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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Patient population
  6. Materials
  7. Determination of endothelins
  8. Determination of nitrite/nitrate concentration
  9. Statistical analysis
  10. Results
  11. Endothelins
  12. Nitrites/nitrates
  13. The effect of UDCA treatment
  14. Hepatic vein endothelins and NOx
  15. Correlations
  16. Discussion
  17. Acknowledgement
  18. References

Background : Endothelins and nitric oxide regulate sinusoidal blood flow and the perfusion of the peribiliary vascular plexus.

Aims : To study the serum and hepatic vein concentration of ET-1, ET-2, ET-3 and nitric oxide in patients with primary biliary cirrhosis and the effect of ursodeoxycholic acid treatment.

Methods : Endothelins and nitrites/nitrates were measured in serum and hepatic vein blood in primary biliary cirrhosis and viral cirrhotic patients prior and after ursodeoxycholic acid therapy and in serum in controls. Endothelins were measured with commercial enzyme-linked immunosorbent assays and nitrites/nitrates with a modification of Griess reaction.

Results : The ET-1 and ET-3 levels were similar in patients and controls. Primary biliary cirrhosis patients had the highest serum ET-2 (P < 0.001) compared with other groups. Nitrites/nitrates was increased in primary biliary cirrhosis (P < 0.05) compared with normal. ET-2 and nitric oxide were similar in all primary biliary cirrhosis stages. Ursodeoxycholic acid significantly decreased ET-2 in all stages (I and II: P < 0.05 and III and IV: P < 0.01) and increased nitric oxide (P < 0.05) in early primary biliary cirrhosis. Hepatic vein ET-1 and ET-3 were higher in viral cirrhosis patients, but only in primary biliary cirrhosis a significant difference for ET-1 and ET-3 between hepatic and peripheral veins was found.

Conclusions : Increased ET-2 is an early defect in primary biliary cirrhosis that is significantly reduced by the ursodeoxycholic acid treatment. The possibility of a more generalized endothelial cell dysfunction in primary biliary cirrhosis requires further investigation.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Patient population
  6. Materials
  7. Determination of endothelins
  8. Determination of nitrite/nitrate concentration
  9. Statistical analysis
  10. Results
  11. Endothelins
  12. Nitrites/nitrates
  13. The effect of UDCA treatment
  14. Hepatic vein endothelins and NOx
  15. Correlations
  16. Discussion
  17. Acknowledgement
  18. References

The endothelins (ETs) consist of a family of three 21 amino acid peptides termed ET-1, ET-2 and ET-3. They are the most potent vasoconstrictor agents so far discovered and their principal role seems to be the regulation of the vascular tone. ET-1 and ET-2 have equal vasoconstrictor effect, while ET-3 has a reduced vasoconstrictor action.1 Apart from vasoregulation, ETs exert multiple other biological actions, being involved in salt and water homeostasis,2 cellular growth3 and differentiation,4 inflammation,5 angiogenesis,6 neural crest development,7 neural function regulation7 and wound healing.8 ETs bind to two different receptors termed ET-A and ET-B. ET-A receptors, found on vascular smooth muscle cells, mediate vasoconstriction, while ET-B receptors, found predominantly on vascular endothelium, mediate vasodilation through nitric oxide (NO) release.9 Production of ETs was initially attributed to endothelial cells, but later it was found that other cell types-like vascular smooth muscle cells, liver stellate cells or macrophages10 can also produce ETs. In liver, ETs play a major role in sinusoidal microcirculation causing contraction of stellate cells9 and are supposed to be responsible, at least in part, for the increased intrahepatic vascular resistance that is observed in liver cirrhosis.9 They are also involved in hepatic fibrogenesis through activation of stellate cells.9, 11

The NO is a ubiquitous messenger involved in multiple biological actions, such as neurotransmission, immune regulation, cytotoxicity and vascular homeostasis.12 It is produced from l-arginine by enzymes called NO synthases (NOS) and are either constitutive or inducible. The inducible NOS (iNOS) is produced in macrophages and other cell types-like hepatocytes, after stimulation by cytokines, lipopolysaccharide and other substances. The constitutive isoforms of NOS are mainly localized at neurons (nNOS) and endothelial cells (eNOS).13, 14 Endothelium-derived NO is a potent vasodilator that acts in a paracrine fashion on the underlying vascular smooth muscle by direct stimulation of soluble guanylate cyclase, leading to increased levels of soluble guanylate cyclase (cGMP) and consequently causing decreased intracellular Ca++ concentration and thus vasorelaxation.15 In liver sinusoidal circulation NO plays the role of the vasoactive agent that counters the contractile activity of the vasoconstrictive compounds, causing direct relaxation to stellate cells.15

