Liver function testing is one of the most fundamental and widely used applications in clinical biochemistry. By measuring enzyme activity, bilirubin, proteins, and other indicators in serum, it systematically evaluates the three core functions of the liver: hepatocellular integrity, biliary excretory function, and hepatic synthetic function. The performance of high-quality liver function testing reagents heavily depends on the proper selection and combination of upstream raw materials. This article analyzes the methodologies, key raw materials, and selection criteria for core liver function testing projects, providing a complete raw material combination recommendation and solutions to common problems.
I. Overview of Liver Function Testing Projects
Liver function tests are primarily used to assess liver metabolism, synthesis, and excretion functions, serving as the core of physical examinations and liver disease diagnosis. They mainly include the following indicators:
| Functional Category | Assessment Content | Representative Indicators |
|---|---|---|
| Hepatocellular Injury | Hepatocellular integrity | ALT, AST |
| Biliary Obstruction | Cholestasis/obstruction | ALP, GGT, TBIL, DBIL |
| Synthetic Function | Protein synthesis capacity | TP, ALB |
II. Core Liver Function Testing Projects and Raw Material Selection
1. Alanine Aminotransferase (ALT)
Alanine aminotransferase is mainly found in the hepatocellular cytoplasm and is one of the most sensitive indicators of hepatocellular injury. When liver cells are damaged, ALT is released into the blood, and serum ALT activity increases.
Key Raw Material List:
| Raw Material Name | CAS No. | Function |
|---|---|---|
| L-Alanine | 56-41-7 | Substrate |
| α-Ketoglutaric acid | 328-50-7 | Substrate |
| NADH (Reduced β-Nicotinamide Adenine Dinucleotide) | 606-68-8 | Core coenzyme |
| LDH (Lactate Dehydrogenase) | 9001-60-9 | Tool enzyme |
| Tris base | 77-86-1 | Buffer |
| Tris-HCl | 1185-53-1 | Buffer |
Raw Material Selection Points:
-
NADH (CAS: 606-68-8) is sensitive to light and prone to oxidative degradation. It is recommended to select high-purity products and store away from light.
-
Reagent shelf life can be extended by adding antioxidants (e.g., Glutathione, CAS: 70-18-8) and chelating agents (e.g., EDTA, CAS: 6381-92-6).
-
Tris buffer (CAS: 77-86-1 / 1185-53-1) does not significantly inhibit ALT/AST activity and is the preferred buffer system.
2. Aspartate Aminotransferase (AST)
Aspartate aminotransferase is found in the myocardium, liver, and skeletal muscle. It is often used in combination with ALT to assess hepatocellular injury.
Key Raw Material List:
| Raw Material Name | CAS No. | Function |
|---|---|---|
| L-Aspartic acid | 56-84-8 | Substrate |
| α-Ketoglutaric acid | 328-50-7 | Substrate |
| NADH (Reduced β-Nicotinamide Adenine Dinucleotide) | 606-68-8 | Core coenzyme |
| MDH (Malate Dehydrogenase) | 9001-64-3 | Tool enzyme |
| LDH (Lactate Dehydrogenase) | 9001-60-9 | Auxiliary tool enzyme |
| Tris base | 77-86-1 | Buffer |
| Tris-HCl | 1185-53-1 | Buffer |
3. Alkaline Phosphatase (ALP)
Alkaline phosphatase is present in the liver, bone, intestines, and other tissues. Elevated serum ALP is commonly seen in biliary obstruction and bone diseases.
Key Raw Material List:
| Raw Material Name | CAS No. | Function |
|---|---|---|
| p-NPP (p-Nitrophenyl Phosphate) | 4264-83-9 | Chromogenic substrate |
| DEA (Diethanolamine) | 111-42-2 | Buffer + Activator |
| MgCl₂·6H₂O (Magnesium chloride) | 7791-18-6 | Cofactor, activates ALP |
| ZnCl₂ (Zinc chloride) | 7646-85-7 | Cofactor, activates ALP |
Critical Warning:
-
Do NOT use Tris buffer: Tris strongly inhibits ALP activity, leading to significantly low test results.
-
Mg²⁺ and Zn²⁺ are mandatory: These divalent ions are ALP activators; their absence will drastically reduce enzyme activity.
-
DEA (CAS: 111-42-2) buffer not only buffers but also activates ALP and is the standard buffer system recommended by the IFCC.
4. Gamma-Glutamyl Transferase (GGT)
Gamma-glutamyl transferase is a sensitive indicator of biliary tract injury. Combined with ALP, it helps differentiate between hepatobiliary and bone diseases.
Key Raw Material List:
| Raw Material Name | CAS No. | Function |
|---|---|---|
| γ-GCANA (L-γ-Glutamyl-p-nitroanilide) | 7300-54-1 | Chromogenic substrate |
| Glycylglycine (Gly-Gly) | 556-50-3 | Acceptor substrate |
| Tris base | 77-86-1 | Buffer |
| Tris-HCl | 1185-53-1 | Buffer |
Raw Material Selection Points:
-
γ-GCANA (CAS: 7300-54-1) is easily hydrolyzed in alkaline solutions. It is recommended to store away from light and refrigerate after preparation.
-
The purity of Glycylglycine (CAS: 556-50-3) affects the reaction rate. High-purity products are recommended.
