Currently, approximately 850 million people worldwide suffer from kidney disease, with the prevalence of chronic kidney disease in China reaching 8.2%—meaning one in every ten people in the country has a kidney condition. Chronic kidney disease has thus become a significant threat to public health.

Creatinine, a byproduct of muscle metabolism, includes both exogenous creatinine, derived from meat, and endogenous creatinine, produced by muscle tissue metabolism within the body. Normally, creatinine is filtered out almost entirely through the glomeruli. However, when kidney function is impaired, creatinine excretion is hindered, leading to elevated creatinine levels in the blood. As such, blood creatinine levels are a vital indicator for assessing kidney function and screening for kidney damage.

Currently, clinical creatinine testing mainly relies on the Jaffe reaction (alkaline picrate method) and enzymatic methods. Compared with the Jaffe method, enzymatic creatinine testing offers superior anti-interference, specificity, accuracy, and a wider linear range.

Enzymatic creatinine testing primarily involves the creatinine amidohydrolase and creatinine oxidase methods. In the creatinine amidohydrolase method, creatinine is hydrolyzed by creatinine deiminase, producing glutamate, which is measured by monitoring the decrease in absorbance at 340 nm. Meanwhile, in the creatinine oxidase method, creatinine is hydrolyzed by creatinine hydrolase to produce creatine, which is then hydrolyzed by creatinase to generate sarcosine and urea. Sarcosine is subsequently oxidized by sarcosine oxidase, resulting in glycine, formaldehyde, and hydrogen peroxide, with quinoneimine pigment in a Trinder reaction providing a colorimetric measurement. Compared with the creatinine amidohydrolase method, the creatinine oxidase method shows better resistance to reducing agents like calcium dobesilate and acetaminophen, despite limitations in reagent stability and supply.

The creatinine oxidase method involves a multi-enzyme cascade system that adds complexity to reagent development, accuracy, stability, and preservative performance. Additionally, as procurement policies emphasize cost-effectiveness, production companies face heightened pressure to control costs and enhance efficiency.

New Release and Performance Upgrade

Creatinase and Catalase Upgrades Enhance Reagent Stability

Creatinase (CR) is a key enzyme in the creatinine detection system and acts as a rate-limiting enzyme, crucial for the accuracy and stability of creatinine test reagents. However, the extensive mutational sequence space from iterative saturation mutagenesis and the influence of higher-order effects complicate creatinase optimization. HZYMES, leveraging enzyme-directed evolution and a high-throughput screening platform, pre-trained a comprehensive protein sequence library with 96 million entries for optimal bacterial host growth. This identified the 13M4 creatinase mutant with enhanced thermal stability and enzyme activity, showing a Tm increase of about 10°C and a half-life at 58°C increased by over 655 times.

To mitigate interference from endogenous creatine in clinical testing, catalase is added to remove it. However, catalase stability issues can cause elevated clinical readings. HZYMES has optimized catalase’s structure and purification process, achieving a 20-fold increase in half-life at 70°C over imported brands, with residual enzyme activity above 90% after 7 days at 37°C.


With catalase upgrades, creatinine reagents can maintain interference elimination even after 14 days of thermal acceleration, ensuring consistent clinical readings.

Enhanced Accuracy and Precision for High-Quality Results

In comparative clinical testing, the HZYMES creatinine test kit showed an average deviation of less than 5% and a correlation coefficient greater than 0.999 when compared to imported brands, demonstrating excellent correlation. When tested with national and U.S. standard frozen human serum samples, results deviated by less than 3% from target values, confirming the high accuracy of HZYMES’ kit.

Broad Linear Range and Simultaneous Blood and Urine Testing

With a linear range of 10 to 8840 μmol/L, the HZYMES creatinine test kit can accommodate both blood and urine samples, reducing the need for sample dilution and enhancing testing efficiency and accuracy.


Advanced Anti-Interference Technology for Enhanced Performance

In 1986, research showed that calcium dobesilate interferes with enzymatic creatinine measurement. Later studies confirmed significant negative interference from calcium dobesilate, especially in patients with impaired renal function. To address this, HZYMES incorporated anti-CaD technology to eliminate interference from calcium dobesilate and made structural changes to the creatinine enzyme, preserving reagent performance while neutralizing interference.

In samples with creatine ≤20 mg/dL, ascorbic acid ≤50 mg/dL, free bilirubin ≤40 mg/dL, conjugated bilirubin ≤20 mg/dL, hemoglobin ≤100 mg/dL, and lipemia ≤1000 mg/dL, the measurement remains unaffected. Additionally, HZYMES creatinine reagents resist interference from calcium dobesilate concentrations ≤20 mg/L and acetaminophen concentrations ≤35 mg/L.

HZYMES specializes in comprehensive creatinine reagent solutions, from high-quality core enzyme selection to customized OEM reagent development and formulation collaboration. Contact us to start your journey towards a personalized creatinine reagent solution.


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Reference

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[2]: Delanaye P, Cavalier E, Pottel H. Serum creatinine: not so simple!. Nephron 2017;136:302–8.

[3]: Peake M, Whiting M. Measurement of serum creatinine–current status and future goals. The Clinical biochemist. Reviews. 2006;27:173–84.

[4]: Tanganelli E, Prencipe L, Bassi D, Cambiaghi S, Murador E. Enzymic assay of creatinine in serum and urine with creatinine iminohydrolase and glutamate dehydrogenase. Clin Chem. 1982 Jul;28(7):1461-

[5]. 戴新华,方向,全灿,等. 血清中肌酐的检测方法及其进展[J]. 分析测试学报,2007,26(5):763-767.

[6]: Guder WG, Hoffmann GE. Multicentre evaluation of enzymatic method for creatinine determination using a sensitive colour reagent.J Clin Chem Clin Biochem. 1986;24:889–902.

[7]: Guo X , Hou L , Cheng X ,et al.Strong Negative Interference by Calcium Dobesilate in Sarcosine Oxidase Assays for Serum Creatinine Involving the Trinder Reaction[J].Medicine, 2015, 94(23):e905.

In the future, Hzymes biotech will always remember its original intention and persist in meticulous cultivation in the IVD field. It will adhere to independent research and development, accelerate the construction of a world-class specialty enzyme production platform, and achieve import substitution of core enzyme raw materials in the field of biomedicine in China. It will collaborate with leading biopharmaceutical companies to expand their global presence and contribute to the advancement of the industry.