We have previously observed that UCB binds to ZnSO4in vitro, and suppresed the biliary bilirubin secretion in the hamster. The aim of this study was designed to investigate whether Zn salts might inhibit the enterohepatic cycling of UCB in subjects with Gilbert´s syndrome. Fifteen patients with Gilbert´s syndrome and 5 normal healthy volunteers were included in this study according to the following criteria: fasting hyperbilirubinemia, no hemolysis, and free of any medication. Patients were randomly assigned to receive acute o chronic treatment. Subjects treated in acute form and normal healthy volunteers were treated with 40 mg of ZnSO4 in a single dose, where as patients treated in chronic form received 100 mg ZnSO4 in a single dose daily for 7 days. The serum UCB levels (mg/dL) decreased from 2.64 ± 1.04 to 2.02 ± 0.87 (p < 0.001) and 1.8 ± 0.36 to 1.48 ± 0.32 (p < 0.005) in subjects treated in acute an chronic form respectively, but not in the control group. Whereas, the serum Zn levels (μg/dL) increased from 96.3 ± 16.8 to 118.8 ± 19. 5, (p < 0.01) and from 117.6 ± 8.5 to 130.7 ± 6.6 (p < 0.03) in subjects treated in acute an chronic form and also in subjects in the control group (98.0 ± 7.3 to 128.0 ± 21.9) p < 0.03. This study showed that acute and chronic oral administration of ZnSO4 decreased serum UCB levels significantly in subjects with Gilbert´s syndrome. Most likely by the inhibition of the “normal” enterohepatic cycling of UCB.
Grants support: This work was partly supported by the National Council of Science and Technology of Mexico (CONACYT),Project number 0817P-M9506 (M.U and N.M-S).
IntroductionThe Gilbert’s syndrome is a chronic disease in which unconjugated bilirubin (UCB) is not efficiently conjugated in the liver and it is accumulated in the blood.1-3 Recently, it has been associated with an extra TA in the promoter region of both alleles for bilirubin UDP-glucuronosyltransferase 1 (UGT1A1).4-8 The resultant 65% decrease in transcription of the (TA)7TAA mutant alleles explains the impaired conjugation of bilirubin found in all Gilbert’s syndrome subjects.9,10
Under normal conditions conjugation of bilirubin takes place in the hepatic cell, and two or one molecules of glucuronic acid leads to form bilirubin diglucuronides (BDG) and bilirubin monoglucuronides (BMG) respectively.11 In humans, BDG constitutes the major conjugate in bile (about 80%). But in subjects with Gilbert’s syndrome the proportion of BDG/BMG is inverted.12 Interestingly, the BMG are the major conjugate species in mice, sheep, pigs, rabbits, guinea pigs, and hamsters.13 According to this information, we believed that both subjects with Gilbert’s syndrome and hamsters have and an EHC of UCB.
On the other hand, we recently obtained evidence for EHC of UCB in rats following distal, but not proximal, small bowel resection.14 We proposed that EHC of UCB was secondary to an interruption in the EHC of bile salts. These findings are consistent with results in patients with Crohn’s disease involving the ileum,15,16 who present with total bilirubin and UCB levels significantly elevated in gallbladder bile.
Furthermore, we recently observed that at physiological pH ZnSO4 adsorb UCB almost entirely from unsaturated micellar bile salt solution in vitro.17 In addition, in hamsters which have principally BMG in bile,13 when we fed them with 1% ZnSO4 for one week, the biliary bilirubin secretion was suppressed suggesting an inhibition of the EHC of UCB.7 The aim of this study was designed to investigate whether Zn salts might inhibit the EHC of UCB in subjects with Gilbert´s syndrome.
MethodsSubjectsWe recruited subjects from one medical center in Mexico City (Medica Sur Clinic & Foundation). Mexican subjects male and female patients 20-46 years of age with Gilbert´s syndrome were included in this study according to the following criteria: a) positive test of fasting hyperbilirubinemia, b) no hemolysis, and c) free of any medication. Normal healthy volunteers acting as controls. Written informed consent was obtained from each subject. The study was approved by the Human Subjects Committee at The Medica Sur Clinic & Foundation as conforming to the ethical guidelines of the 1975 Declaration of Helsinki.