The role of NO and ETs has already been studied extensively in liver cirrhosis and portal hypertension. The aim of our study was to investigate the serum levels of these vasoactive mediators in patients with primary biliary cirrhosis (PBC). Previous studies16–20 report evidence of an increased NO production in PBC that was mainly related with the development of inflammation and liver injury. There is no current information concerning circulating levels of ETs in PBC patients. In this study, we measured serum levels of nitrites/nitrates (NOx; the last NO converted metabolites)20 and ET-1, ET-2, ET-3 in patients with PBC of different histological stages and compared them with healthy controls, patients with liver cirrhosis and patients with chronic hepatitis C. The effect of ursodeoxycholic acid (UDCA) therapy in the levels of these vascular mediators in PBC patients was also evaluated.

Patient population

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Patient population
  6. Materials
  7. Determination of endothelins
  8. Determination of nitrite/nitrate concentration
  9. Statistical analysis
  10. Results
  11. Endothelins
  12. Nitrites/nitrates
  13. The effect of UDCA treatment
  14. Hepatic vein endothelins and NOx
  15. Correlations
  16. Discussion
  17. Acknowledgement
  18. References

The study included 41 Greek patients with PBC (35 women, six men, median age 62, range: 31–85) followed up at the Department of Gastroenterology of the University Hospital of Heraklion, Crete. Thirty-seven of them were positive for antimitochondrial antibody (AMA)-M2 testing by immunofluorescence and by enzyme-linked immunosorbent assay (ELISA). All patients had typical biochemical pattern. Liver histology consistent with PBC was available in 38 patients. Three patients with clinical evidence of portal hypertension (oesophageal varices) and positive AMA-M2 were not submitted to liver biopsy. At the time of diagnosis, 25 patients (median age 58, range: 31–71) were in histological stage I or II and 16 patients (median age 67, range: 54–85) were in histological stage III or IV, according to Ludwig et al. criteria.21 Laboratory data and clinical details at the time of diagnosis and prior to initiation of UDCA therapy are shown in Table 1. All patients were negative for markers of hepatitis B and C. Patients with cardiac, renal or respiratory disorders had been excluded since these conditions have been associated with increased serum levels of ETs.22

Table 1.  Baseline characteristics of PBC patients (data expressed as mean ± s.d.)
 Stages I and IIStages III and IV
  1. GGT, γ-glutamyl transpeptidase; norm, normal range; PBC, primary biliary cirrhosis; SGOT, serum glutamic-oxaloacetic transaminase; SGPT, serum glutamic-pyruvic transaminase; IgM, immunoglobulin M.

n2516
Age (year)56 ± 11.566.75 ± 8.1
Pruritus (presence)105
Fatigue (presence)53
Bilirubin (mg/dL)0.77 ± 0.421.32 ± 0.97
Albumin (g/dL)4.44 ± 0.433.65 ± 0.5
Prothrombin time (s)12.5 ± 113.7 ± 1.6
GGT (IU/L) (norm: 10–75)203.8 ± 216209.3 ± 207
Alkaline phosphatase (IU/L) (norm: 35–125)224 ± 203.8289 ± 237.6
SGOT (IU/L) (norm: 5–40)61.3 ± 6854.8 ± 32.5
SGPT (IU/L) (norm: 5–40)62.45 ± 8650.5 ± 38.3
IgM (mg/dL) (norm: 25–170)437.9 ± 405.8339 ± 148

The UDCA at a dose of 15 mg/kg/day was administered to all patients. Serum levels of ETs and NOx were measured in all patients prior to initiation of UDCA therapy. In 24 patients (15 in stages I and II and nine in stages III and IV) ETs and NOx were also measured after 6 months of UDCA therapy. No patients were withdrawn from treatment during this 6-month period.

A group of 13 patients (eight men, five women, median age 53, range: 41–64) with biopsy-proven postviral (hepatitis C virus, HCV) liver cirrhosis was also enrolled. Another group of 10 patients (five men, five women, median age 36, range: 22–49), with biopsy-proven chronic hepatitis C was also included in the study as disease controls. Finally, 15 healthy volunteers, matched to the PBC population for age and sex, served as a reference group.

