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Glucuronidated bilirubin: Significantly increased in hepatic encephalopathy
CHAPTER TWENTY-TWO
Glucuronidated bilirubin: Significantly increased in hepatic encephalopathy Limin Tanga, Meng Zhangb, Xiulian Lib, Lijuan Zhangb,* a
Department of Radiology, Affiliated Hospital of Qingdao University, Qingdao, China Systems Biology and Medicine Center for Complex Diseases, Affiliated Hospital of Qingdao University, Qingdao, China *Corresponding author: e-mail address: [email protected] b
Contents 1. Introduction 2. Serum bilirubin measurement 3. Serum direct bilirubin levels in 38 different types of human diseases and healthy controls 3.1 Methods 3.2 Statistical analysis 3.3 Results 4. Discussions and conclusions Acknowledgment Conflict of interest References
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Abstract Bilirubin is produced by the breakdown of hemoglobin in senescent erythrocytes by macrophages and carried by albumin from blood circulation to the liver for removal in normal physiology. Glucuronic acid modification of bilirubin by UDPglucuronyltransferase in the liver is the key event for its subsequent elimination from human body. Conditions that accelerate the breakdown of erythrocytes may cause an elevated blood level of unconjugated bilirubin whereas the factors affect the glucuronidated bilirubin formation and subsequent elimination may cause decreased or increased blood level of glucuronidated bilirubin, the water soluble “direct bilirubin” measured by clinical blood test. Studies showed that increased total serum bilirubin has a protective effect on cardiovascular and other related diseases, but it is unknown how direct bilirubin levels were related to different diseases. By taking advantage of the data collected in the clinical laboratory of our hospital, the direct bilirubin data from 192,535 patients with 72 clinically defined diseases were compared to that of healthy controls (10,497). Based on the mean, median, and p values, we found that patients with hepatic encephalopathy had the highest serum direct bilirubin level, which resembled acute
Progress in Molecular Biology and Translational Science, Volume 162 ISSN 1877-1173 https://doi.org/10.1016/bs.pmbts.2018.12.009
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hepatic encephalopathy caused by increased serum direct bilirubin level in neonates. In contrast, patients with uremia, nephrotic syndrome, and preeclampsia had significantly lower levels of serum direct bilirubin. Taken together, our data revealed that serum direct bilirubin levels were either increased or decreased in a disease-dependent manner. The possible molecular mechanisms of increased direct bilirubin levels in patients suffering hepatic encephalopathy are discussed.
1. Introduction Bilirubin is a waste product produced by the breakdown of heme, a component of hemoglobin, when macrophages either in spleen or liver remove senescent erythrocytes. Heme consists of a Fe2+ ion in the center of porphyrin. The production of biliverdin begins when heme oxygenase-I metabolizes heme1, which is followed by the enzyme biliverdin reductase that converts biliverdin into bilirubin.2 The water insoluble bilirubin released by macrophages of the spleen is transported by albumin from blood circulation to the liver. UDP-glucuronosyl transferase family 1 member A13 in the liver makes bilirubin soluble by modifying bilirubin with one or two covalently linked glucuronic acids, which is the key event for subsequent elimination of bilirubin from the body4. Interestingly, only glucuronidated bilirubin can react with albumin nonenzymatically to form δ-bilirubin, a covalently linked albumin–bilirubin conjugate found in human serum.5–9 Thus, serum total bilirubins include unconjugated bilirubin and the water soluble bilirubins containing monoglucuronidated bilirubin, diglucuronidated bilirubin, and δ-bilirubin. Since δ-bilirubin formation uses glucuronidated bilirubins as substrates, the so-called “direct bilirubin” is the three glucuronidation-related water soluble bilirubins. The UGT1A1 gene is part of a complex locus that encodes several UDPglucuronosyl transferases.10,11 The locus includes 13 unique alternate first exons followed by four common exons. Four of the alternate first exons are considered pseudogenes. Each of the remaining nine 50 exons may be spliced to the four common exons, resulting in nine UDP-glucuronosyl transferases with different N-termini and identical C-termini. Each first exon encodes the substrate binding site that makes each UDPglucuronosyltransferase unique. The preferred substrate for UDPglucuronosyltransferase family 1 member A1 is bilirubin, although it also has moderate activity with simple phenols, flavones, and steroids. Mutations
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in this gene result in Crigler–Najjar syndromes types I and II12–14 and in Gilbert syndrome,15–17 which are severe to mild liver disorders. A series of studies indicate that glucuronidation of bilirubin is a rate-limiting step for the disposal of bilirubin in animals and the cause of various diseases.18–20 Approximately 250–350 mg of bilirubin is produced daily in healthy adults. Humans are naturally hyperbilirubinemia compared to other mammals with normal ranges of serum total bilirubin levels from 1 to 10 μmol/L. In general, increased unconjugated bilirubin level may be associated with unusual hemolysis or when the liver is unable to uptake or process bilirubin. Conversely, increased “direct” bilirubin level may indicate that the liver is able to modify bilirubin with glucuronic acid but is not able to pass the glucuronidated bilirubin to the bile for removal. In clinic, bilirubin is used to monitor diseases such as cirrhosis, hepatitis, or gallstones and to determine the causes of hemolytic anemia.21,22 Since almost every newborn is hyperbilirubinemia with bilirubin level great than 30 μmol/L during the first week of life, a rapid laboratory tests for both total and direct bilirubin are required in order to start therapy to prevent acute bilirubin encephalopathy in neonates.23,24 Since the discoveries that moderately increased levels of serum bilirubin are correlated with a significantly decreased risk of coronary artery disease in individuals with Gilbert syndrome,25–27 the antioxidant, antiinflammation, and antitumor properties of bilirubin in blood circulation and its beneficial effects in aging and different diseases have been reported.28–39 However, it has not been reported if direct bilirubin levels were related to different diseases except for a few cases.40,41 Most importantly, only direct bilirubin can pass blood brain barrier and is neurotoxic at sufficiently high concentrations24. It will be important to know if adult hepatic encephalopathy was associated with increased serum direct bilirubin level as well. By taking advantage of the data collected in the clinical laboratory of our hospital, the direct bilirubin data from 192,535 patients with 72 clinically defined diseases were compared to that of normal controls (10,497) with at least 100 datasets for each type of disease. Based on the mean, median, and p values, we found that patients with hepatic encephalopathy had the highest serum direct bilirubin level. In contrast, patients with uremia, nephrotic syndrome, and preeclampsia had significantly lower levels of serum direct bilirubin. Taken together, our data revealed that serum direct bilirubin levels were either increased or decreased significantly in a diseasespecific manner.
