Fifth module: Unconjugated vs conjugated bilirubin, and the signifigance of antioxidant power of the bilverdin-bilirubin cycle
Fifth module: Unconjugated vs conjugated bilirubin, and the signifigance of antioxidant power of the bilverdin-bilirubin cycle
Learn how bilirubin metabolism works, the limitations of bilirubin testing, and why jaundice is Nature's answer to stress and adverse conditions
It turns out, bilirubin is a powerful antioxidant! Our adult bodies, and our babies' bodies, use bilirubin to counter oxidative stress. In that context, bilirubin is more powerful than alpha-tocopherol (alpha-tocopherol is usually thought of as the most effective lipid-soluble antioxidant in the human species). Bilirubin is also thought to work in partnership with glutathione, another powerful antioxidant the body makes1.
When a baby is undergoing cellular stress (which all babies do thanks to the transition from placenta-supported womb-life to breathing oxygen-rich air), it makes sense for the baby’s body to make use of bilirubin. Bilirubin is readily available from the breakdown of blood cells (which all babies have to do), and the bilirubin cleans up some of that oxidative stress on it’s way out.
Here, we run into another paradigm shift.
Current models, everything shared so far, mainly account for the production and excretion of bilirubin, with excretion always being the main goal. The current view sees the re-entry of bilirubin through the enterohepatic pathway (see the graphic below) as an impediment to the excretion process. However, a newer view of of jaundice is emerging in the latest research—and in this view, the important physiological role bilirubin plays in maintaining the balance of oxidant and antioxidant forces is recognized.
For example, in a review published in 2023, the authors state:
Because bilirubin is involved in maintaining the balance between pro-oxidant and antioxidant agents, the downregulation of bilirubin levels is not always desirable.
Wang, J et al
If you trust Nature, you must trust that the pathway for bilirubin to re-enter a baby’s circulation exists for a good reason. Here’s our current understanding of how it works:
In this module, we are going to look at the right side of the above graphic, and then the left side. By the end, you should have a much more accurate picture of what is actually happening in each step of physiological jaundice. Knowing the ins and outs of each of these steps also means you will understand the what is in (and out of) our control in terms of supporting a baby who is going through jaundice. It will also help you understand the power and limitation of bilirubin testing.
Bilirubin creation and excretion (the right side of the graphic)
Bilirubin is made from old blood cells breaking down, ineffective red blood cell creation, and enzyme activity. There are a couple of forms bilirubin takes:
Indirect (unconjugated or free) bilirubin: water-resistant, this form can pass through the blood-brain barrier. In order to make it out of the body, it must bind to the protein albumin and be taken to the liver for further processing.
Direct (conjugated) bilirubin: water-friendly, this form is created in the liver and can combine with the bile and go further through the digestive system where intestinal bacteria will transform it into pigments that can be excreted in the urine and feces.
Total Serum Bilirubin (TSB): this is the sum of both forms of bilirubin. Even though conjugated bilirubin can’t pass through cell membranes or the blood-brain barrier, it is still included in clinical decision-making.
Factors that impair the process by which unconjugated bilirubin becomes conjugated
Unconjugated free bilirubin is the most dangerous, because it can directly pass through cell membranes and the blood-brain barrier. Here are the factors that reduce the body’s ability to conjugate it:
low albumin level or reduced-albumin binding capacity (certain drugs compete with bilirubin for albumin-binding, including sulfonamides, ceftriaxone, ibuprofen, and certain antibiotics—always ask about this possibility before accepting any new drugs for a newborn with jaundice)
trouble establishing breastfeeding
high-lipid parenteral diet in preterm infants (the fatty acids used can displace bilirubin and prevent it from being bound by albumin)
Bilirubin re-circulation (the left side of the graphic)
Under certain conditions (below), conjugated bilirubin can transform back into unconjugated bilirubin. When this happens, that unconjugated free bilirubin goes back into circulation instead of being excreted in the urine and feces. Here are the conditions that can increase the rate at which conjugated bilirubin goes back into circulation as unconjugated bilirubin:
breastfeeding
adverse PH (acidosis)
adverse temperatures (hypothermia)
hypoxia or asphyxia
certain drugs (including sulfonamides, ceftriaxone, ibuprofen and certain antibiotics)
It would be easy to think this was a mistake based on the last 4 of these conditions, but Nature doesn’t make mistakes, and breastfeeding should not trigger adverse events. So, what is going on?
Why would adverse conditions cause the body to re-uptake bilirubin?