In 10 cirrhotic PBC patients and in all 13 controls cirrhotics ET serum levels estimations were also made in blood taken from the hepatic vein during catheterization for the measurement of wedged hepatic pressure done for assessment of portal hypertension. All PBC patients catheterized were receiving UDCA treatment for at least 1 year.

Blood was collected from all patients and controls and was immediately centrifuged at 4 °C. All serum samples were stored at −70 °C until assayed. The study was approved by the local hospital Ethics committee, and written informed consent was obtained from all patients.

Materials

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Patient population
  6. Materials
  7. Determination of endothelins
  8. Determination of nitrite/nitrate concentration
  9. Statistical analysis
  10. Results
  11. Endothelins
  12. Nitrites/nitrates
  13. The effect of UDCA treatment
  14. Hepatic vein endothelins and NOx
  15. Correlations
  16. Discussion
  17. Acknowledgement
  18. References

The HEPES and trifluoroacetic acid (TFA) were purchased from Merck (Darmstadt, Germany). Nitrate reductase (from Aspergilus species), l-lactic dehydrogenase, pyruvic acid (sodium salt), NADPH (reduced form, tetrasodium salt), FAD (disodium salt), N-(1-napthyl)ethylenediamine, acetonitrile and sulphanilamide were purchased from Sigma (Munchen, Germany). Endothelin-1 ELISA assay, Endothelin-2 ELISA assay and Endothelin-3 ELISA assay, were purchased from IBL Co. (Fujioka-Shi, Japan). C18 Sep-Pak columns were purchased from Waters Corp. (Milford, MA, USA).

Determination of endothelins

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Patient population
  6. Materials
  7. Determination of endothelins
  8. Determination of nitrite/nitrate concentration
  9. Statistical analysis
  10. Results
  11. Endothelins
  12. Nitrites/nitrates
  13. The effect of UDCA treatment
  14. Hepatic vein endothelins and NOx
  15. Correlations
  16. Discussion
  17. Acknowledgement
  18. References

Each ET was determined with a separate commercially available ELISA assay kit (IBL Co.) according to the manufacturer's instructions. In a pilot study, plasma levels of all three ETs were compared with serum levels and there was a very good correlation (r = 0.91, P < 0.001). Briefly, to extract each ET from serum we used a protocol that involved the use of 200 mg C18 Sep-Pak column. The columns were equilibrated by washing with 60% acetonitrile in 0.1% TFA followed by 0.1% TFA in water. Equal volume of 0.1% TFA in water was added to the serum and was centrifuged at 3000 g for 15 min at 4 °C. The supernatant was then applied to the column and washed with 0.1% TFA in water. The sample was then eluted slowly by applying 60% acetonitrile in 0.1% TFA in water. The eluant was collected in plastic tubes and dried down in a centrifugal evaporator under vacuum. It was then stored at −20 °C. The pellet was reconstituted with assay buffer and measured immediately.

Determination of nitrite/nitrate concentration

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Patient population
  6. Materials
  7. Determination of endothelins
  8. Determination of nitrite/nitrate concentration
  9. Statistical analysis
  10. Results
  11. Endothelins
  12. Nitrites/nitrates
  13. The effect of UDCA treatment
  14. Hepatic vein endothelins and NOx
  15. Correlations
  16. Discussion
  17. Acknowledgement
  18. References

Total nitrite plus nitrate concentration was assayed with a spectrophotometric method that uses a modification of the Griess reaction as described before.23 Briefly, 100 μL of serum sample was incubated for 30 min at 37 °C in the presence of 0.2 U/mL Aspergillus nitrate reductase, 50 mm HEPES buffer, 5 μm FAD and 0.1 mm NADPH in a total volume of 500 μL. Following the incubation, 5 μL of lactate dehydrogenase (1500 U/mL) and 50 μL of 100 mm pyruvic acid were added to each tube to oxidize any unreacted NADPH (reduced pyridine nucleotides strongly inhibit the Griess reaction). Samples were then incubated for an additional 10 min at 37 °C. Finally, 1 mL of premixed Griess’ regent was added to each tube. After 10 min of incubation at room temperature, the absorbance of each sample was determined at 543 nm in a Hitachi U-2000 Spectrophotometer (Tokyo, Japan).