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2. Serum bilirubin measurement Total serum bilirubins comprise unconjugated bilirubin of the insoluble fraction that is attached to serum albumin no-covalently and conjugated bilirubins of the water soluble bilirubin including monoglucuronide, bilirubin diglucuronides, and δ-bilirubin (covalently linked bilirubin–albumin conjugate). Currently, most clinical laboratories rely on automated analyzers for rapid bilirubin determinations for multiple samples42. Two forms of bilirubin in the serum are measured by the clinical tests: “direct” bilirubin level (conjugated/glucuronidated bilirubin and δ-bilirubin) and total bilirubin level (unconjugated plus direct bilirubin). Clinical test also provides the “indirect” level of unconjugated bilirubin by subtracting direct bilirubin level from the total bilirubin level. All methods used for the measurements of serum bilirubin are based on one of the two properties of bilirubin: (1) The intrinsic absorption spectra of different bilirubin fractions with the absorption maximum of 463 nm for unconjugated bilirubin and 453 nm for conjugated bilirubin and loss of the absorption after reducing bilirubin to biliverdin by oxidase or vanadate. (2) The novel absorption spectra of bilirubin after the diazo reaction. Based on the detection method, bilirubin fractions can be measured by highperformance liquid chromatography (HPLC) method, direct spectrophotometry method, the diazo derivatization method, and oxidation method, respectively9. The diazo reaction has been used for serum bilirubin quantification since 1950s and still is the most widely used method. In this method, the color of azobilirubin formed by the reaction of the porphyrin rings of bilirubin with a diazo compound is spectrophotometrically measured. Because unconjugated bilirubin reacts slowly, accelerators such as methanol, ethanol, or caffeine, are used to release unconjugated bilirubin from the protein carrier albumin, the total bilirubin level is then measured. In the absence of these accelerators, the director bilirubin level can be measured by this method.43,44 However, a small portion of unconjugated bilirubin is also detected by the diazo method as direct bilirubin.45,46 Moreover, only 76%–89% of direct bilirubin is reported to be detected as direct bilirubin5. The accuracy of bilirubin measurements by the diazo method is influenced by coexistent of other serum proteins or compounds, such as hemoglobin, immunoglobulins, and ascorbic acid.47–51 Therefore, the accuracy of direct
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bilirubin measurement by the diazo assay has been an issue needed to be addressed52. HPLC method, which can separate all four bilirubin fractions, is a reliable method for bilirubin quantification in theory, but impractical for clinical use because of labor costs and long turnaround time6. Efforts to develop alternative clinical assays for bilirubin quantification have led to the use of bilirubin oxidase to measure direct bilirubin.53–56 In this method, bilirubin oxidase catalyzes the oxidation of bilirubin to biliverdin, which causes the characteristic absorption for bilirubin to disappear. Thus, direct bilirubin can be exclusively detected by bilirubin oxidase method. The vanadate method is based on oxidation of bilirubin to biliverdin by the chemical vanadate as an oxidizing agent. In this method, all bilirubins are oxidized by vanadate in the presence of detergent to produce colorless products. In the absence of the detergent, only direct bilirubin in serum is oxidized by the vanadate and measured. The precision studies reveal that the vanadate method has comparable between-run and within-run CVs to those of the diazo method57. The vanadate method is less interfered by the coexisting serum proteins and other compounds. Thus, the direct bilirubin measurement is more reliable than that of the diazo method.
3. Serum direct bilirubin levels in 38 different types of human diseases and healthy controls 3.1 Methods Serum samples for the detection of direct bilirubin levels were obtained retrospectively from the clinical laboratory of The Affiliated Hospital of Qingdao University over the past 5 years (2012–17). The serum direct bilirubin levels were analyzed using an autoanalyzer (Beckman Coulter AU5800, USA) with assay kit purchased from Beijing Lidman Biochemical Co., Ltd. It is a vanadate assay kit and this assay is more reliable in quantifying serum direct bilirubin levels compared to that of diazo assay. The normal values of total bilirubin are 1.71–17.1 μmol/L and direct bilirubin are 1.71–7.0 μmol/L in healthy controls. The current study including the direct bilirubin data from 192,535 patients with 72 clinically defined diseases were compared to that of normal controls (10,497) with at least 100 datasets for each type of disease.
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3.2 Statistical analysis Statistical analysis was performed by SPSS version 19 (Inc., Chicago, USA) and all p values were two side. The Mann–Whitney test was used for the nonnormal distribution data, with p values <0.05 considered significant. All data were presented as the median (interquartile range) or mean (SD).
3.3 Results The clinical laboratory of our hospital measured the serum direct and total bilirubin levels routinely for either patients requested by the doctors and healthy populations during their annual physical examination. To this end, we collected all the clinical serum direct bilirubin assay data from the clinical laboratory of our hospital during the past 5 years. As a result, we obtained the direct bilirubin data from 192,535 patients with 72 clinically defined diseases and 10,479 healthy controls during their annual physical examination. Based on these data, we calculated and listed the mean, median, and p values of direct bilirubin levels for each type of disease in Table 1. In order to make the results more intuitive, we plotted a scatter diagram of direct bilirubin values for each type of disease with lower quartile (25%), median (50%), and upper quartile (75%) ranges marked. As shown in Fig. 1, the median values of direct bilirubin in 12 