Total Bile Acids: The Value of Fifth Generation Tests
Bile acids are water-soluble, amphipathic end products of cholesterol metabolism and are involved in liver, biliary and intestinal diseases. They are formed in the liver and are absorbed in the small intestine before being excreted. The fundamental role of bile acids is to aid in the digestion and absorption of fats and fat-soluble vitamins in the small intestine.1
Intrahepatic Cholestasis of Pregnancy
Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder. It can be indicated by pruritus, jaundice, elevated total bile acids and/or serum transaminases and usually affects women during the second and third trimester of pregnancy.2,3
Intrahepatic Cholestasis of pregnancy or Obstetric Cholestasis is a condition that restricts the flow of bile through the gallbladder resulting in a build-up of bile acids in the liver.3 Due to the build-up, bile acids leak into the bloodstream where they are detected at concerning levels. It is an extremely serious complication of pregnancy that can lead to the increased risk of premature birth or even stillbirth, as such it is vital that women with the disease are monitored carefully.
In healthy pregnancies, there is very little increase in total bile acid levels although a slight increase is likely to be seen in the third trimester. Measurement of total bile acids in serum is thought to be the most suitable method of diagnosing and monitoring ICP.6
According to several reports total bile acid levels in ICP can reach as high as 100 times the upper limit of a normal pregnancy. It has been reported that a doubling in maternal serum bile acids, results in a 200% increased risk of stillbirth with total bile acids thought to trigger the onset of preterm labour. Additionally, bile acids can affect the foetal cardiovascular system as it has been found that there are often cardiac rhythm disturbances in the foetus due to the elevated bile acids in circulation.5
Although it is a rare condition, with only 0.3-0.5% of women likely to develop ICP, it can have extreme risks and so it is important to properly diagnose and monitor the condition.6 ICP increases the risk of meconium staining of the amniotic fluid and is reported to be a sign of foetal distress. This complication is found in 16-58% of all ICP cases, worryingly 100% of cases have resulted in foetal death. The frequency of this condition is found to be greater in pregnancies with higher levels of serum total bile acids.
There are several risk factors associated with ICP such as a family history of ICP, use of oral contraceptives, assisted reproduction techniques and multiple gestation. Genetic influence accounts for approximately 15% of ICP cases. Dietary selenium is a contributing environmental factor as serum selenium levels often decrease throughout pregnancy. Further to this, incidences of ICP rise in the winter months, most likely due to the fact selenium levels are naturally less during these months.7,8
Total Bile Acids
In addition to ICP, bile acid levels are also measured in the diagnosis of other liver disorders. The bile acids test in an extremely sensitive indicator of liver function, capable of detecting changes in hepatic function before clinical symptoms arise, thus providing valuable information that standard liver function tests cannot. As a result of its high sensitivity, bile acids can be used to assess liver function in transplant patients, allowing monitoring of the transplant success and of antirejection therapy. The bile acids test is most beneficial when used in conjunction with standard liver function tests such as ALT and AST which are markers of liver damage rather than liver function.
Measurement of Total Bile Acids
The enzyme cycling method, also known as the Fifth Generation Bile Acids test, is a method that allows for signal amplification through cycled regeneration reactions as can be seen in Figure 1. In the presence of Thio-NAD, the enzyme 3-α hydroxysteroid dehydrogenase (3-α HSD) converts bile acids to 3-keto steroids and Thio-NADH. The reaction is reversible and 3-α HSD can convert 3-keto steroids and Thio-NADH to bile acids and Thio-NAD. In the presence of excess NADH, the enzyme cycling occurs efficiently and the rate of formation of Thio-NADH is determined by measuring specific change of absorbance at 405 nm and is proportional to the amount of total bile acids in the sample. The analysing capability of the fifth generation total bile acids assay is far beyond the performance of conventional bile acid tests.10,11
Figure 1: The assay principle⁹
Inadequacies of Traditional Bile Acids Assays
Determining the cause and extent of liver damage is important in guiding treatment decisions and preventing disease progression. Standard liver function tests include; ALT, AST, ALP, GGT and Bilirubin. The measurement of TBA is most beneficial in conjunction with these standard liver tests and offers unrivalled sensitivity allowing identification of early stage liver dysfunction. There are several commercial methods available for the detection and measurement of TBA in serum. Traditional TBA tests based on the enzymatic method use nitrotetrazolium blue (NBT) to form a formazan dye. The reaction is measured at 546nm and the intensity of the colour is proportional to the concentration of bile acids.
Newer methods such as the enzyme cycling method or fifth generation methods offer many advantages including greater sensitivity, liquid reagents, small sample volumes and reduced instrument contamination from formazan dye. Additionally, the fifth generation assay does not suffer from interference from lipaemic or haemolytic samples. Both lipemia and haemolysis are common in new-borns and pregnant women, so this further supports that the fifth generation test is more sensitive for these sample types.12
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 The continuing importance of bile acids in liver and intestinal disease. A.f., Hofmann. 1999, Arch Intern Med, pp. 2647-2658.