Statistical analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Patient population
  6. Materials
  7. Determination of endothelins
  8. Determination of nitrite/nitrate concentration
  9. Statistical analysis
  10. Results
  11. Endothelins
  12. Nitrites/nitrates
  13. The effect of UDCA treatment
  14. Hepatic vein endothelins and NOx
  15. Correlations
  16. Discussion
  17. Acknowledgement
  18. References

All results are expressed as mean ± S.E.M. Student's paired and unpaired t-test were used for comparisons between groups where applicable. Pearson correlation was used for analysis of correlation between variables. A value of P < 0.05 was considered statistically significant.

Endothelins

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Patient population
  6. Materials
  7. Determination of endothelins
  8. Determination of nitrite/nitrate concentration
  9. Statistical analysis
  10. Results
  11. Endothelins
  12. Nitrites/nitrates
  13. The effect of UDCA treatment
  14. Hepatic vein endothelins and NOx
  15. Correlations
  16. Discussion
  17. Acknowledgement
  18. References

Mean serum levels of ET-1 were higher in PBC patients (16.21 ± 2.14 pg/mL) compared with the other groups, but there was no statistical significance. There was also no statistical significance between the two groups of PBC (16.07 ± 3.34 pg/m: in stages I and II and 16.43 ± 1.72 pg/mL in stages III and IV). Levels in both early and late-PBC were significantly higher compared with chronic hepatitis C (P < 0.003 and P < 0.001, respectively) (Table 2).

Table 2.  Serum levels of endothelins and NOx in patients and controls (data expressed as mean ± S.E.M.)
 Patients (n)ET-1 (pg/mL)ET-2 (pg/mL)ET-3 (pg/mL)NOx (μm)
  1. CAH, chronic active hepatitis C; NOx, nitrites/nitrates; PBC, primary biliary cirrhosis; ET, endothelin.

Controls1513.03 ± 2.5321.19 ± 3.8813.16 ± 2.2742.99 ± 9.79
Cirrhotics1312.22 ± 212.04 ± 2.2318.05 ± 1.1383.12 ± 10.84
CAH107.72 ± 1.4213.78 ± 2.616.29 ± 1.5666.95 ± 7.9
PBC4116.21 ± 2.1448.44 ± 6.0215.03 ± 2.0483.46 ± 8.99
PBC I and II2516.07 ± 3.3451.98 ± 8.711.57 ± 1.9673.52 ± 8.77
PBC III and IV1616.43 ± 1.7241.87 ± 5.9718.76 ± 3.4100.66 ± 18.95

Mean levels of ET-2 in PBC (48.44 ± 6.02 pg/mL) were significantly higher compared with hepatitis C patients (13.78 ± 2.6 pg/mL, P < 0.001), cirrhotics (12.04 ± 2.23 pg/mL, P < 0.001) and healthy controls (21.19 ± 3.88 pg/mL, P < 0.001). The difference between PBC stages I and II (51.98 ± 8.7 pg/mL) and stages III and IV (41.87 ± 5.97 pg/mL) was not significant (Figure 1).

image

Figure 1. Levels of serum ET-2 in healthy controls, patients with viral cirrhosis, primary biliary cirrhosis (PBC) patients and patients with chronic hepatitis C (CAH C) (*P < 0.001).

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Mean levels of ET-3 in patients with PBC stages III and IV (18.76 ± 3.4 pg/mL) were higher compared with hepatitis C patients (16.29 ± 1.56 pg/mL), cirrhotic patients (18.05 ± 1.13 pg/mL), healthy controls (13.16 ± 2.27 pg/mL) and PBC patients with stages I and II (11.57 ± 1.96 pg/mL), but there was no statistical significance between the various groups (Table 2).

Nitrites/nitrates

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Patient population
  6. Materials
  7. Determination of endothelins
  8. Determination of nitrite/nitrate concentration
  9. Statistical analysis
  10. Results
  11. Endothelins
  12. Nitrites/nitrates
  13. The effect of UDCA treatment
  14. Hepatic vein endothelins and NOx
  15. Correlations
  16. Discussion
  17. Acknowledgement
  18. References

Serum levels of NOx were found to be increased in patients with PBC (83.46 ± 8.99 μm) compared with healthy controls (42.99 ± 9.79 μm, P < 0.05). There was no statistical difference between PBC stages I and II (73.52 ± 8.77 μm) compared with stages III and IV (100.66 ± 18.95 μm). Cirrhotic patients, when compared with healthy controls, were also found to have significantly higher NOx (83.12 ± 10.84 μm, P < 0.05) (Figure 2). Results are collectively summarized in Table 2.

image

Figure 2. Levels of serum nitrites/nitrates (NOx) in healthy controls, patients with viral cirrhosis, primary biliary cirrhosis (PBC) patients and patients with chronic hepatitis C (CAH C) (*P < 0.05).

