Methods for Serum Total Bile Acids Test

Several assays have been used to determine both total or individual bile acids in biological fluids. The methods that have beenused specifically to analyze serum TBA are gas-liquid chromatography  (GLC), High Performance Liquid Chromatography (HPLC), enzymatic assays and enzyme cycling assays. GLC and HPLC methods are not commonly used in clinical laboratories where automated clinical chemistry analyzers are used for most of chemistry tests including TBA testing. The enzymatic assay (so called third generation TBA assay) is now mainly used in small laboratories where manual operations are allowed as the reagents of the 3rd generation TBA test are in lyophilized powder form, and manual reconstitution steps are needed before use. At present, the most widely used TBA test in clinical laboratories is the enzyme cycling method (also called the 5th generation TBA assay). That is a liquid-stable assay and ready to use for all types of automated chemistry analyzers.

1. Enzymatic method (3rd generation):


The enzymatic TBA assay method, as depicted in Figure 4, uses an enzyme, 3-a-hydroxysteroid dehydrogenase (3a-HSD), to catalyze the oxidation reaction converting 3-ahydroxyl group of all bile acids to 3-keto group with concomitant formation of a co-enzyme NADH from NAD+. The NADHformed is further reacted with nitrotetrazolium blue (NBT) toform a formazan dye in the presence of diaphorase enzyme. Thedye formation is monitored by measuring the absorbance at 540nm, which is directly proportional to the bile acids concentration in the serum sample.

2. Enzyme Cycling Amplification Method (5th generation):


The enzyme cycling assay is depicted in Figure 5 and is a method that allows for signal amplification through cycled regeneration reactions. In the enzyme cycling based TBA assay, serum bile acids molecules are repeatedly oxidized and reduced by the enzyme 3-a-hydroxysteroid dehydrogenase (3-a-HSD) with a concomitant accumulation of reduced co-enzyme thio-NADH that is detected at a specific wavelength (405 nm). As shown in the reaction scheme below, in the forward reaction, the enzyme catalyzes the oxidation reaction in the presence of co-enzyme thio-NAD+ to form oxidized bile acids and reduced co-enzyme thio-NADH. On the other hand, in the reverse reaction, the enzyme catalyzes the reduction reaction in the presence of excess co-enzyme NADH to convert oxidized bile acids back to bile acids which are then ready for the next round of forward oxidization reaction. The innovative use of this paired co-enzyme and co-enzyme analog enables a significant signal amplification, and therefore leads to a much higher detection sensitivity of the assay. The rate of thio-NADH formation is detected at 405 nm, and is proportional to the amount of TBA in the sample. The enzyme cycling amplification TBA assay offers analytical performance far beyond the capabilities of conventional bile acids test methods.

Total Bile Acid Assay Methods Comparison

Table 2 lists the advantages and disadvantages of enzymatic and enzyme cycling methods for TBA assay. The major advantages of the enzyme cycling assays over the conventional enzymatic assays are:

  • Liquid stable, ready to use
  • High detection sensitivity
  • Less interference from lipemic and hemolytic samples
  • Less sample volume needed
  • No instrumentation contamination by formazan dye

 

Diazyme Enzymatic
Signal Amplification
(Thio-NAD)

Conventional Methods

Comments

Reagent format

Liquid Stable

Powder

Diazymes liquid stable formulation requires no reagent preparation and is always ready to use

Sample volume

3-5 uL

20 uL

Smaller sample size is ideal for pediatric and veterinary application 

Interference by
lipemic samples

No

Yes

Diazymes method is recommended for post-parenial  specimens

Interference by hemolytic samples

No

Yes

 Improved confidence with challenging  pediatric and veterinary samples    

Instrument
contamination

No

Yes

Older MBT dyes can stain instrument cuvetts and reagent lines


Table 2. TBA assay methods comparison

Total Bile Acid Assay Methods Comparison
Interference Graphs
Figure 6
Figure 7
Figures 6 and 7 show the effects of triglyceride and hemoglobin on TBA assays of enzymatic colorimetric method (NBT method) and enzyme cycling method (thio-NAD method). As seen from these figures, the enzyme cycling based TBA assay method has significantly less lipemic and hemolytic interferences.