 Diagnostic and Therapeutic Profiles of Serum Bile Acids in Women with Intrahepatic Cholestasis of Pregnancy – A Pseudo-Targeted Metabolomics Study. Cui, Yue. Xu, Biao. Zhang, Xiaoqing. He, Yifan. Shao, Yong. Ding, Min. s.l. : Clinica Chimica, 2018, Vol. 483.
 Randox Laboratories. Bile Acids Test for Obstetric Cholestasis – A serious complication of pregnancy. 2012.
 British Liver Trust (2019) Facts about Liver Disease, Available at: https://www.britishlivertrust.org.uk/about-us/media-centre/facts-about-liver-disease/ (Accessed: 18th June 2019).
 .Geenes, Victoria. Williamson, Catherine. 17, s.l. : World J Gastroenterol, 2009, Vol. 15.
 Howland, Genevieve. Cholestasis of Pregnancy: Why You Can’t Ditch the Itch. Mama Natural. [Online] December 22, 2018. [Cited: February 19, 2019.] https://www.mamanatural.com/cholestasis-of-pregnancy/.
 Bile Acid Levels and Risk of Adverse Perinatal Outcomes in Intrahepatic Cholestasis of Pregnancy: A Meta-Analysis. Cui, Donghua, et al.
 Intrahepatic Cholestasis of Pregnancy. Chivers, Sian. Williamson, Catherine. 7, 2018, Vol. 28.
 Masoud, N; Neill, S.H. Serum bile acids as a sensitive biological marker for evaluating hepatic effects of organic solvents. Available from URL: https://www.ncbi.nlm.nih.gov/pubmed/23885947 [Accessed 1 November 2018]
 Microassay of Serum Bile Acids by an Enzymatic Cycling Method. Komiyama, Y, et al. 10, s.l. : Chemical and Pharmaceutical Bulletin, 1982, Vol. 30.
 Evaluation of a Colorimetric Enzymatic Procedure for Determining the Total Bile Acids in the Blood. Agape, V, et al. 3, s.l. : Minerva Gastroenterologica e Dietologica, 1989, Vol. 35.
 Total Bile Acids Test & Clinical Diagnosis. Diazyme. 2019.
The aim of Biomedical Science Day is to raise the public’s awareness of the importance of biomedical science and the vital role it plays in the world. Randox are dedicated to improving healthcare worldwide through placing a major focus on research and development. The Randox scientists work in pioneering research into a range of common illnesses such as cancer, cardiovascular disease and Alzheimer’s disease.
A recent blog from Doris-Ann Williams, the Chief Executive at BIVDA, explains how “increased funding is not enough to sustain the NHS” and how “we need to make better use of in vitro diagnostics to ensure a successful future”.
The National Health Service (NHS) is a publicly funded, primarily taxation, national healthcare system in the United Kingdom. It was first set-up on July 5th, 1948 by Aneurin Bevan as he believed that everyone, regardless of wealth, should have access to good healthcare. Whilst the NHS is an extremely important aspect of healthcare in the UK, in vitro diagnostics are the heart and soul of the healthcare system as healthcare professionals not only rely on blood tests to diagnose and treat patients, but also to rule out the different contributing causes to a disease state. In vitro diagnostics also plays a key role in monitoring chronic disease states. In vitro diagnostics can also aid in reducing hospital stays, reduce misdiagnosis and support patients in looking after their own health and to deliver personalised treatment plans.
The Randox scientists have developed several niche assays to improve patient diagnosis, monitor treatment and eliminate misdiagnosis.
Adiponectin is a protein hormone secreted by adipocytes with anti-inflammatory and insulin-sensitising properties. It plays an important role in a number of metabolic processes including glucose regulation and fatty acid oxidation. Adiponectin levels are inversely correlated with abdominal visceral fat which have proven to be a strong predictor of several pathologies, including: metabolic syndrome, type 2 diabetes mellitus (T2DM), cancers and cardiovascular disease (CVD). For more information on the importance of testing Adiponectin levels, check out our Adiponectin Whitepaper.
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Small-dense LDL Cholesterol (sdLDL-C)
LDL Cholesterol (LDL-C) consists of two parts: the large and buoyant LDL Cholesterol and the small and dense LDL Cholesterol. Whilst all LDL-C transports triglycerides and cholesterol to bodily tissues, their atherogensis varies according to their size. As sdLDL-C is small and dense, they can more readily permeate the arterial wall and are more susceptible to oxidation. Research indicates that individuals with a predominance of sdLDL-C have a 3-fold increased risk of myocardial infarction. It has been noted that sdLDL-C carries less Cholesterol than large LDL, therefore a patient with predominately sdLDL-C particle may require nearly 70% more sdLDL-C particles to carry the same amount of cholesterol as the patient with predominately LDL-C particles. For more information on the importance of testing sdLDL-C levels, check out our sdLDL-C Whitepaper.
These three niche in vitro diagnostics tests developed by Randox scientists can aid in reducing NHS costs due to their higher performance compared to the traditional tests. Randox are constantly striving to improve healthcare worldwide.
For more information on the extensive range of Randox third-party in vitro diagnostic reagents, visit: https://www.randox.com/diagnostic-reagents/ or contact firstname.lastname@example.org.