Serum Bilirubin
Blood Tests

Author: alessandra pelle
Date: 08/02/2012



A short yet comprehensive description

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Specificity, sensitivity etc.

Diagnostic Algorithms


Open Questions

Chianale Valentina
Pelle Alessandra


We made some hypothesis about this topic :
What Happens after meals ? Does it change anything?

The answer is yes. Glycemia raises after eating but it’s not the explanation because after the parenteral nutrition bilirubinemia raises . In fact with long-term parenteral nutrition there are some changes in the normal mechanism responsible for bile production and flow that can causes hyperbilirubinemia.

Hepatobiliary dysfunction in infants and children associated with long-term total parenteral nutrition. A clinico-pathologic study.

The mechanism involved in bilirubin decreasing after meals are four:

  1. intestinal motility
  2. glucagon and heme oxydase
  3. insulin and MRP2
  4. Free oxygen radicals and NF-KB

Now we are going to explain them step by step.

1. Fasting decreases intestinal motility:

During fasting the peristalsis decreases and brings to a major time interval during which the bilirubin conyugated and bile remain in contact. Bile cuts the bond between glucuronic group and bilirubin changing conyugated bilirubin in unconjugated. Unconjugated bilirubin crosses the plasmatic membrane of enterocites and goes to the circulate. Fasting decreases intestinal motility and elimination of bile pigments. Accumulation of bilirubin in the intestine during fasting allows enhanced enterohepatic circulation and results in an increased reflux to plasma. This seems to be a major factor involved in fasting-induced hyperbilirubinemia.

Fasting-related hyperbilirubinemia in rats: the effect of decreased intestinal motility.1996

2. Glucagon and heme - oxydase

A possible mechanism that may be advanced to account for the positive relationship between fasting or hypoglycemia and the rise in serum bilirubin, is enhancement of bilirubin formation from increased heme turnover in the liver. In fact some studies about hepatic HO activity in rats show that HO activity increases progressively, reaching approximately 3 times the control value after 72 hours of fasting.
A study valuates HO activity in relation with some hormones that are enhanced during hypoglycemia and starvation and reduced after meals: glucagon, insulin, epinephrine, arginine (which triggers the release of endogenous glucagon). The results of this study are below.
Glucagon injection doubled the hepatic HO activity, while splenic HO activity remained unaffected (Table II). Glucose given together with glucagon did not abolish the stimulatory effect of the hormone (Table II). In animals whose hepatic HO had been stimulated by 48 hr fasting, glucagon had no additional effect on the enzyme activity (Fig. 2). However, on prolonged starvation, glucagon given at 72 and 92 hr doubled and tripled HO activity as compared to that in untreated rats fasted for the same length of time. Arginine, caused a fivefold increase in hepatic HO activity (Table II). This rise in activity exceeded the enzyme stimulation obtained with individual glucagon injections at 7 and 5, or 5 and 3 hr before assay (Table II). Epinephrine (0.1 mg/100 g rat) and glucagon (1 mg/100 g rat) given together in two i.p. injections 7 and 5 hr before enzyme assay produced additive enhancement of hepatic enzyme activity (Table II)
Insulin-induced hypoglycemia was associated with an increase in hepatic HO activity that was similar to that seen in fasted animals (Table I). This stimulatory effect of insulin was blunted or abolished when the injected insulin was "covered" with glucose administered simultaneously (Table I), which indicates that insulin by itself does not stimulate the enzyme.

Metabolic Regulation of Heme Catabolism and Bilirubin Production I.1972

TABLE I: Hepatic Heme Oxygenase Activity: Effect of Hypoglycemia

Treatment*n°of animalsenzyme activity
Saline60.05 4-0.02
Insulin, 1 IU40.12 :410.02
Insulin, 12 IU40.34 :10.04
Insulin, 12 IU plus glucose40.13 :10.03
Insulin, 1 IU plus glucose50.06 :10.02
Mannose50.33 :4:0.05
  • For experimental details, see text.
    $ nmole bilirubin formed per min per 10 mg protein A:SD.

    FIGURE II: Hepatic heme oxygenase activity in rats treated with epinephrine or glucagon after fasting for 48, 72, or 92 hr. Enzyme activity in untreated fasted rats is indicated by the continuous line.

3. Insulin and MRP2

Another mechanism that could be involved in the decreasing of bilirubin after meals could be the insulinic regulation. In fact after meals there is a peak of insulin to regulate glycemia that influences bilirubin levels. Now we explain why insulin regulates the expression of MRP2.
Insulin is involved in the expression of the Mrp2 transporter, an ABC trasporter.


In fact there is a consistent reduction of normal levels of Mrp2 in insulinopenic animals like Zucker rats (animals with defective brain leptin dependent signal transduction due to either lack of leptin production or a dysfunctional leptin receptor, resulting in markedly increased food intake and decreased energy expenditure, which are associated with obesity, insulin resistance, and fatty liver).

Figure I: Indirect immunofluorescent localization of multidrug resistant associated protein 2 (Mrp2) in 14 week old control and obese Zucker rats (ZR). Frozen liver sections from control (A) and obese (B) ZR were used to assess qualitative distribution of Mrp2 by indirect immunofluorescence. Decreased labelling of canalicular membranes was observed accounting for reduction in Mrp2 protein expression in obese ZR.

Figure II: shows representative images of immunostaining of Mrp2, which was downregulated in obese ZR, demonstrating decreased labelling of the canalicular membrane in livers from obese rats.
Thus it seems likely that insulin resistance is involved in the observed downregulation of Mrp2 in obese ZR.
Mrp2 downregulation appeared to involve both transcriptional and post-transcriptional events, because no changes in mrp2 mRNA levels were detected by northern blot analysis in eight week old animals but a significant decrease in Mrp2 message was seen in older animals (fig 3).