Study designThis was a clinical trial carried out in fifteen patients with Gilbert´s syndrome and 5 normal healthy volunteers acting as controls. Subjects who met the entrance criteria were randomly assigned to acute o chronic treatments, subjects treated in acute form and normal healthy volunteers were treated with 40 mg of ZnSO4 in a single dose. Whereas patients treated in chronic form received 100 mg of ZnSO4 in a single dose daily for 7 days.
Clinical monitoringSubjects treated in acute form (n = 10) and normal healthy volunteers B (n = 5) were hospitalized one day before treatment in order to avoid physical activities. After a period of 14 h overnight (baseline) they received a carbohydrate (CHD)-rich breakfast (600 kcal) and two h later a single 40 mg oral dose of ZnSO4. Subjects treated in chronic form (n = 5) were treated as outpatients with a single 100 mg oral dose of ZnSO4 daily for 7 days.
Analytical proceduresSerum UCB and Zn levels were monitored by HPLC18and atomic absorption spectroscopy at baseline and after oral administration of ZnSO4, and fecal UCB concentrations were determined before and 24 h after oral administration of ZnSO4 by HPLC.18
Statistical analysisAll values are expressed as means ± SD, the paired “t” Student and Wilcoxon tests were used to compare the serum UCB and Zn levels. The differences were considered as statistically significant when p was < 0.05.19
ResultsSubjects with Gilbert´s syndrome treated in acute form and normal healthy volunteers were directly observed by the investigators, indicated that they had good tolerance of the ZnSO4. All other subjects treated as out-patients do not referred side effects attributed to ZnSO4. Clinical characteristics of all groups are given in Table I.
Characteristics of the three treated groups at entry.
| Acute | Chronic | Control | p Value | |
|---|---|---|---|---|
| Clinical | ||||
| No. patients | 10 | 5 | 5 | - |
| Age (yr) | 33.3 ± 5.8 | 31.8 ± 7.9 | 25.4 ± 1.5 | NS |
| Sex (M/F) | (8/2) | (4/1) | (4/1) | 0.001 |
| BMI | 22.6 ± 2.7a | 23.9 ± 2.56 | 27.4 ± 2.0 | 0.05 |
| Serum | ||||
| UCB (mg/dL) | 2.64 ± 1.04b | 2.22 ± 0.8b | 0.9 ± 0.17 | 0.05 |
BMI, body mass index UCB, unconjugated bilirubin
Figure 1 shows the effect of the ZnSO4 on serum UCB levels (mg/dL) in subjects with Gilbert’s syndrome in acute and chronic forms. The UCB levels range from 2.66 ± 0.91 at the baseline to 2.84 ± 0.89 and after administration of ZnSO4 there was an decrease to 2.34 ± 0.90 (p < 0.0003). Whereas serum UCB levels in subjects with Gilbert’s syndrome. treated in a chronic form decreased from 1.8 ± 0.36 to 1.48 ± 0.32 (p < 0.005). The serum Zn levels (μg/dL) in acute an chronic form. The levels increased from 101.51 ± 12.38 to 122.08 ± 20.0, (p < 0.04) and from 117.64 ± to 130.7 ± 6.6, (p < 0.03) in subjects with Gilbert’s syndrome.
Shows the effect of ZnSO4 on serum UCB levels in subjects with Gilbert’s syndrome, in acute and chronic form. In both forms of treatment there were a significant decrease in serum UCB levels, The UCB levels range from 2.66 ± 0.91 at the baseline to 2.84 ± 0.89 and after administration of ZnSO4 there was an decrease to 2.34 ± 0.90 (p < 0.0003). Whereas serum UCB levels in subjects treated in a chronic form decreased from 1.8 ± 0.36 to 1.48 ± 0.32 (p < 0.005). The serum Zn levels (μg/dL) in acute an chronic form. The levels increased from 101.51 ± 12.38 to 122.08 ± 20.0, (p < 0.04) and from 117.64 ± to 130.7 ± 6.6, (p < 0.03). Values are means ± SD.
