Creatinine-related enzymes can convert creatinine into measurable products such as ammonia and hydrogen peroxide through direct deamination or multienzyme cascades, providing the reaction basis for biochemical renal function testing and enzyme sensor construction.

Keywords: creatinine; creatinine deiminase; creatininase; creatinase; sarcosine oxidase; renal function testing; enzyme-coupled reaction; enzyme sensor

1 Creatinine Formation, Metabolism, and Clinical Significance

1.1 Creatinine Formation

Creatinine is a nitrogen-containing metabolite formed through the spontaneous dehydration and cyclization of creatine and phosphocreatine. Most creatine in the body is distributed in skeletal muscle and participates in energy buffering through the creatine–phosphocreatine system. A small proportion of creatine and phosphocreatine continuously undergoes nonenzymatic conversion to creatinine, which then enters the bloodstream. Creatinine production is associated with total muscle mass, muscle metabolic status, and dietary intake.

1.2 Fate and Excretion of Creatinine In Vivo

Creatinine undergoes little further conversion in the body. It is mainly filtered through the glomeruli into the urine and is generally not substantially reabsorbed by the renal tubules, although a certain degree of tubular secretion may occur. When the glomerular filtration rate decreases, creatinine clearance is reduced and creatinine accumulates in the blood. Therefore, serum or plasma creatinine concentrations usually increase as renal function declines.

Figure 1. Creatine synthesis, creatine–phosphocreatine cycling, and creatinine formation and renal excretion

1.3 Clinical Significance of Creatinine Testing

Serum creatinine is a commonly used indicator for evaluating renal function and estimating the glomerular filtration rate. Urinary creatinine can be used to calculate creatinine clearance and to normalize the concentrations of urinary protein, urinary albumin, and other urinary analytes. Creatinine concentrations are also affected by age, sex, muscle mass, diet, exercise, muscle injury, and certain medications. A single creatinine result cannot fully represent glomerular filtration function and should be interpreted together with the estimated glomerular filtration rate and other renal function indicators.

1.4 Major Methods for Creatinine Testing

Laboratory methods for creatinine testing mainly include the picrate method and enzymatic methods. The picrate method is simple and relatively inexpensive but is readily affected by ketone bodies, glucose, proteins, bilirubin, and certain medications. Enzymatic methods use creatinine deiminase or a cascade consisting of creatininase, creatinase, and sarcosine oxidase to generate measurable signals and generally provide better analytical specificity.

2 Molecular Characteristics and Catalytic Mechanism of Creatinine Deiminase

2.1 Enzyme Sources and Reaction Type

Creatinine deiminase, also known as creatinine deaminase or creatinine iminohydrolase, is mainly derived from microorganisms capable of utilizing creatinine. This enzyme directly catalyzes the hydrolytic deamination of creatinine without first converting creatinine into creatine:

Creatinine + H₂O → N-methylhydantoin + NH₃

The ammonia generated by the reaction can be detected through colorimetric, potentiometric, pH-based, or enzyme-coupled methods. Compared with multienzyme cascade methods, the creatinine deiminase method involves fewer reaction steps and is suitable for rapid analysis and the construction of immobilized-enzyme sensors.

2.2 Substrate Binding and Catalytic Process

After creatinine enters the substrate-binding pocket of the enzyme, its cyclic nitrogen-containing structure is positioned within the catalytic center. An activated water molecule attacks the reactive carbon atom of the creatinine ring, followed by proton transfer and C–N bond cleavage, producing N-methylhydantoin and releasing ammonia. Some microbial creatinine deiminases are metal-dependent hydrolases in which metal ions participate in water activation, substrate positioning, and stabilization of reaction intermediates. Because enzymes from different sources differ in subunit composition, metal dependence, and catalytic residues, their responses to EDTA, buffer components, and externally added metal ions are not identical.

2.3 Conversion of the Ammonia Signal

The ammonia generated by creatinine deiminase can be coupled to a glutamate dehydrogenase reaction. In the presence of α-ketoglutarate and NADH or NADPH, ammonia is converted into glutamate while the reduced coenzyme is oxidized. Creatinine content can therefore be calculated by monitoring the decrease in absorbance near 340 nm.

Ammonia can also be detected using an ammonium ion-selective electrode, a pH-sensitive element, or an ammonia color reaction. Because pre-existing ammonia in samples, reagents, and the laboratory environment also generates a detection signal, sample blanks, reagent blanks, or pretreatment procedures are required.

3 Substrate Specificity and Enzymatic Properties of Creatinine Deiminase

3.1 Substrate Specificity

Creatinine deiminase primarily recognizes the cyclic nitrogen-containing structure and spatial conformation of creatinine. Creatine is an open-chain compound and generally cannot enter the active site in the same manner. Certain cyclic nitrogen-containing compounds may compete for the substrate-binding site. Therefore, when establishing a detection method, the cross-reactivity of creatine, urea, uric acid, ammonium ions, and structurally related analogues should be evaluated.

