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.
On a global scale, 1.62 billion people are affected by anaemia which is equivalent to 24.8% of the population ₁. According to a review carried out by WHO of various national surveys, anaemia affects approximately 42% of pregnant women worldwide and it is also estimated that at least 50% of all anaemia cases are due to iron deficiency.
Anaemia caused by iron deficiency is usually expected during pregnancy. This is due to several reasons: the increased demand for iron by a pregnant woman’s body from increased total blood cell volume, requirements of the foetus and placenta as well as mass blood loss during labour₂. Although iron cost is unbalanced by the lack of loss of menstrual blood during pregnancy, the net cost is still high enough that iron recommendations are higher than in non-pregnant women. Also, iron is critical during pregnancy considering its involvement in foetal growth: 600-800mg of iron is required during pregnancy with around 300mg needed just for the foetus, a minimum of 25mg for the placenta and almost 500mg due to the increase in volume of red blood cells. ₃
Iron deficiency is the most common micronutrient deficiency in pregnant women leading to iron deficiency anaemia if left untreated. However, iron deficiency can be difficult to measure in some populations due to the lack of availability of field-specific biomarkers. For example, anaemia can affect up to 56% of pregnant women in developing countries, which suggests a high prevalence of iron deficiency anaemia: around 25%. In settings with endemic malaria, such as certain countries in Africa, the number of pregnant women with anaemia is much higher: around 65%.
There are various factors that may increase the risks of iron deficiency anaemia. For example, a diet influenced by religious beliefs can cause a lack of iron in the diet, such as vegetarianism which is common in countries such as India where religious beliefs dictate this. Iron levels can also be affected by consumption of nutrients which inhibit proper absorption of iron, such as calcium or ones that promote iron absorption, such as vitamin C. Other circumstantial risks include infections, multiple pregnancies and adolescent pregnancy while socioeconomic factors and access to healthcare mean some women won’t have access to anaemia control programs, iron supplements or even access to information about iron deficiency anaemia during pregnancy.
To prevent iron deficiency, international guidelines state that iron supplementation to manage iron deficiency is recommended during pregnancy. ₄ However, this is not always available, especially in developing countries.
Iron deficiency anaemia during pregnancy can cause several complications for the mother including:
- Increased fatigue
- Short-term memory loss
- Decreased attention span
- Increased pressure on the cardiovascular system due to insufficient haemoglobin and blood oxygen levels
- Lower resistance to infections
- Reduced tolerance to significant blood loss and surgical implications during labour.
As expected, neonates with mothers who suffered from iron deficiency anaemia during pregnancy will also be confronted with risks and, even if iron deficiency is only mild to moderate, can result in a premature birth, complications with foetal brain development, low birth weight and even foetal death. Additionally, it has been proven that cognitive and behavioural abnormalities can be seen in children for up to ten years after iron insufficiency in the womb.
Randox Reagents offer a Soluble Transferrin Receptor assay to expand upon our current iron testing offering.
In iron deficiency anaemia, soluble transferrin receptor levels are significantly increased, however, remain normal in acute phase conditions including: chronic diseases and inflammation. As such, sTfR measurements are useful in the differential diagnosis of anaemia: anaemia of chronic disease or iron deficiency anaemia.
In iron deficiency anaemia, increased sTfR levels have also been observed in haemolytic anaemia, sickle cell anaemia and B12 deficiency.
The benefits of the Randox Soluble Transferrin Receptor (sTfR) Reagent include:
- Latex enhanced immunoturbidimetric method facilitating testing on biochemistry analysers and eliminating the need for dedicated equipment.
- Liquid ready-to-use reagents for convenience and ease-of-use
- Stable to expiry date when stored at +2 to +8 °C
- Excellent measuring range of 0.5 – 11.77mg/L, comfortably detecting levels outside of the normal health range of 0.65 – 1.88mg/L
- Excellent correlation coefficient of r=0.977 when compared against other commercially available methods
- Applications available detailing instrument-specific settings for a wide range of clinical chemistry analysers
Find out more at: https://www.randox.com/stfr/
- de Benoist B et al., eds.Worldwide prevalence of anaemia 1993-2005. WHO Global Database on Anaemia Geneva, World Health Organization, 2008.
- Harvey et al, Assessment of Iron Deficiency and Anemia in Pregnant Women: An Observational French Study, Women’s Health, Vol 12 Issue 1, 2016
- Burke et al, Identification, Prevention and Treatment of Iron Deficiency during the First 1000 Days, Nutrients, Vol 6 Issue 10, 2014
- Guideline: Daily Iron and Folic Acid Supplementation in Pregnant Women. World Health Organization; Geneva, Switzerland: 2012
With this year’s World Kidney Day theme focusing on women’s health and in particular, their kidney health, the campaign is drawing attention to the need for a higher awareness, timely diagnosis and proper follow-up of kidney issues amongst women.
One key area being highlighted by the campaign is the close links between pregnancy and kidney health problems. The two are intrinsically connected – with CKD considered a high-risk factor for problematic pregnancies and reduced fertility, and in turn, pregnancy-related complications, including preeclampsia, can increase the risk of kidney disease.
Although not commonly known, women who have Chronic Kidney Disease are at increased risk of hypertensive disorders and premature births – which can be devastating for all involved.
Women with Chronic Kidney Disease who become pregnant also usually have mild kidney dysfunction, the severity of which will depend on the stage the CKD is at.
It is clear therefore that there is a need for increased awareness of Chronic Kidney Disease in pregnancy, to timely identify its existence before conception, and to monitor its progress before, during and after birth.
With a comprehensive panel of kidney health tests, Randox are working to ensure timely diagnosis of kidney function problems, to ensure that necessary treatment is administered at the earliest possible stage, when it is most likely to be successful.
Pregnant women, or women hoping to get pregnant in the future, can therefore determine their kidney health and be empowered to embark upon the necessary lifestyle changes or treatment required to ensure a safe and healthy pregnancy.
For example, the Randox test for albumin, low concentrations of which are the earliest marker of kidney damage, can identify individuals with diabetic nephropathy (damage to the kidneys caused by diabetes) around 10 years earlier than standard protein tests. The Randox albumin test can therefore enable preventative measures to be taken to reduce your risk of developing kidney disease.
In addition to albumin, there are a number of other highly specific and sensitive tests for kidney health, which are available as part of a Randox Health Check at our Randox Health Clinics. These include;
- Estimated Glomerular Filtration Rate, which is an equation that considers age, gender, blood and protein levels to determine how well the kidneys are functioning.
- Creatinine, which is a waste product produced by muscle tissue, and removed by the kidneys. When kidney function is diminished, creatinine levels increase.
- Other proteins within the body which should be filtered by the kidneys, and are therefore measured to determine kidney function, include;
– Cystatin C
– Microalbumin, which is not usually found in urine, but can appear when normal kidney function is impaired.
- Minerals processed by the kidneys and analysed by Randox Health include;
Both World Kidney Day and Randox are working towards improving healthcare worldwide. With access to these high-performance kidney health tests, expectant mothers with kidney problems can be diagnosed early, before the condition develops into something more serious – keeping both you, and your baby healthy.
With early diagnosis we can improve patient treatment outcomes and reduce the number of people across the world suffering with kidney health problems.
If you want to find out the status of your own Kidney Health, book a health check with Randox Health today. Speak to our team by phoning 0800 2545 130.