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The effect of UDCA treatment

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Patient population
  6. Materials
  7. Determination of endothelins
  8. Determination of nitrite/nitrate concentration
  9. Statistical analysis
  10. Results
  11. Endothelins
  12. Nitrites/nitrates
  13. The effect of UDCA treatment
  14. Hepatic vein endothelins and NOx
  15. Correlations
  16. Discussion
  17. Acknowledgement
  18. References

Regarding the effect of UDCA therapy in the subgroup of 24 PBC patients, we found that after 6 months of treatment, there were significant alterations in the levels of circulating ETs and NOx. In patients with PBC stages I and II (Figure 3) the post-treatment levels of ET-1 (4.69 ± 0.7 pg/mL) and ET-2 (17.6 ± 2 pg/mL) have been decreased compared with pre-treatment levels (ET-1: 19.3 ± 8.2 pg/mL and ET-2: 64 ± 17.2 pg/mL), but this is significant only for ET-2 (P < 0.05). On the contrary, in these patients (stages I and II) the post-treatment serum levels of ET-3 (17.6 ± 3 pg/mL) and NOx (90.6 ± 9.05 μm), were increased compared with the pre-treatment levels (ET-3: 12.2 ± 2.1 pg/mL, N.S. and NOx:73.7 ± 11.9 μm, P < 0.05).

image

Figure 3. Levels of endothelins and nitrites/nitrates (NOx) in primary biliary cirrhosis (PBC) patients with histological stages I and II, before and after treatment with ursodeoxycholic acid (UDCA) (*P < 0.05).

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In patients with PBC stages III and IV (Figure 4) post-treatment mean levels of ET-2 were significantly decreased (13.5 ± 3.9 pg/mL vs. 47.3 ± 6.2 pg/mL, P < 0.01). A decrease in the post-treatment levels of NOx (80.7 ± 7.6 μm vs. 105.7 ± 24.1 μm) was also noticed, but was not statistically significant. In these patients, there was also an non-significant post-treatment increase of ET-3 (27.3 ± 5.8 pg/mL vs. 21.4 ± 4.7 pg/mL), while levels of pre- and post-treatment of ET-1 were almost unchanged (14.9 ± 2.3 pg/mL vs. 14.2 ± 1.9 pg/mL respectively).

image

Figure 4. Levels of endothelins and nitrites/nitrates (NOx) in primary biliary cirrhosis (PBC) patients with histological stages III and IV, before and after treatment with ursodeoxycholic acid (UDCA) (*P < 0.01).

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Hepatic vein endothelins and NOx

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Patient population
  6. Materials
  7. Determination of endothelins
  8. Determination of nitrite/nitrate concentration
  9. Statistical analysis
  10. Results
  11. Endothelins
  12. Nitrites/nitrates
  13. The effect of UDCA treatment
  14. Hepatic vein endothelins and NOx
  15. Correlations
  16. Discussion
  17. Acknowledgement
  18. References

Comparisons of the levels of ETs between the hepatic and peripheral veins demonstrated that ET-1 was higher in the periphery in PBC patients (P < 0.01), where hepatic ET-1 was barely detectable (8.96 ± 1.97 and 1.22 ± 0.86 respectively). Similar findings were noted for ET-3 (17.9 ± 2.8 and 8.3 ± 2.2 respectively, P < 0.05, Figure 5). By contrast, levels for ET-2 were similar in the periphery and the hepatic vein (13.41 ± 5.53 and 15.11 ± 3.19 respectively). Equally, similar levels of NOx were noted between the peripheral and portal veins (Figure 5). In viral cirrhosis, levels for ETs are similar between the hepatic and peripheral veins. Although, direct comparisons between hepatic vein levels in PBC and viral cirrhosis cannot be made, since in PBC, all measurements were done after treatment with UDCA, it should be noted that ET-1 and ET-3 levels were significantly higher in viral cirrhosis (1.22 ± 0.86 vs. 5.19 ± 1.25 P < 0.05 for ET-1 and 8.30 ± 2.2 vs. 17.83 ± 4.31 for ET-3, P < 0.05) (Figure 6).

image

Figure 5. Levels of endothelins and nitrites/nitrates (NOx) in hepatic and peripheral blood in patients with primary biliary cirrhosis (PBC).