In this experiment the effects of reduction of insulin resistance in obese animals was studied after administration of the proliferator activated receptor c (PPAR-c) agonist rosiglitazone.
Treatment of obese ZR with rosiglitazone reversed some features of insulin resistance, and significantly increased Mrp2 protein mass by twofold.
(In this study TNF- a was also indagated in the role of the mrp2 expression. Etanercept, a TNF-a binding moiety derived from soluble TNF-a receptor subunits was given to obese ZR to study the effects of neutralisation of this inflammatory cytokine on bile secretory function. After administration of etanercept we did not found any significant changes Mrp2 protein levels in etanercept treated rats compared with non-treated obese ZR (fig 5)).

Figure III: Effect of etanercept or rosiglitazone administration on multidrug resistant associated protein 2 (Mrp2) protein mass in obese Zucker rats (ZR).
The anti-tumour necrosis factor a inhibitor etanercept (ETN) was given to 14 week old obese ZR in a single intraperitonealinjection of 8 mg/kg. Mrp2 protein mass was assessed 72 hours later.
Rosiglitazone (RGZ) was given by gavage at a dose of 3 mg/kg/day over 10 days. Mrp2 protein levels in plasma membrane fractions of treated and untreated obese rat livers were measured as described in materials and methods. (A) Each band represents the results of a single animal. Representative experiments with three rats per group are shown. (B) Bar diagram showing western blot band volume as per cent of control animals. A significant increase in Mrp2 protein levels was seen after RGZ treatment while no significant changes were observed after etanercept administration.

Bile secretory function in the obese Zucker rat: evidence of cholestasis and altered canalicular transport function 2004
Another study which prove this theory is this one:
Spontaneously diabetic biobreeding rats and impai... [Hepatology. 1992] - PubMed - NCBI.

4. ROS and bilirubin

Because oxygen consumption or energy expenditure increases after a meal (the thermic effect of food), it would be reasonable to postulate a food-induced increase in free radical (ROS and/or RNS) production from the increased oxygen metabolism and thus a food-induced decrease in serum antioxidant capacity. ROS activate IKK/IKB pathway which activates NFKB.

Antioxidants e ROS after meals.2001

Why Hyperbilirubinemia can be reduced by activation of Nfkb?
NFKB regulates the expression of UGT1A1 in fact when NFKB is inactivated UGT1A1 is suppressed . After meals NFKB increases so UGT1A1 raises and bilirubin is glucuronated by this enzime so bilirubin is eliminated with bile pigments and is not adsorbed anymore.
This hypothesis is proved in part by a study in mice about breast milk. In this study mices with human UGT 1A1 were fed with human breast milk and it was examined inflammatory signaling pathways IκB-kinase–α and IκB kinase–β in the intestinal epithelium. .
In comparing UGT1A1 gene expression by quantitative real-time reverse transcription PCR, the levels of UGT1A1 RNA gene transcript are nearly 5-fold greater in GI fetal tissue on embryonic day 20 (E20) when compared to levels observed after birth when the newborn mice have been nursing for 12 hours (Figure 1A). These findings suggest that exposure to breast milk leads to a reduction or suppression of intestinal UGT1A1 gene expression.
Breast milk appeared to suppress intestinal IκB kinase α and β, resulting in inactivation of nuclear factor–κB and loss of expression of UGT1A1, leading to hyperbilirubinemia

Figure: Effects of breast milk and formula on serum bilirubin levels and gene expression in hUGT1 mice. (A) RNA was isolated from intestinal tissue from hUGT1 mice at embryonic day 20 (E20) and 12 hours after birth (12 hr). Quantitative real-time reverse transcription PCR was performed to measure relative expression for UGT1A1. Fold induction of the genes is expressed as compared to E20 mice. (B) Newborn pups were nursed (N), fed with formula (F) or human breast milk (HBM) for 5 days. At 14 days, serum bilirubin levels were measured. © Gene expression by quantitative real-time reverse transcription PCR of UGT1A1, Cyp2b10, and Cyp3a11 were determined from small intestine RNA isolated after 5 days of formula and HBM treatment. Fold induction of the genes in the formula-fed mice is expressed as compared to nursing mice. (D) Immunoblot of microsomal human UGT1A1, mouse CYP2B10, and CYP3A11 from small intestinal tissue is shown after formula and HBM treatment. The density of the bands was also quantified. (E) Body weight of mice was determined. (F) Nine-day-old hUGT1 mice were orally treated with 50 mg/kg

Reduced Expression of UGT1A1 in Intestines of Humanized UGT1 Mice via Inactivation of NF-κB Leads to Hyperbilirubinemia. 2012

Physiological Antioxidative Network of the Bilirubin System in Aging and Age-Related Diseases, 2012

In this regard, the biliverdin to bilirubin conversion pathway, via biliverdin reductase (BVR), is suggested to be another major protective mechanism that scavenges lipophilic oxidants because of the lipophilic nature of bilirubin.

Working Hypothesis

Fasting-related hyperbilirubinemia in rats: the effect of decreased intestinal motility. 1996"
|Pubmed |Metabolic Regulation of Heme Catabolism and Bilirubin Production I 1972|
|Pubmed |Bile secretory function in the obese Zucker rat 2004|
|Pubmed |Antioxidants e ROS after meals. 2001|
|Pubmed |Reduced Expression of UGT1A1 in Intestines of Humanized 2012|

Working Hypothesis

Oral treatment of unconjugated hyperbilirubinemia, Groningen 2006

Lavori interessanti bilirubin and inflammation

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