diseases were higher than that of healthy controls. Among all the diseases, patients suffering hepatic encephalopathy, cirrhosis, pancreatic cancer, pancreatitis, and liver cancer had direct bilirubin levels higher that of healthy controls. Unexpectedly, patients with uremia, nephrotic syndrome, preeclampsia, lupus erythematous, diabetic nephropathy, breast cancer, and nephritis had significantly lower levels of direct bilirubin compared to that of healthy controls. Moreover, certain patients after gastric surgery, with hepatitis, or suffering sepsis had very higher levels of serum direct bilirubin compared to patients with other diseases. Using the median values, we then calculated the p values as shown in Fig. 2. The p value is used in the context of null hypothesis testing in order to quantify the idea of statistical significance of evidence. Null hypothesis testing is a reduction argument adapted to statistics. In essence, the statistical significance is assumed valid when the p values are far apart from that of control values. Unexpectedly, patients with cirrhosis, pancreatitis, pancreatic cancer, hepatic encephalopathy, liver cancer, myeloproliferative disorders, and acute cerebral infarction had highest log10 p values
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Table 1 The mean, median, and log10 p values of serum direct bilirubin levels for healthy controls and patients with cancer and other diseases. No. of 2Log10 p DBIL cases Mean (SD) Median value
Hepatic encephalopathy
213
39.00 (47.70) 24.90
137.23
Cirrhosis
8467
26.50 (38.21) 12.00
>176
Pancreatic cancer
1050
30.99 (52.45)
7.00
110.35
Pancreatitis
1636
10.52 (13.19)
6.22
139.14
Liver cancer
258
12.76 (33.81)
6.20
40.07
Acute myocardial infarction
2298
5.89 (3.06)
5.20
55.48
Myeloproliferative disorder
1162
5.97 (3.40)
5.10
22.88
Bladder stone
151
5.30 (1.84)
5.00
4.64
Healthy controls >65 years old
1209
4.91 (1.57)
4.80
7.32
Acute cerebral infarction
8657
5.02 (2.10)
4.62
12.27
Healthy controls
10,497
4.65 (1.55)
4.56
0.00
Gastric ulcer
270
4.74 (2.16)
4.51
0.51
Gastric cancer
12,000
5.12 (2.87)
4.50
0.99
Bone fracture
1808
4.91 (2.26)
4.50
0.54
Coronary heart disease
19,828
4.86 (2.29)
4.40
1.56
Sepsis
273
7.46 (10.22)
4.37
0.62
Cerebrovascular disease
4147
4.63 (1.92)
4.32
5.69
Gout
1341
4.51 (1.81)
4.22
6.73
Hepatitis
6940
6.45 (7.60)
4.20
6.70
Intracranial hemorrhage
3532
4.83 (2.54)
4.20
5.75
Chronic obstructive PD
1464
4.75 (2.45)
4.20
5.02
Esophagus cancer
3547
4.58 (2.13)
4.20
13.86
Rectum cancer
7600
4.55 (2.15)
4.20
24.20
Cerebral ischemia
2097
4.42 (1.79)
4.20
13.76
Renal cyst
476
4.39 (1.49)
4.20
3.69
Alzheimer’s disease
94
4.60 (2.26)
4.17
1.38 Continued
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Table 1 The mean, median, and log10 p values of serum direct bilirubin levels for healthy controls and patients with cancer and other diseases.—cont’d No. of 2Log10 p DBIL cases Mean (SD) Median value
Anemia
2499
5.17 (3.70)
4.11
7.88
Bladder cancer
982
4.43 (2.05)
4.10
12.16
Colon cancer
5920
4.50 (2.27)
4.04
37.98
Aplastic anemia
1203
4.62 (2.77)
3.97
12.87
Brain trauma
639
4.37 (2.44)
3.90
14.00
Encephalitis
672
4.29 (2.61)
3.90
18.14
Hyperuricemia
147
4.07 (1.70)
3.84
5.56
Leukemia
8601
4.26 (2.33)
3.80
137.12
Bone cancer
101
4.01 (1.95)
3.72
5.87
Lung fibrosis
305
4.00 (1.88)
3.67
14.46
Cerebral arteriosclerosis
739
3.87 (1.69)
3.60
40.28
Gastritis
3091
3.90 (1.93)
3.58
140.28
Lymphoma
4441
4.01 (2.22)
3.54
167.59
Type 2 diabetes mellitus
10,424
3.78 (1.74)
3.50
>176
Asthma
576
3.84 (1.76)
3.48
37.58
Cystitis
63
3.57 (1.39)
3.46
7.17
Osteoporosis
241
3.64 (1.72)
3.40
22.02
Multiple myeloma
2050
3.71 (2.00)
3.37
150.33
Wilms’ tumor
248
3.29 (1.45)
3.30
36.51
Breast lumps
100
3.36 (1.86)
3.25
13.71
Psoriasis
135
3.51 (1.82)
3.21
15.51
Lung cancer
9740
3.50 (1.63)
3.20
>176
Kidney cancer
1419
3.36 (1.59)
3.10
171.78
Necrosis of femoral head
172
3.17 (0.98)
3.07
36.60
Osteoarthritis
78
3.66 (2.11)
3.00
9.78
Endometrial cancer
1090
3.17 (1.37)
3.00
175.86
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Table 1 The mean, median, and log10 p values of serum direct bilirubin levels for healthy controls and patients with cancer and other diseases.—cont’d No. of 2Log10 p DBIL cases Mean (SD) Median value
Knee-joint degenerative diseases
442
3.07 (1.07)
2.90
98.60
Cervical cancer
2050
3.02 (1.33)
2.89
>176
Azotemia
441
2.84 (1.47)
2.70
107.57
Postbreast surgery
38
7.33 (25.32)
2.69
6.65
Rheumatic arthritis
419
3.05 (1.95)
2.66
81.89
Ovarian cancer
2171
2.83 (1.28)
2.61
>176
Ankylosing spondylitis
77
2.89 (1.40)
2.60
20.08
Nephritis
2173
2.76 (1.51)
2.56
>176
Breast cancer
4923
2.60 (1.29)
2.40
>176
Diabetic nephropathy
540
2.53 (1.55)
2.20
158.75
Lupus erythematosus
1272
2.40 (1.66)
2.09
>176
Preeclampsia
877
2.25 (1.35)
1.99
>176
Nephrotic syndrome
3877
2.14 (1.28)
1.90
>176
Uremia
5759
2.03 (1.30)
1.79
>176
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Fig. 1 The direct bilirubin values in 50 different types of clinical diseases. The data were sorted in a descending order of the median values. Direct bilirubin values for each type of disease with lower quartile (25%), median (50%), and upper quartile (75%) ranges were marked in red.
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Fig. 2 Log10 p values for direct bilirubin in 50 different types of clinical diseases compared to that of healthy controls. When the serum direct bilirubin levels were lower than that of healthy controls, blue color was used in the plot whereas when the serum direct bilirubin levels were higher than that of healthy controls, red color was used in the plot.
compared to that of healthy controls, which made direct bilirubin level an outstanding biomarkers for these diseases. Taken together, both median and p values supported the facts that increased direct bilirubin levels were useful for diagnosis of multiple diseases, especially for hepatic encephalopathy. In addition, the decreased serum bilirubin levels based on the p values could be served as biomarkers for many different types of human diseases, not limited to uremia, nephrotic syndrome, preeclampsia, nephritis, ovarian cancer, cervical cancer, lung cancer, type 2 diabetes, and endometrial cancer.