As we started this module learning, bilirubin can act as a powerful antioxidant in the body. It makes sense, then, that if the body is going through an adverse event that increases oxidative stress that the body would start taking up more of the bilirubin to counteract this stress.
Here’s a reading that will put all of this into greater clarity. Notice where things we have been talking about, such as oxidative stress, mitochondrial health, and cellular respiration (ie, the electron transport chain) come up in this reading.
Antioxidant effect of bilirubin (excerpt from Bilirubin: The toxic mechanisms of an antioxidant molecule by Constanza P. Soto Conti, M.D.)
The best-known biological effects of bilirubin are its harmful effects on the Central Nervous System (CNS) of New Born Infants (NBIs) when it reaches high serum levels. However, molecular biology studies in adults have evidenced that bilirubin is a cytoprotective antioxidant.
Free Radicals (FRs) originate in the mitochondrial respiratory chain and are responsible for cellular oxidative damage. In aerobic organisms, cellular respiration originates ATP and water molecules.
In oxygen deprivation situations, this metabolic function generates FRs (superoxide anion, hydrogen peroxide, hydroxyl radical), which cause the lipid peroxidation of cell membranes and are involved in cancer pathophysiology, aging, heart disease, chronic inflammation, and infections. In Premature New Born Infants (PNBIs), FRs participate in disease pathogenesis, such as bronchopulmonary dysplasia, periventricular leukomalacia, necrotizing enterocolitis, patent ductus arteriosus, and retinopathy of prematurity.
Birth is an event that causes oxidation due to the change in oxygen pressures. PNBIs also experience a high tissue growth rate and immature or absent antioxidant molecules, such as superoxide dismutase, catalase, vitamins E and C, and beta-carotenes.
In 1937, Najib-Farah was the first person to postulate that bilirubin was part of a protective mechanism designed to overcome infections. In 1989, Glazer presented a hypothesis about the role of end products of metabolic pathways as cell protection agents (uric acid, the end product of purine metabolism, and taurine, the end product of cysteine metabolism). In this context, Stocker observed, in an in vitro study, that the antioxidant activity of bilirubin increased as oxygen levels decreased from 21 % (environmental concentration) to 2 % (physiologically relevant concentration, corresponding to that of tissues). Stocker documented that bilirubin had a greater action than alpha-tocopherol, the most effective lipid-soluble antioxidant in the human species. Stocker proposed the beneficial role of bilirubin as a product of metabolic waste, disruptor of the oxidation biological chain.
The bilirubin metabolism level involved in antioxidant functions is that where Heme Oxygenase (HO) and Biliverdin reductase (BVR) enzymes act. HO, which is induced by heme and proinflammatory lesions, has antiapoptotic, antioxidant, and anti-inflammatory effects. It transforms the heme molecule into biliverdin. The metabolic pathway is completed once biliverdin is reduced by BVR to bilirubin.
New hypotheses have suggested that, based on the extent of oxidation to which cells are exposed and in order to maintain their antioxidant homeostasis, they have bilirubin recovery pathways. These include the biliverdin-bilirubin redox cycle in the endoplasmic reticulum system and the cytochrome P450 pathway in the mitochondria, both with a reverse action compared to bilirubin metabolism.
In these metabolic pathways, bilirubin is oxidized to biliverdin, which will be once again reduced to bilirubin through the BVR enzyme. Based on such redox sequences, bilirubin captures FRs, detoxifies up to 10,000 times FR excess, and protects membrane lipids from peroxidation.
In amphibians, reptiles, and birds, heme metabolism stops at the level of biliverdin. In mammals, the antioxidant power of bilirubin justifies that the heme metabolic pathway progresses to bilirubin formation, although it requires a greater amount of enzymatic agents, a greater energy consumption, and albumin binding so it is transported and conjugated for excretion.
In adults, several publications established a relation between the antioxidant property of bilirubin and a better prognosis in diseases that cause oxidative stress: Alzheimer’s disease, ischemia-reperfusion injuries, cardiovascular disease, diabetes, inflammation, cancer. In Gilbert’s syndrome, which causes high indirect/unconjugated bilirubin (IB) levels, the frequency of ischemic heart disease is lower than in the general population (2% versus 12 %).
Science has made advances in the study of cellular damage caused by oxidative stress. Accurately establishing the role of FRs and molecules that are part of antioxidant defenses is a major challenge. In this context, a better understanding of bilirubin’s antioxidant capacity, its intermediate metabolites, and its complex enzymatic structure, will probably allow to take advantage of its potential benefit for PNBIs.