3.2 pH, Temperature, and Metal Ions

Creatinine deiminase generally exhibits relatively high activity under neutral to mildly alkaline conditions, although the specific optimum range depends on the enzyme source. The pH affects both the protonation states of catalytic residues and the ratio between ammonia and ammonium ions. Increasing the temperature can accelerate the reaction, but excessively high temperatures may reduce enzyme stability. Metal-dependent creatinine deiminases may be inhibited by chelating agents such as EDTA. Whether external metal ions are required should be determined according to the specific enzyme preparation.

3.3 Reaction Kinetics and Stability

Within a relatively low creatinine concentration range, the reaction rate increases as the substrate concentration increases. As the enzyme gradually approaches saturation, the reaction rate reaches a plateau. For quantitative testing, the sample concentration should remain within the linear range of the method, and the enzyme amount and reaction time should be sufficient to achieve stable substrate conversion.

Storage temperature, the number of freeze–thaw cycles, buffer composition, and immobilization method also affect residual enzyme activity and lot-to-lot consistency. In coupled assays, the downstream signal-generating reaction should not become the rate-limiting step.

4 Cascade Reactions of Creatininase, Creatinase, and Sarcosine Oxidase

4.1 Multienzyme Cascade Route

The multienzyme-coupled method sequentially uses creatininase, creatinase, and sarcosine oxidase to convert creatinine into creatine, sarcosine, and hydrogen peroxide. The hydrogen peroxide is then detected by peroxidase-mediated color development or an electrochemical method. The basic route is:

Creatinine → Creatine → Sarcosine → H₂O₂ → Colorimetric or electrochemical signal

Insufficient activity of any enzyme in the cascade may cause the accumulation of intermediates or a decrease in the final signal. Therefore, downstream enzymes are generally maintained in relative excess so that the overall reaction rate primarily reflects the creatinine content of the sample.

4.2 Individual Enzymatic Reactions

（1）Creatininase Reaction

Creatininase catalyzes the hydrolysis of the creatinine ring to generate creatine:

Creatinine + H₂O → Creatine

This reaction is the first step through which creatinine enters the creatine metabolic pathway in the multienzyme method. Insufficient creatininase activity causes incomplete creatinine conversion and directly limits the strength of the downstream signal.

（2）Creatinase Reaction

Creatine amidinohydrolase, also known as creatinase, further catalyzes the hydrolysis of creatine to generate sarcosine and urea:

Creatine + H₂O → Sarcosine + Urea

Creatine originally present in the sample can also enter this reaction. Therefore, detection systems generally use a prereaction, a two-reagent design, or a sample blank to subtract the signal generated by endogenous creatine.

（3）Sarcosine Oxidase Reaction

Sarcosine oxidase uses molecular oxygen to oxidize sarcosine, producing glycine, formaldehyde, and hydrogen peroxide:

Sarcosine + O₂ + H₂O → Glycine + Formaldehyde + H₂O₂

This step depends on oxygen. Sample viscosity, mixing of the reaction solution, and oxygen diffusion through an immobilized-enzyme membrane can all affect the rate of hydrogen peroxide generation. Endogenous sarcosine in the sample may also generate background signals unrelated to creatinine.

（4）Peroxidase Color Reaction

The generated hydrogen peroxide can oxidize a chromogen in the presence of peroxidase to form a colored product. The color intensity can then be used to calculate the creatinine concentration. Ascorbic acid, glutathione, and certain medications may consume hydrogen peroxide or reduce the colored product, resulting in falsely low results.

5 Comparison Between the Creatinine Deiminase Method and the Multienzyme-Coupled Method

5.1 Major Differences Between the Two Detection Routes

5.2 Method Selection and Result Interpretation

The creatinine deiminase method reduces problems associated with multienzyme activity ratios and incomplete conversion of intermediates. However, without effective ammonia blank correction, it may produce a systematic positive bias. The multienzyme-coupled method is suitable for automated biochemical analyzers but requires correction for endogenous creatine and sarcosine and appropriate matching of the enzymatic activities and color-development reactions at each step.

The performance of the two methods cannot be judged solely by the number of enzymes involved. Method selection should comprehensively consider the sample type, creatinine concentration range, instrument platform, reaction speed, and major interfering substances. Results should be verified using calibrators, quality-control materials, and spike-recovery experiments.

6 Reaction Conditions and Interfering Factors in Enzymatic Creatinine Testing

6.1 Control of Reaction Conditions

（1）pH, Temperature, and Reaction Time

The optimal pH values of creatininase, creatinase, sarcosine oxidase, and peroxidase are not completely identical. The reaction system therefore requires a compromise condition that maintains effective activity of all enzymes. Increasing the temperature can accelerate the reaction but may also reduce enzyme stability and increase blank color development. An endpoint method must allow the reaction to reach a stable state, whereas a kinetic method requires a time window in which the reaction rate maintains a stable relationship with the creatinine concentration.

（2）Enzyme Activity Ratios

Creatininase determines the efficiency with which creatinine enters the cascade pathway. Creatinase and sarcosine oxidase should rapidly convert the generated products to prevent the accumulation of creatine or sarcosine. Downstream enzymes are generally maintained in relative excess to ensure that the final signal is primarily controlled by the creatinine concentration.