4. Discussions and conclusions The current study demonstrated that patients with hepatic encephalopathy had the highest serum direct bilirubin level. In contrast, patients with uremia, nephrotic syndrome, and preeclampsia had significantly lower levels of serum direct bilirubin. The significantly low p value shown in Table 1 indicated that serum glucuronidated bilirubin level might be sufficient to serve as a biomarker for patients suffering hepatic encephalopathy and other diseases. Hepatic encephalopathy is an altered level of consciousness due to liver failure58. Increased total bilirubin levels have been closely monitored to prevent neonatal hepatic encephalopathy. Moreover, a series of elegant studies indicate that high direct bilirubin levels but not unconjugated bilirubin levels are responsible for the observed neuron toxicity both in vitro and in vivo.24,59–64 In contrast, for adult hepatic encephalopathy, the role of direct bilirubin played is largely ignored. The underlying molecular mechanism for adult is believed to be involved in the buildup of blood ammonia that cause damage of brain in adult.65 Our data shown in Table 1 and Figs. 1
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and 2 suggested that the increased direct bilirubin levels might be responsible for adult hepatic encephalopathy as well. In theory, conditions affect the glucuronidated bilirubin formation may cause decreased blood level of glucuronidated bilirubin whereas conditions affect the glucuronidated bilirubin elimination may cause increased blood level of glucuronidated bilirubin. Paradoxically, the data presented in Table 1 and Figs. 1 and 2 showed that hepatic encephalopathy, cirrhosis, pancreatic cancer, pancreatitis, and liver cancer were conditions associated with increased direct bilirubin levels whereas uremia, nephrotic syndrome, diabetic nephropathy, and nephritis were conditions associated with decreased direct bilirubin levels. There results indicated that the regulations of serum direct bilirubin levels might not be as intuitive as previously thought. It is known that the accuracy of bilirubin measurements by the clinical diazo method is influenced by the coexistent of hemoglobin, immunoglobulins, and ascorbic acid and the diazo method is high sensitivity to hemolysis.66,67 The reliability of clinical method in measuring direct bilirubin has constantly been challenged52. The vanadate method used in our clinical test is less interfered by the coexisting serum proteins and other compounds, the direct bilirubin data presented in Table 1 and Figs. 1 and 2 should be reliable. However, our current study is retrospective, prospective study is required to test if direct bilirubin levels could serve as biomarkers for hepatic encephalopathy and other diseases.
Acknowledgment This research was supported by the Natural Science Foundation of China (Grant 81672585), Key Technology Fund of Shandong Province (Grant 2016ZDJS07A07), the Taishan Scholar Fellowship, and the “Double First Class fund” of Shandong Province in China to L.Z.
Conflict of interest The authors declare no conflict of interest.
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27. Schwertner HA, Vitek L. Gilbert syndrome, UGT1A1*28 allele, and cardiovascular disease risk: possible protective effects and therapeutic applications of bilirubin. Atherosclerosis. 2008;198(1):1–11. 28. Kao TW, Chou CH, Wang CC, Chou CC, Hu J, Chen WL. Associations between serum total bilirubin levels and functional dependence in the elderly. Intern Med J. 2012;42(11):1199–1207. 29. Fujiwara R, Haag M, Schaeffeler E, Nies AT, Zanger UM, Schwab M. Systemic regulation of bilirubin homeostasis: potential benefits of hyperbilirubinemia. Hepatology. 2018;67(4):1609–1619. 30. Sedlak TW, Snyder SH. Bilirubin benefits: cellular protection by a biliverdin reductase antioxidant cycle. Pediatrics. 2004;113(6):1776–1782. 31. Lin JP, Vitek L, Schwertner HA. Serum bilirubin and genes controlling bilirubin concentrations as biomarkers for cardiovascular disease. Clin Chem. 2010;56(10):1535–1543. 32. Tomaro ML, Batlle AM. Bilirubin: its role in cytoprotection against oxidative stress. Int J Biochem Cell Biol. 2002;34(3):216–220. 33. Liu M, Li Y, Li J, Lv X, He Y. Elevated serum total bilirubin levels are negatively associated with major diabetic complications among Chinese senile diabetic patients. J Diabetes Complications. 2017;31(1):213–217. 34. Li N, Xu M, Cai MY, et al. Elevated serum bilirubin levels are associated with improved survival in patients with curatively resected non-small-cell lung cancer. Cancer Epidemiol. 2015;39(5):763–768. 35. Kang SJ, Kim D, Park HE, et al. Elevated serum bilirubin levels are inversely associated with coronary artery atherosclerosis. Atherosclerosis. 2013;230(2):242–248. 36. Jang BK. Elevated serum bilirubin levels are inversely associated with nonalcoholic fatty liver disease. Clin Mol Hepatol. 2012;18(4):357–359. 37. Zheng J, Wu Y, Li Z, et al. Low serum total bilirubin concentration was associated with increased high sensitive C reactive protein level in patients with impaired glucose tolerance and type 2 diabetes mellitus subjects. Clin Lab. 2016;62(5):901–907. 38. Erdogan T, Cicek Y, Kocaman SA, et al. Increased serum bilirubin level is related to good collateral development in patients with chronic total coronary occlusion. Intern Med. 2012;51(3):249–255. 39. Liu C, Zhong C, Zhou X, et al. Higher direct bilirubin levels during mid-pregnancy are associated with lower risk of gestational diabetes mellitus. Endocrine. 2017;55(1):165–172. 40. Xu XQ, Lv ZC, Liu QQ, et al. Direct bilirubin: a new risk factor of adverse outcome in idiopathic pulmonary arterial hypertension. Int J Cardiol. 2017;228:895–899. 41. Gao C, Fang L, Li JT, Zhao HC. Significance and prognostic value of increased serum direct bilirubin level for lymph node metastasis in Chinese rectal cancer patients. World J Gastroenterol. 2016;22(8):2576–2584. 42. Doumas BT, Kwok-Cheung PP, Perry BW, et al. Candidate reference method for determination of total bilirubin in serum: development and validation. Clin Chem. 1985;31(11):1779–1789. 43. Watson D, Rogers JA. A study of six representative methods of plasma bilirubin analysis. J Clin Pathol. 1961;14:271–278. 44. Rolinski B, Kuster H, Ugele B, Gruber R, Horn K. Total bilirubin measurement by photometry on a blood gas analyzer: potential for use in neonatal testing at the point of care. Clin Chem. 2001;47(10):1845–1847. 45. Chan KM, Scott MG, Wu TW, et al. Inaccurate values for direct bilirubin with some commonly used direct bilirubin procedures. Clin Chem. 1985;31(9):1560–1563. 46. Mullon CJ, Langer R. Determination of conjugated and total bilirubin in serum of neonates, with use of bilirubin oxidase. Clin Chem. 1987;33(10):1822–1825. 47. Cascavilla N, Falcone A, Sanpaolo G, D’Arena G. Increased serum bilirubin level without jaundice in patients with monoclonal gammopathy. Leuk Lymphoma. 2009;50(8):1392–1394.
