecules like carbohydrates and proteins.

The study examines these interferences, as summarized in Table 2, confirming that the method can accurately measure Glo I activity despite potential disruptors. A key consideration is the matrix effect, where non-target components can distort results. To mitigate this, a control test tube containing all assay components except the Glo I substrate allows the absorbance measurement from interfering compounds. By comparing the absorbance of samples and controls, researchers can isolate the contribution of unreacted substrates, leading to a more accurate assessment of Glo I activity. This meticulous approach enhances result precision, ensuring accurate measurements while minimizing biases. Ultimately, the DNPH-Glo I assay yields reliable and reproducible results in biological studies, enhancing enzymatic activity measurement.

Practical applications

The DNPH-Glo I method’s reproducibility and precision were evaluated through a rigorous comparison with the established UV-Glo I method for measuring Glo I activity in animal tissue samples. Statistical analysis revealed a strong good agreement between the two methods, as illustrated in Table 3, which presents a comparative analysis of Glo I activity across different tissue types. The data indicate that the DNPH-Glo I method produced comparable results to the UV-Glo I method, with intraday and inter-day analyses showing mean values and standard deviations that reflect minimal variability. For instance, in spinal cord samples, the DNPH-Glo I method yielded an intra-day mean of 483 ± 9 U/g protein, while the UV-Glo I method reported 491 ± 11 U/g protein, demonstrating the method’s reliability. The comparative analysis also shows low RSD% values for intraday and inter-day measurements, indicating high precision, with the spinal cord samples demonstrating an RSD% of 1.86% for the DNPH-Glo I method. Although inter-day RSD% values are slightly higher, they remain within acceptable limits, suggesting consistent performance over multiple testing sessions. The data reveal variability in Glo I activity across different tissues, reflecting physiological differences and emphasizing the importance of context in result interpretation. Overall, these findings underscore the DNPH-Glo I method’s potential as a precise and reproducible alternative for measuring Glo I activity in various biological contexts, making it a reliable tool for researchers studying metabolic disorders where accurate measurements are essential.

Conclusion

The current study introduces a robust, reliable method for measuring Glo I enzyme activity. The DNPH-Glo I method offers high sensitivity, reproducibility, and accuracy, comparable to established methods like the UV-Glo I method. It is particularly effective in complex biological samples containing potential interfering substances, ensuring accurate measurements. The current assay presents a valuable tool for researchers studying the role of Glo I in various biological processes, advancing our understanding of metabolic disorders and related health conditions.

Author contributions

Mahmoud Hussein Hadwan (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology development, Project administration, Resource management, Software implementation, Supervision, Validation, Visualization, Writing—original draft, and Writing—review and editing). Mohammed Alaa Kadhum (Data curation, Formal Analysis, Investigation, Methodology, Software development, Validation, Visualization, and Writing—review and editing)

Conflict of interest statement. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Funding

None declared.

Data availability

The authors declare that all data supporting this study’s findings are included in the article. Further data are available from the corresponding author upon request.