Figure 2 shows the effect of ZnSO4 on serum UCB and Zn levels in the control group. There was no change in UCB levels after administration of ZnSO4. Whereas Zn levels increased from 98.0 ± 7.3 to 128.0 ± 21.9) p < 0.03. Fecal UCB concentration (μg/g dry) was increased after oral administration of ZnSO4 from 27.25 ± 21.4 to 37.2 ± 23.08, p < 0.03 in subjects treated in acute form, but not in the control group (Table II).
The results of this study showed that acute and chronic oral administration of ZnSO4 decreases serum UCB levels significantly in subjects with Gilbert´s syndrome. Most likely by inhibition of the EHC of UCB.
How can we explain the effect of ZnSO4 on the serum UCB concentration in subjects with Gilbert´s syndrome? First, we have shown in our in vitro studies that Zn salts at physiological pH and above adsorb UCB essentially completely from unsaturated BS micellar solutions. This indicate that there is a physico-chemical interaction between UCB and Zn salts.17 The interaction probably is via the carboxyl groups of UCB, as occurs with other divalent cations such as Ca2+20-22 leading to the formation of calcium bilirubinate [Ca(BH)2].
Second, we have also found that ZnSO4 reduced biliary bilirubin secretion in hamsters in vivo.17 One explanation for the reduced secretion of biliary bilirubin observed in the hamsters fed ZnSO4 is that this is probably the result of the interaction between UCB and ZnSO4 at the distal intestinal lumen where the pH is ~ 8, and we know from our in vitro experiments that at this pH, UCB might interact with ZnSO4.17 Then this interaction leads to formation of a flocculated material, which in turn, may be excreted in the fecal content as we shown in this study. As a resulted of this interaction, the final event is an interruption of the EHC of UCB by ZnSO4.
Third, to look the most potent effect of ZnSO4 was seen when we used a carbohydrate-rich diet which is known is able to increase the serum UCB levels.23,24 In fact, we saw that the effect of ZnSO4 was more evident after a 14-hour fasting period and a carbohydrate-rich diet. The serum UCB levels decreased significantly after the administration of a 40 mg of ZnSO4 given acutely (see Figure 1), and these findings were resembled after oral administration of 100 mg for seven days. Interestingly, the low dose was probably potent enough to decrease the serum UCB concentration.
Four, serum Zn levels increased 20% and 13% in both acute and chronic administration of ZnSO4, suggesting an effect of this salt on serum bilirubin. Whereas fecal UCB concentration was increased after oral administration of ZnSO4 in subjects treated in acute form, but not in the control group.
On the other hand, we believed that ZnSO4 might act as insoluble calcium phosphate which has been proposed as a good cheletor of UCB from bile salts solutions25 and in fact, oral calcium phosphate/carbonate has been suggested as effective in treating hyperoxaluria in patients with ileal resection.26-28 In a recent paper, insoluble calcium salts were shown to suppress serum bilirubin levels in patients with Crigler Najjär Type II syndrome.29 According with the results of the present study Zn which is the least toxic of the trace metals, may be used in the following conditions: a) to inhibit EHC of bilirubin associated with bile salt absorption [distal ileal diseases (Crohn’s, AIDS, resection, bypass, and immaturity)], b) to suppress serum bilirubin levels in patients with Crigler Najjär Type II syndrome, c) in patients with chronic cholestasis diseases (primary biliary cirrhosis, primary sclerosing cholangitis), and d) chronic liver diseases (hepatic cirrhosis) interestingly, Dashti et al30 observed in experimental liver cirrhosis produced by thioacetamide that zinc sulphate-treated animals showed a restoration of normal hepatic and plasma zinc and copper levels plasma. Similarly, plasma levels of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl aminotransferase, and total bilirubin decreased significantly. Light microscopic studies showed that most of the hepatocytes appeared normal in zinc-treated as compared with untreated cirrhotic animals.
In conlusion, this study showed that acute and chronic administration of oral ZnSO4 decreases serum UCB levels significantly in subjects with Gilbert´s syndrome. This observation suggests inhibition of a “normal” EHC of UCB, and strongly support the potential usefulness of ZnSO4 in the treatment a series of pathological conditions that are characterized by hyperbilirubinemia.