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48. Shull BC, Lees H, Li PK. Mechanism of interference by hemoglobin in the determination of total bilirubin. II. Method of Jendrassik–Grof. Clin Chem. 1980;26(1):26–29. 49. Shull BC, Lees H, Li PK. Mechanism of interference by hemoglobin in the determination of total bilirubin. I. Method of Malloy–Evelyn. Clin Chem. 1980;26(1):22–25. 50. Yang Y, Howanitz PJ, Howanitz JH, Gorfajn H, Wong K. Paraproteins are a common cause of interferences with automated chemistry methods. Arch Pathol Lab Med. 2008;132(2):217–223. 51. Martinello F, da Silva EL. Ascorbic acid interference in the measurement of serum biochemical parameters: in vivo and in vitro studies. Clin Biochem. 2006;39(4):396–403. 52. Ball M, Miller I, Cotten SW. Direct bilirubin higher than total bilirubin? Clin Chem. 2015;61(6):889. 53. Andreu Y, Ostra M, Ubide C, Galban J, de Marcos S, Castillo JR. Study of a fluorometric–enzymatic method for bilirubin based on chemically modified bilirubinoxidase and multivariate calibration. Talanta. 2002;57(2):343–353. 54. Doumas BT, Yein F, Perry B, Jendrzejczak B, Kessner A. Determination of the sum of bilirubin sugar conjugates in plasma by bilirubin oxidase. Clin Chem. 1999;45(8 pt 1):1255–1260. 55. Kurosaka K, Senba S, Tsubota H, Kondo HA. New enzymatic assay for selectively measuring conjugated bilirubin concentration in serum with use of bilirubin oxidase. Clin Chim Acta. 1998;269(2):125–136. 56. Morimoto Y, Ishihara T, Takayama M, Kaito M, Adachi Y. Novel assay for measuring serum conjugated bilirubin and its clinical relevance. J Clin Lab Anal. 2000;14(1):27–31. 57. Ameri M, Schnaars H, Sibley J, Honor D. Comparison of the vanadate oxidase method with the diazo method for serum bilirubin determination in dog, monkey, and rat. J Vet Diagn Invest. 2011;23(1):120–123. 58. Bajaj JS. Review article: the modern management of hepatic encephalopathy. Aliment Pharmacol Ther. 2010;31(5):537–547. 59. Wennberg RP, Ahlfors CE, Bhutani VK, Johnson LH, Shapiro SM. Toward understanding kernicterus: a challenge to improve the management of jaundiced newborns. Pediatrics. 2006;117(2):474–485. 60. Oh W, Stevenson DK, Tyson JE, et al. Influence of clinical status on the association between plasma total and unbound bilirubin and death or adverse neurodevelopmental outcomes in extremely low birth weight infants. Acta Paediatr. 2010;99(5):673–678. 61. Ahlfors CE, Amin SB, Parker AE. Unbound bilirubin predicts abnormal automated auditory brainstem response in a diverse newborn population. J Perinatol. 2009;29(4):305–309. 62. Wennberg RP, Ahlfors CE, Aravkin AY. Intervention guidelines for neonatal hyperbilirubinemia: an evidence based quagmire. Curr Pharm Des. 2009;15(25):2939–2945. 63. Ostrow JD, Pascolo L, Tiribelli C. Reassessment of the unbound concentrations of unconjugated bilirubin in relation to neurotoxicity in vitro. Pediatr Res. 2003;54(6):926. 64. Calligaris SD, Bellarosa C, Giraudi P, Wennberg RP, Ostrow JD, Tiribelli C. Cytotoxicity is predicted by unbound and not total bilirubin concentration. Pediatr Res. 2007;62(5):576–580. 65. Cash WJ, McConville P, McDermott E, McCormick PA, Callender ME, McDougall NI. Current concepts in the assessment and treatment of hepatic encephalopathy. QJM. 2010;103(1):9–16. 66. Devgun MS, Richardson C. Direct bilirubin in clinical practice—interpretation and haemolysis interference guidance reassessed. Clin Biochem. 2016;49(18):1351–1353. 67. Greene DN, Holmes DT, Lin MJ, Liang JY, Lorey TS, Schmidt RL. Development of an equation to correct for hemolysis in direct bilirubin measurements. Clin Chim Acta. 2014;429:194–197.
Glucuronidated bilirubin: Significantly increased in hepatic encephalopathy Limin Tanga, Meng Zhangb, Xiulian Lib, Lijuan Zhangb,* a
Department of Radiology, Affiliated Hospital of Qingdao University, Qingdao, China Systems Biology and Medicine Center for Complex Diseases, Affiliated Hospital of Qingdao University, Qingdao, China *Corresponding author: e-mail address: [email protected] b
Contents 1. Introduction 2. Serum bilirubin measurement 3. Serum direct bilirubin levels in 38 different types of human diseases and healthy controls 3.1 Methods 3.2 Statistical analysis 3.3 Results 4. Discussions and conclusions Acknowledgment Conflict of interest References
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Abstract Bilirubin is produced by the breakdown of hemoglobin in senescent erythrocytes by macrophages and carried by albumin from blood circulation to the liver for removal in normal physiology. Glucuronic acid modification of bilirubin by UDPglucuronyltransferase in the liver is the key event for its subsequent elimination from human body. Conditions that accelerate the breakdown of erythrocytes may cause an elevated blood level of unconjugated bilirubin whereas the factors affect the glucuronidated bilirubin formation and subsequent elimination may cause decreased or increased blood level of glucuronidated bilirubin, the water soluble “direct bilirubin” measured by clinical blood test. Studies showed that increased total serum bilirubin has a protective effect on cardiovascular and other related diseases, but it is unknown how direct bilirubin levels were related to different diseases. By taking advantage of the data collected in the clinical laboratory of our hospital, the direct bilirubin data from 192,535 patients with 72 clinically defined diseases were compared to that of healthy controls (10,497). Based on the mean, median, and p values, we found that patients with hepatic encephalopathy had the highest serum direct bilirubin level, which resembled acute
Progress in Molecular Biology and Translational Science, Volume 162 ISSN 1877-1173 https://doi.org/10.1016/bs.pmbts.2018.12.009
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hepatic encephalopathy caused by increased serum direct bilirubin level in neonates. In contrast, patients with uremia, nephrotic syndrome, and preeclampsia had significantly lower levels of serum direct bilirubin. Taken together, our data revealed that serum direct bilirubin levels were either increased or decreased in a disease-dependent manner. The possible molecular mechanisms of increased direct bilirubin levels in patients suffering hepatic encephalopathy are discussed.