5. Total Bilirubin (TBIL) and Direct Bilirubin (DBIL)
Bilirubin is a metabolite of hemoglobin. The measurement of total and direct bilirubin is used for the differential diagnosis of jaundice.
Key Raw Material List:
| Raw Material Name | CAS No. | Function |
|---|---|---|
| Caffeine | 58-08-2 | Accelerator (TBIL) |
| Sodium metavanadate | 13718-26-8 | Oxidizing agent |
| Sodium dihydrogen phosphate | 13472-35-0 | Buffer |
| Disodium hydrogen phosphate | 7558-79-4 | Buffer |
| 3,5-Dichlorophenyldiazonium salt | — | Chromogenic agent (Diazonium method) |
Method Comparison and Selection Suggestions:
| Comparison Item | Diazonium Method | Vanadate Oxidation Method |
|---|---|---|
| Advantages | Classical method, low cost | Non-toxic waste, good automation compatibility |
| Disadvantages | Caffeine is toxic, waste disposal is troublesome | Slightly higher reagent cost |
| Suitable Scenarios | Manual method, low throughput | Automated biochemical analyzers |
Raw Material Selection Points:
-
Bilirubin is light-sensitive. Both standards and reagents should be protected from light.
-
Caffeine (CAS: 58-08-2) is a classic accelerator, but its toxicity must be considered.
6. Total Protein (TP)
Total protein measurement is used to assess liver synthetic function, nutritional status, and certain diseases (e.g., nephropathy, multiple myeloma).
Key Raw Material List:
| Raw Material Name | CAS No. | Function |
|---|---|---|
| Copper(II) sulfate pentahydrate (CuSO₄·5H₂O) | 7758-99-8 | Source of copper ions |
| Potassium sodium tartrate | 304-59-6 | Stabilizes copper ions, prevents precipitation |
| Potassium iodide (KI) | 7681-11-0 | Antioxidant |
| Sodium hydroxide (NaOH) | 1310-73-2 | Provides alkaline environment |
Raw Material Selection Points:
-
The key role of Potassium sodium tartrate (CAS: 304-59-6) is to prevent Cu(OH)₂ precipitation under alkaline conditions, ensuring reagent stability.
-
Potassium iodide (CAS: 7681-11-0) prevents the oxidation of Cu⁺, extending reagent shelf life.
7. Albumin (ALB)
Albumin is the most abundant protein in serum, synthesized by the liver. Low albumin levels are commonly seen in liver disease, kidney disease, and malnutrition.
Key Raw Material List:
| Raw Material Name | CAS No. | Function |
|---|---|---|
| Bromocresol Green (BCG) | 76-60-8 | Chromogenic agent |
| Succinic acid (Butanedioic acid) | 110-15-6 | Buffer |
| Brij-35 | 9002-92-0 | Detergent, reduces non-specific adsorption |
Raw Material Selection Points:
-
The BCG method is extremely sensitive to pH. A high-quality buffer must be used, and the pH must be precisely controlled at 4.0-4.2.
-
Non-ionic detergents like Brij-35 (CAS: 9002-92-0) can significantly reduce background signals and improve detection sensitivity.
III.Common Problems and Solutions
Problem 1: Rapid Degradation of NADH in ALT/AST Reagents, Leading to Short Shelf Life
Cause Analysis:
-
NADH (CAS: 606-68-8) is light-sensitive and prone to oxidation when exposed to light.
-
Presence of heavy metal ions (e.g., Fe²⁺, Cu²⁺) in the solution catalyzes the oxidation reaction.
Solutions:
| Measure | Specific Operation | Raw Materials Involved |
|---|---|---|
| Add antioxidant | Add reduced Glutathione 0.5-1 mM | Glutathione (CAS: 70-18-8) |
| Add chelating agent | Add EDTA 1-2 mM | EDTA·2Na (CAS: 6381-92-6) |
| Packaging suggestion | Use brown bottles, store away from light at 2-8°C | — |
Problem 2: Low ALP Test Results
Cause Analysis:
-
Tris buffer was used incorrectly (strong inhibition).
-
Mg²⁺/Zn²⁺ activators were not added.
Solutions:
| Measure | Specific Operation | Raw Materials Involved |
|---|---|---|
| Check buffer type | Confirm use of DEA buffer, not Tris | DEA (CAS: 111-42-2) |
| Confirm activator addition | Ensure Mg²⁺ and Zn²⁺ have been added | MgCl₂ (CAS: 7791-18-6), ZnCl₂ (CAS: 7646-85-7) |
Problem 3: High Reagent Blank for Albumin (BCG Method)
Cause Analysis:
-
Low purity of Bromocresol Green (CAS: 76-60-8).
-
Inaccurate buffer pH.
Solutions:
| Measure | Specific Operation | Raw Materials Involved |
|---|---|---|
| Replace BCG batch | Select ≥95% high-purity Bromocresol Green | Bromocresol Green (CAS: 76-60-8) |
| Calibrate pH | Strictly prepare succinate buffer, ensure pH = 4.0-4.2 | Succinic acid (CAS: 110-15-6) |
IV.Summary
As a core area of clinical chemistry, liver function testing demands exceptionally high standards for raw material quality and supply stability. Through a one-stop raw material solution, you can not only significantly improve product development efficiency but also enhance the accuracy and reliability of end-user testing.
If you are developing or optimizing a liver function testing reagent system, choosing a professional, stable, and customizable partner like J&K Scientific is a key step to enhancing your market competitiveness.
艳 李
