Creatinine, which is widely used as a marker for evaluating renal function and muscle disorders, has long been measured using the Jaffé method. Developed by Max Jaffé in 1886, this method is based on the reaction of creatinine with alkaline picrate. Although this method is simple and cost-effective, it is susceptible to interference from substances such as cephalosporin antibiotics, leading to concerns regarding analytical accuracy. Accordingly, the development of more specific enzymatic methods began in the 1970s. In the early 1970s, methods combining creatininase with the Jaffé reaction were used to eliminate the influence of endogenous creatine in blood samples. Around 1980, fully enzymatic creatinine assays became commercially available. These assays used creatininase, creatine kinase, pyruvate kinase, and lactate dehydrogenase, with measurements based on changes in NADH absorbance.1) Subsequently, additional assay systems using different enzyme combinations were developed, but all relied on NAD(P)H as the detection system(Figure 1). ²⁾

Figure 1. Various enzymatic methods for creatinine measurement

During the same period, hydrogen peroxide-based detection methods gained attention as useful enzymatic assay systems. The use of Trinder reagents enabled the generation of chromogenic products with higher molar absorptivity than NADH, enabling highly sensitive detection. Consequently, these methods were widely used in the late 1970s to detect various clinical chemistry analytes. However, no suitable oxidase had yet been identified for creatinine measurement systems, thus preventing the application of Trinder chemistry. Notably, sarcosine dehydrogenase was the only known enzyme capable of acting on the sarcosine generated from the sequential reactions of creatininase and creatinase. Therefore, a new reaction system capable of producing hydrogen peroxide was needed. To address this challenge, Kikkoman researchers conducted an extensive screening of a wide range of microorganisms and were the first to discover sarcosine oxidase in 1979. 3) In 1981, they reported its enzymatic properties and proposed its application in creatinine measurement(Figure 2).

Figure 2. Creatinine measurement method using sarcosine oxidase

The discovery of sarcosine oxidase enabled the generation of hydrogen peroxide from sarcosine, thereby paving the way for the development of highly sensitive creatinine assays based on Trinder chemistry. In addition to developing the enzyme itself, our company focused on the formulation of creatinine measurement reagents. For example, to minimize the interference from endogenous creatine, we propose a reagent system in which the first reagent contains both creatinase and sarcosine oxidase. ⁴⁾ This concept has been incorporated in modern creatinine assays. Our commitment to research and development extends beyond enzyme discovery and improvement to the design of practical assay systems and applications, a philosophy that continues to guide our current work.

Consequently, we established technologies to manufacture creatininase, creatinase, and sarcosine oxidase. Using these enzymes, KAINOS Laboratories, Inc. developed an enzymatic creatinine assay based on hydrogen peroxide detection, which was successfully commercialized in 1983. Currently, these enzymes are used in creatinine measurement reagents produced by diagnostic manufacturers worldwide, and we continue to supply them with raw materials for diagnostic reagents. Since the discovery of sarcosine oxidase, which enabled enzymatic creatinine measurement, the assay has undergone continuous improvement. In parallel, we remain committed to enhancing enzyme performance to support the ongoing evolution of creatinine testing.

What Is Creatinine? ^{5) 6)}

Creatinine is an end-product of metabolism that is generated non-enzymatically from creatine and phosphocreatine, which play essential roles in muscle contraction. After entering the bloodstream, creatinine is filtered by the glomeruli of the kidneys and excreted in the urine with minimal reabsorption. Because of these characteristics, blood creatinine concentration is widely used as a standard marker for evaluating renal function. Since creatinine production depends on muscle mass, serum creatinine concentrations vary among individuals even when renal function is the same. People with lower muscle mass generally have lower serum creatinine levels. In routine clinical practice, renal function is assessed using estimated glomerular filtration rate(eGFR), which is calculated based on serum creatinine levels and individual factors such as age and gender. For this reason, accurate measurement of creatinine is essential for the reliable assessment of renal function.

References

1. The Journal of Clinical Laboratory Instruments and Reagents 3 (2), 118–122 (1980)
2. Journal of Clinical Laboratory Medicine, 22 (11), 1331–1338 (1978)
3. Seikagaku, 51 (8), 788 (1979)
4. Clinica Chimica Acta, 143, 147–155 (1984)
5. Journal of the Japanese Society of Internal Medicine, 109(12), 2466–2470 (2020)
6. CKD Clinical Practice Guide 2024, edited by the Japanese Society of Nephrology