1. Introduction Bilirubin is a waste product produced by the breakdown of heme, a component of hemoglobin, when macrophages either in spleen or liver remove senescent erythrocytes. Heme consists of a Fe2+ ion in the center of porphyrin. The production of biliverdin begins when heme oxygenase-I metabolizes heme1, which is followed by the enzyme biliverdin reductase that converts biliverdin into bilirubin.2 The water insoluble bilirubin released by macrophages of the spleen is transported by albumin from blood circulation to the liver. UDP-glucuronosyl transferase family 1 member A13 in the liver makes bilirubin soluble by modifying bilirubin with one or two covalently linked glucuronic acids, which is the key event for subsequent elimination of bilirubin from the body4. Interestingly, only glucuronidated bilirubin can react with albumin nonenzymatically to form δ-bilirubin, a covalently linked albumin–bilirubin conjugate found in human serum.5–9 Thus, serum total bilirubins include unconjugated bilirubin and the water soluble bilirubins containing monoglucuronidated bilirubin, diglucuronidated bilirubin, and δ-bilirubin. Since δ-bilirubin formation uses glucuronidated bilirubins as substrates, the so-called “direct bilirubin” is the three glucuronidation-related water soluble bilirubins. The UGT1A1 gene is part of a complex locus that encodes several UDPglucuronosyl transferases.10,11 The locus includes 13 unique alternate first exons followed by four common exons. Four of the alternate first exons are considered pseudogenes. Each of the remaining nine 50 exons may be spliced to the four common exons, resulting in nine UDP-glucuronosyl transferases with different N-termini and identical C-termini. Each first exon encodes the substrate binding site that makes each UDPglucuronosyltransferase unique. The preferred substrate for UDPglucuronosyltransferase family 1 member A1 is bilirubin, although it also has moderate activity with simple phenols, flavones, and steroids. Mutations
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in this gene result in Crigler–Najjar syndromes types I and II12–14 and in Gilbert syndrome,15–17 which are severe to mild liver disorders. A series of studies indicate that glucuronidation of bilirubin is a rate-limiting step for the disposal of bilirubin in animals and the cause of various diseases.18–20 Approximately 250–350 mg of bilirubin is produced daily in healthy adults. Humans are naturally hyperbilirubinemia compared to other mammals with normal ranges of serum total bilirubin levels from 1 to 10 μmol/L. In general, increased unconjugated bilirubin level may be associated with unusual hemolysis or when the liver is unable to uptake or process bilirubin. Conversely, increased “direct” bilirubin level may indicate that the liver is able to modify bilirubin with glucuronic acid but is not able to pass the glucuronidated bilirubin to the bile for removal. In clinic, bilirubin is used to monitor diseases such as cirrhosis, hepatitis, or gallstones and to determine the causes of hemolytic anemia.21,22 Since almost every newborn is hyperbilirubinemia with bilirubin level great than 30 μmol/L during the first week of life, a rapid laboratory tests for both total and direct bilirubin are required in order to start therapy to prevent acute bilirubin encephalopathy in neonates.23,24 Since the discoveries that moderately increased levels of serum bilirubin are correlated with a significantly decreased risk of coronary artery disease in individuals with Gilbert syndrome,25–27 the antioxidant, antiinflammation, and antitumor properties of bilirubin in blood circulation and its beneficial effects in aging and different diseases have been reported.28–39 However, it has not been reported if direct bilirubin levels were related to different diseases except for a few cases.40,41 Most importantly, only direct bilirubin can pass blood brain barrier and is neurotoxic at sufficiently high concentrations24. It will be important to know if adult hepatic encephalopathy was associated with increased serum direct bilirubin level as well. By taking advantage of the data collected in the clinical laboratory of our hospital, the direct bilirubin data from 192,535 patients with 72 clinically defined diseases were compared to that of normal controls (10,497) with at least 100 datasets for each type of disease. Based on the mean, median, and p values, we found that patients with hepatic encephalopathy had the highest serum direct bilirubin level. In contrast, patients with uremia, nephrotic syndrome, and preeclampsia had significantly lower levels of serum direct bilirubin. Taken together, our data revealed that serum direct bilirubin levels were either increased or decreased significantly in a diseasespecific manner.
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2. Serum bilirubin measurement Total serum bilirubins comprise unconjugated bilirubin of the insoluble fraction that is attached to serum albumin no-covalently and conjugated bilirubins of the water soluble bilirubin including monoglucuronide, bilirubin diglucuronides, and δ-bilirubin (covalently linked bilirubin–albumin conjugate). Currently, most clinical laboratories rely on automated analyzers for rapid bilirubin determinations for multiple samples42. Two forms of bilirubin in the serum are measured by the clinical tests: “direct” bilirubin level (conjugated/glucuronidated bilirubin and δ-bilirubin) and total bilirubin level (unconjugated plus direct bilirubin). Clinical test also provides the “indirect” level of unconjugated bilirubin by subtracting direct bilirubin level from the total bilirubin level. All methods used for the measurements of serum bilirubin are based on one of the two properties of bilirubin: (1) The intrinsic absorption spectra of different bilirubin fractions with the absorption maximum of 463 nm for unconjugated bilirubin and 453 nm for conjugated bilirubin and loss of the absorption after reducing bilirubin to biliverdin by oxidase or vanadate. (2) The novel absorption spectra of bilirubin after the diazo reaction. Based on the detection method, bilirubin fractions can be measured by highperformance liquid chromatography (HPLC) method, direct spectrophotometry method, the diazo derivatization method, and oxidation method, respectively9. The diazo reaction has been used for serum bilirubin quantification since 1950s and still is the most widely used method. In this method, the color of azobilirubin formed by the reaction of the porphyrin rings of bilirubin with a diazo compound is spectrophotometrically measured. Because unconjugated bilirubin reacts slowly, accelerators such as methanol, ethanol, or caffeine, are used to release unconjugated bilirubin from the protein carrier albumin, the total bilirubin level is then measured. In the absence of these accelerators, the director bilirubin level can be measured by this method.43,44 However, a small portion of unconjugated bilirubin is also detected by the diazo method as direct bilirubin.45,46 Moreover, only 76%–89% of direct bilirubin is reported to be detected as direct bilirubin5. The accuracy of bilirubin measurements by the diazo method is influenced by coexistent of other serum proteins or compounds, such as hemoglobin, immunoglobulins, and ascorbic acid.47–51 Therefore, the accuracy of direct
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bilirubin measurement by the diazo assay has been an issue needed to be addressed52. HPLC method, which can separate all four bilirubin fractions, is a reliable method for bilirubin quantification in theory, but impractical for clinical use because of labor costs and long turnaround time6. Efforts to develop alternative clinical assays for bilirubin quantification have led to the use of bilirubin oxidase to measure direct bilirubin.53–56 In this method, bilirubin oxidase catalyzes the oxidation of bilirubin to biliverdin, which causes the characteristic absorption for bilirubin to disappear. Thus, direct bilirubin can be exclusively detected by bilirubin oxidase method. The vanadate method is based on oxidation of bilirubin to biliverdin by the chemical vanadate as an oxidizing agent. In this method, all bilirubins are oxidized by vanadate in the presence of detergent to produce colorless products. In the absence of the detergent, only direct bilirubin in serum is oxidized by the vanadate and measured. The precision studies reveal that the vanadate method has comparable between-run and within-run CVs to those of the diazo method57. The vanadate method is less interfered by the coexisting serum proteins and other compounds. Thus, the direct bilirubin measurement is more reliable than that of the diazo method.