(Published in 2021)
So, why does modern jaundice treatment ignore the antioxidant activity of bilirubin? If bilirubin is being re-absorbed in order to counter oxidative stress, doesn’t it make sense to try to reduce oxidative stress as part of the treatment plan?
We know from many angles that balancing oxidative stress is a challenge for the health of both mamas and babies.
Unfortunately, the conventional model lacks good ways of dealing with oxidative stress in the newborn context. Therefore, it is not a big focus in current treatment plans.
For example, supplementing dietary antioxidants to newborns actually increases their risk of sepsis! This is because babies have an immature immune system that uses oxidants to fight invading pathogens. So, even though we know oxidative stress can be damaging, clinically there aren’t a lot of good options for measuring or treating it at present. However, lifestyle-wise, as we have been discussing, there is a lot we can do! We’ll get even more into this in the final modules of this course. For now…
Doesn’t it make sense that there is an interplay between:
A. The oxidative forces the newborn uses to fight germs and that naturally arise in the newborn period thanks to the transition to breathing air rather than being supported in the womb
and
B. The bilirubin (and other endogenous antioxidants) that helps prevent those same oxidative forces from damaging the baby’s own body?
It’s a fascinating balance.
And this is where that beautiful, cellular-made water from a healthy mitochondrial electron transport chain comes back into the picture in a huge way.
The latest research is showing that this mitochondria-produced water is not just any water, but actually organizes itself into a charge-based pattern.
The water we drink, liquid water (also called bulk water), is constantly moving. The water made inside our cells forms a matrix that is more like a gel or liquid crystal. Some are calling this gel-like water the Fourth Phase of Water, structured water, and Exclusion Zone (EZ) water. It shapes itself so that one side that is negative, and the other side is positive (like all water, its net charge is neutral). Some estimate that only 13% of the bulk water we drink turns into EZ water, with the rest of the EZ water being made directly inside of cells from free oxygen, hydrogen and electrons.
This special water transports electrons (antioxidant forces) and protons (oxidative forces) anywhere in the body. So, in this way of supporting hydration you can nurture both sides of the antioxidant-oxidant dance.
Mainstream science calls this dance redox. Because just like “keeping bilirubin down” isn’t as effective as working with the body’s physiological process, we also don’t want to just “keep oxidative stress down.” Oxidants also have biological roles. What we want is for the body to be able to fluidly balance the negative and positive charges (redox) wherever in the body they are needed.
(This powerful cellular hydration is a result of healthy mitochondria, which, as you will learn in the next module, is also imperative to preventing the cascade that can lead to brain damage and even death.)
So, with a healthy circadian environment supporting the production and recycling of cells at the right times and with excellent cellular hydration supporting redox, there remains one final thing that needs a fresh look: the blood-brain barrier. That is what the next module will be about.
Bilirubin testing
The lab test to measure unconjugated bilirubin is not widely used in clinical practice2. Instead, the more widely available TSB test values are used to make clinical decisions. TSB values include the conjugated bilirubin that does not pass through cell membranes. There is new evidence coming out that measuring the albumin-bilirubin ratio might be a better method of determining risk, but so far, testing of this also remains mostly unavailable in the clinical setting. Additionally, there is a known circadian control of bilirubin detoxification (meaning there will be a predictable high-low variation across the day). However, this has yet to be put into practice in terms of deliberately timing testing at a predictably high or low peak of the day.
Reading for Module 5
Compare and contrast the following two readings:
Cochrane review on jaundice treatment: https://www.evidentlycochrane.net/neonatal-jaundice/
Put your questions, aha moments, or key takeaways in the comments, then like this post to mark it as complete.
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Sedlak, T. W., Saleh, M., Higginson, D. S., Paul, B. D., Juluri, K. R., & Snyder, S. H. (2009). Bilirubin and glutathione have complementary antioxidant and cytoprotective roles. Proceedings of the National Academy of Sciences of the United States of America, 106(13), 5171–5176. https://doi.org/10.1073/pnas.0813132106
Lee, Z. M., Chang, L. S., Kuo, K. C., Lin, M. C., & Yu, H. R. (2023). Impact of Protein Binding Capacity and Daily Dosage of a Drug on Total Serum Bilirubin Levels in Susceptible Infants. Children (Basel, Switzerland), 10(6), 926. https://doi.org/10.3390/children10060926