（3）Oxygen Supply and Color-Development System

Sarcosine oxidase depends on molecular oxygen. Increased sample viscosity, inadequate mixing, or an excessively thick immobilized-enzyme layer may limit oxygen diffusion and cause falsely low results. The concentrations of peroxidase and the chromogenic substrate should be sufficient to respond to the generated hydrogen peroxide while avoiding increased blank values caused by chromogen auto-oxidation.

6.2 Major Interfering Factors

（1）Endogenous Creatine and Sarcosine

Creatine in a sample can directly enter the creatinase reaction, whereas sarcosine can directly enter the sarcosine oxidase reaction. Both generate background signals unrelated to creatinine. In a two-reagent method, the signal generated by endogenous creatine and sarcosine can first be measured, after which creatininase is added to initiate the complete cascade and the difference is calculated.

（2）Reducing Substances

Ascorbic acid, glutathione, and certain medications may consume hydrogen peroxide or interfere with color development, producing falsely low results. The addition of ascorbate oxidase can reduce interference from ascorbic acid, although the effects of other reducing substances on the reaction system should still be evaluated.

（3）Bilirubin, Hemoglobin, and Lipemia

Bilirubin and hemoglobin may absorb light near the detection wavelength, whereas lipemia increases light scattering. Sample blanks, dual-wavelength measurements, and auxiliary reactions can reduce these effects but cannot replace appropriate quality assessment of icteric, hemolyzed, and lipemic samples.

6.3 Sample Types and Method Validation

Serum, plasma, and urine differ in creatinine concentration ranges and matrix composition. Their linear ranges, dilution conditions, and quality-control criteria should therefore be established separately. Method validation should include precision, linearity, recovery, detection capability, interference resistance, and reagent stability. Enzymatic methods using different enzyme sources, reaction routes, and calibration systems may also exhibit method-dependent biases.

7 Applications of Creatinine Deiminase in Biochemical Testing and Enzyme Sensors

7.1 Biochemical Testing

（1）Coenzyme Absorbance Method

Creatinine deiminase can be coupled with glutamate dehydrogenase to quantify creatinine by monitoring the decrease in NADH or NADPH absorbance. This method does not depend on peroxidase-mediated color development and may reduce certain interferences related to hydrogen peroxide and chromogens. However, endogenous ammonia and background coenzyme absorbance in the sample must be controlled.

（2）Ammonia Colorimetric Method

Ammonia generated by creatinine deiminase can be detected through a color reaction. This method is suitable for microplate, flow-analysis, and dry-chemistry systems. Although the reaction route is relatively short, ammonia-containing reagents, environmental ammonia, and reagent-container sealing can affect blank stability.

7.2 Enzyme Sensors

（1）Ammonium Ion Potentiometric Sensor

Creatinine deiminase can be immobilized near an ammonium ion-sensitive membrane, allowing creatinine to be measured by detecting the potential change caused by generated ammonium ions. Ammonium ions originally present in the sample produce a background signal. Therefore, a reference channel, blank reaction, or pretreatment procedure is required.

（2）pH-Based Enzyme Sensor

The creatinine deimination reaction can alter the local acid–base equilibrium. A pH-sensitive electrode, ion-sensitive field-effect transistor, or optical pH probe can convert this change into a detection signal. Excessively high buffer capacity weakens the response, whereas excessively low buffer capacity makes the system more susceptible to the intrinsic pH of the sample.

（3）Multienzyme Electrochemical Sensor

Creatininase, creatinase, and sarcosine oxidase can be co-immobilized on an electrode surface, and creatinine can be determined by measuring the electrical current generated by hydrogen peroxide. Enzyme-layer thickness, oxygen diffusion, hydrogen peroxide mass transfer, and electrode-surface fouling all affect response speed and the linear range.

7.3 Evaluation of Immobilized-Enzyme Performance

Immobilization can improve enzyme reusability and storage stability but may also alter enzyme conformation and substrate diffusion. In addition to sensitivity and linear range, sensor evaluation should examine selectivity, response time, drift, number of reuse cycles, lot-to-lot variation, and storage stability. Enzyme-free controls, substrate-free controls, and spike experiments using actual samples should be performed to confirm the source of the signal.

8 Products

8.1 Core Enzymes for Creatinine Testing and Cascade-Reaction-Related Products

8.2 Creatinine, Creatine, and Sarcosine Standards and Substrate-Related Products

8.3 Products for Creatinine and Creatine Content Testing

The reliability of creatinine testing methods depends on enzyme substrate specificity, cascade-reaction efficiency, and the signal-conversion method. Stable and interpretable renal function test results can be obtained only by selecting an appropriate detection route according to the sample type and major interfering substances and by simultaneously controlling blanks, enzyme activity ratios, and the calibration system.

For more related articles, please see below:

[1] Creatininase-Based Creatinine Detection: Reaction Principles, Method Systems, and Result Quality Control

[2] Creatinine Content Detection: Main Methods, Reaction Principles, and Result Control

[3] Experimental determination of serum creatinine (Cr) by the deproteinization endpoint method