3. Serum direct bilirubin levels in 38 different types of human diseases and healthy controls 3.1 Methods Serum samples for the detection of direct bilirubin levels were obtained retrospectively from the clinical laboratory of The Affiliated Hospital of Qingdao University over the past 5 years (2012–17). The serum direct bilirubin levels were analyzed using an autoanalyzer (Beckman Coulter AU5800, USA) with assay kit purchased from Beijing Lidman Biochemical Co., Ltd. It is a vanadate assay kit and this assay is more reliable in quantifying serum direct bilirubin levels compared to that of diazo assay. The normal values of total bilirubin are 1.71–17.1 μmol/L and direct bilirubin are 1.71–7.0 μmol/L in healthy controls. The current study including the direct bilirubin data from 192,535 patients with 72 clinically defined diseases were compared to that of normal controls (10,497) with at least 100 datasets for each type of disease.
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3.2 Statistical analysis Statistical analysis was performed by SPSS version 19 (Inc., Chicago, USA) and all p values were two side. The Mann–Whitney test was used for the nonnormal distribution data, with p values <0.05 considered significant. All data were presented as the median (interquartile range) or mean (SD).
3.3 Results The clinical laboratory of our hospital measured the serum direct and total bilirubin levels routinely for either patients requested by the doctors and healthy populations during their annual physical examination. To this end, we collected all the clinical serum direct bilirubin assay data from the clinical laboratory of our hospital during the past 5 years. As a result, we obtained the direct bilirubin data from 192,535 patients with 72 clinically defined diseases and 10,479 healthy controls during their annual physical examination. Based on these data, we calculated and listed the mean, median, and p values of direct bilirubin levels for each type of disease in Table 1. In order to make the results more intuitive, we plotted a scatter diagram of direct bilirubin values for each type of disease with lower quartile (25%), median (50%), and upper quartile (75%) ranges marked. As shown in Fig. 1, the median values of direct bilirubin in 12 diseases were higher than that of healthy controls. Among all the diseases, patients suffering hepatic encephalopathy, cirrhosis, pancreatic cancer, pancreatitis, and liver cancer had direct bilirubin levels higher that of healthy controls. Unexpectedly, patients with uremia, nephrotic syndrome, preeclampsia, lupus erythematous, diabetic nephropathy, breast cancer, and nephritis had significantly lower levels of direct bilirubin compared to that of healthy controls. Moreover, certain patients after gastric surgery, with hepatitis, or suffering sepsis had very higher levels of serum direct bilirubin compared to patients with other diseases. Using the median values, we then calculated the p values as shown in Fig. 2. The p value is used in the context of null hypothesis testing in order to quantify the idea of statistical significance of evidence. Null hypothesis testing is a reduction argument adapted to statistics. In essence, the statistical significance is assumed valid when the p values are far apart from that of control values. Unexpectedly, patients with cirrhosis, pancreatitis, pancreatic cancer, hepatic encephalopathy, liver cancer, myeloproliferative disorders, and acute cerebral infarction had highest log10 p values
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Table 1 The mean, median, and log10 p values of serum direct bilirubin levels for healthy controls and patients with cancer and other diseases. No. of 2Log10 p DBIL cases Mean (SD) Median value
Hepatic encephalopathy
213
39.00 (47.70) 24.90
137.23
Cirrhosis
8467
26.50 (38.21) 12.00
>176
Pancreatic cancer
1050
30.99 (52.45)
7.00
110.35
Pancreatitis
1636
10.52 (13.19)
6.22
139.14
Liver cancer
258
12.76 (33.81)
6.20
40.07
Acute myocardial infarction
2298
5.89 (3.06)
5.20
55.48
Myeloproliferative disorder
1162
5.97 (3.40)
5.10
22.88
Bladder stone
151
5.30 (1.84)
5.00
4.64
Healthy controls >65 years old
1209
4.91 (1.57)
4.80
7.32
Acute cerebral infarction
8657
5.02 (2.10)
4.62
12.27
Healthy controls
10,497
4.65 (1.55)
4.56
0.00
Gastric ulcer
270
4.74 (2.16)
4.51
0.51
Gastric cancer
12,000
5.12 (2.87)
4.50
0.99
Bone fracture
1808
4.91 (2.26)
4.50
0.54
Coronary heart disease
19,828
4.86 (2.29)
4.40
1.56
Sepsis
273
7.46 (10.22)
4.37
0.62
Cerebrovascular disease
4147
4.63 (1.92)
4.32
5.69
Gout
1341
4.51 (1.81)
4.22
6.73
Hepatitis
6940
6.45 (7.60)
4.20
6.70
Intracranial hemorrhage
3532
4.83 (2.54)
4.20
5.75
Chronic obstructive PD
1464
4.75 (2.45)
4.20
5.02
Esophagus cancer
3547
4.58 (2.13)
4.20
13.86
Rectum cancer
7600
4.55 (2.15)
4.20
24.20
Cerebral ischemia
2097
4.42 (1.79)
4.20
13.76
Renal cyst
476
4.39 (1.49)
4.20
3.69
Alzheimer’s disease
94
4.60 (2.26)
4.17
1.38 Continued
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Table 1 The mean, median, and log10 p values of serum direct bilirubin levels for healthy controls and patients with cancer and other diseases.—cont’d No. of 2Log10 p DBIL cases Mean (SD) Median value
Anemia
2499
5.17 (3.70)
4.11
7.88
Bladder cancer
982
4.43 (2.05)
4.10
12.16
Colon cancer
5920
4.50 (2.27)
4.04
37.98
Aplastic anemia
1203
4.62 (2.77)
3.97
12.87
Brain trauma
639
4.37 (2.44)
3.90
14.00
Encephalitis
672
4.29 (2.61)
3.90
18.14
Hyperuricemia
147
4.07 (1.70)
3.84
5.56
Leukemia
8601
4.26 (2.33)
3.80
137.12
Bone cancer
101
4.01 (1.95)
3.72
5.87
Lung fibrosis
305
4.00 (1.88)
3.67
14.46
Cerebral arteriosclerosis
739
3.87 (1.69)
3.60
40.28
Gastritis
3091
3.90 (1.93)
3.58
140.28
Lymphoma
4441
4.01 (2.22)
3.54
167.59
Type 2 diabetes mellitus
10,424
3.78 (1.74)
3.50
>176
Asthma
576
3.84 (1.76)
3.48
37.58
Cystitis
63
3.57 (1.39)
3.46
7.17
Osteoporosis
241
3.64 (1.72)
3.40
22.02
Multiple myeloma
2050
3.71 (2.00)
3.37
150.33
Wilms’ tumor
248
3.29 (1.45)
3.30
36.51
Breast lumps
100
3.36 (1.86)
3.25
13.71
Psoriasis
135
3.51 (1.82)
3.21
15.51
Lung cancer
9740
3.50 (1.63)
3.20
>176
Kidney cancer
1419
3.36 (1.59)
3.10
171.78
Necrosis of femoral head
172
3.17 (0.98)
3.07
36.60
Osteoarthritis
78
3.66 (2.11)
3.00
9.78
Endometrial cancer
1090
3.17 (1.37)
3.00
175.86
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Table 1 The mean, median, and log10 p values of serum direct bilirubin levels for healthy controls and patients with cancer and other diseases.—cont’d No. of 2Log10 p DBIL cases Mean (SD) Median value
Knee-joint degenerative diseases
442
3.07 (1.07)
2.90
98.60
Cervical cancer
2050
3.02 (1.33)
2.89
>176
Azotemia
441
2.84 (1.47)
2.70
107.57
Postbreast surgery
38
7.33 (25.32)
2.69
6.65
Rheumatic arthritis
419
3.05 (1.95)
2.66
81.89
Ovarian cancer
2171
2.83 (1.28)
2.61
>176
Ankylosing spondylitis
77
2.89 (1.40)
2.60
20.08
Nephritis
2173
2.76 (1.51)
2.56
>176
Breast cancer
4923
2.60 (1.29)
2.40
>176
Diabetic nephropathy
540
2.53 (1.55)
2.20
158.75
Lupus erythematosus
1272
2.40 (1.66)
2.09
>176
Preeclampsia
877
2.25 (1.35)
1.99
>176
Nephrotic syndrome
3877
2.14 (1.28)
1.90
>176
Uremia
5759
2.03 (1.30)
1.79
>176
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Fig. 1 The direct bilirubin values in 50 different types of clinical diseases. The data were sorted in a descending order of the median values. Direct bilirubin values for each type of disease with lower quartile (25%), median (50%), and upper quartile (75%) ranges were marked in red.
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Fig. 2 Log10 p values for direct bilirubin in 50 different types of clinical diseases compared to that of healthy controls. When the serum direct bilirubin levels were lower than that of healthy controls, blue color was used in the plot whereas when the serum direct bilirubin levels were higher than that of healthy controls, red color was used in the plot.
compared to that of healthy controls, which made direct bilirubin level an outstanding biomarkers for these diseases. Taken together, both median and p values supported the facts that increased direct bilirubin levels were useful for diagnosis of multiple diseases, especially for hepatic encephalopathy. In addition, the decreased serum bilirubin levels based on the p values could be served as biomarkers for many different types of human diseases, not limited to uremia, nephrotic syndrome, preeclampsia, nephritis, ovarian cancer, cervical cancer, lung cancer, type 2 diabetes, and endometrial cancer.
4. Discussions and conclusions The current study demonstrated that patients with hepatic encephalopathy had the highest serum direct bilirubin level. In contrast, patients with uremia, nephrotic syndrome, and preeclampsia had significantly lower levels of serum direct bilirubin. The significantly low p value shown in Table 1 indicated that serum glucuronidated bilirubin level might be sufficient to serve as a biomarker for patients suffering hepatic encephalopathy and other diseases. Hepatic encephalopathy is an altered level of consciousness due to liver failure58. Increased total bilirubin levels have been closely monitored to prevent neonatal hepatic encephalopathy. Moreover, a series of elegant studies indicate that high direct bilirubin levels but not unconjugated bilirubin levels are responsible for the observed neuron toxicity both in vitro and in vivo.24,59–64 In contrast, for adult hepatic encephalopathy, the role of direct bilirubin played is largely ignored. The underlying molecular mechanism for adult is believed to be involved in the buildup of blood ammonia that cause damage of brain in adult.65 Our data shown in Table 1 and Figs. 1
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and 2 suggested that the increased direct bilirubin levels might be responsible for adult hepatic encephalopathy as well. In theory, conditions affect the glucuronidated bilirubin formation may cause decreased blood level of glucuronidated bilirubin whereas conditions affect the glucuronidated bilirubin elimination may cause increased blood level of glucuronidated bilirubin. Paradoxically, the data presented in Table 1 and Figs. 1 and 2 showed that hepatic encephalopathy, cirrhosis, pancreatic cancer, pancreatitis, and liver cancer were conditions associated with increased direct bilirubin levels whereas uremia, nephrotic syndrome, diabetic nephropathy, and nephritis were conditions associated with decreased direct bilirubin levels. There results indicated that the regulations of serum direct bilirubin levels might not be as intuitive as previously thought. It is known that the accuracy of bilirubin measurements by the clinical diazo method is influenced by the coexistent of hemoglobin, immunoglobulins, and ascorbic acid and the diazo method is high sensitivity to hemolysis.66,67 The reliability of clinical method in measuring direct bilirubin has constantly been challenged52. The vanadate method used in our clinical test is less interfered by the coexisting serum proteins and other compounds, the direct bilirubin data presented in Table 1 and Figs. 1 and 2 should be reliable. However, our current study is retrospective, prospective study is required to test if direct bilirubin levels could serve as biomarkers for hepatic encephalopathy and other diseases.
Acknowledgment This research was supported by the Natural Science Foundation of China (Grant 81672585), Key Technology Fund of Shandong Province (Grant 2016ZDJS07A07), the Taishan Scholar Fellowship, and the “Double First Class fund” of Shandong Province in China to L.Z.
Conflict of interest The authors declare no conflict of interest.
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