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image

Figure 6. Levels of endothelins and nitrites/nitrates (NOx) in hepatic and peripheral blood in patients with viral cirrhosis.

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Correlations

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Patient population
  6. Materials
  7. Determination of endothelins
  8. Determination of nitrite/nitrate concentration
  9. Statistical analysis
  10. Results
  11. Endothelins
  12. Nitrites/nitrates
  13. The effect of UDCA treatment
  14. Hepatic vein endothelins and NOx
  15. Correlations
  16. Discussion
  17. Acknowledgement
  18. References

There was a significant positive correlation between levels of immunoglobulin M (IgM) and ET-2 in PBC patients (r = 0.44, P < 0.05). Also, there was a significant positive correlation between levels of ET-3 and NOx in PBC patients (r = 0.28, P < 0.05). No other significant correlations between ETs, NOx and biochemical parameters were identified.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Patient population
  6. Materials
  7. Determination of endothelins
  8. Determination of nitrite/nitrate concentration
  9. Statistical analysis
  10. Results
  11. Endothelins
  12. Nitrites/nitrates
  13. The effect of UDCA treatment
  14. Hepatic vein endothelins and NOx
  15. Correlations
  16. Discussion
  17. Acknowledgement
  18. References

In liver sinusoidal microcirculation, NO and ETs seem to be the major vasoactive agents who regulate the blood flow. In liver cirrhosis, because of sinusoidal endothelial dysfunction, there is a deficiency of endothelial NO production in the intrahepatic microcirculation. On the contrary, splanchnic and systemic vascular NO production is increased, causing systemic vasodilation and thus the hyperdynamic circulation syndrome. Additionally, the activated stellate cells, along with the endothelial cells, seem to overproduce ETs.9, 11, 15, 24–26

In our study, no significant difference was found in serum ET-1 between patients with cirrhosis and healthy controls. Previous studies are conflicting. There are papers reporting that cirrhotic patients have significantly higher levels of ETs and this increase is mostly due to an increase in ET-1 and ET-3 in some cases in proportion to the severity of liver damage.27–33 By contrast, other studies report that this difference is not significant31, 34 in patients without ascites. As reported elsewhere,23 these discrepancies could be due to either methodological differences or inclusion of cirrhotics of varying aetiology and severity.

Few studies have estimated NO production in PBC,16–19and so far no other studies have evaluated the role of ETs in this disease. In a previous report,35 we presented preliminary data indicating an increased level of ET-1 and NO in patients with both early and late-PBC. However, the ELISA we used then for measurement of ET-1 had a cross-reactivity of over 100% with ET-2. Therefore, we extended our study using specific ELISAs for each of the three known ETs. It has been previously reported that ET-2 is not present in human plasma.27, 28 However, in the present study we found increased circulating levels of ET-2 in patients with PBC, both in early (I and II) and late (III and IV) stages of the disease, as well as in both the normal and disease controls. It is possible that the antibodies used in these earlier studies were not capable of separately detecting ET-2. It is interesting that, in our study, increased ET-2 was found even in early stage PBC, indicating that this might be a primary event. Therefore, it could possibly be involved in PBC pathophysiology.

Serum levels of ET-1 were found to be similar in all PBC stages, and higher but not significantly so, from healthy controls. However, even the non-significant increase of ET-1 as observed in early PBC, might add to the ischaemic injury of perivascular plexus (PVP) of the small intrahepatic ductules. It is possible that an endothelial dysfunction is the reason for the increased ETs in PBC patients,36, 37 a situation that has been previously reported in ischaemic heart disease.38

The serum levels of ET-3 in PBC patients were found to be similar to healthy controls. Yet, when this result was further analysed, we noticed that patients with PBC stages III and IV had higher levels of ET-3 compared with healthy controls or patients with histological stages I and II, but this difference did not reach statistical significance. This increase that parallels disease progression, was also found in the group of cirrhotic patients but again there was no statistical significance. Therefore, it seems that this increase is secondary to liver damage. Elevated circulating levels of ET-3 in cirrhotic patients have been previously reported27, 30, 34 and attributed, at least in part, to increased splanchnic release.

Serum levels of NOx in all stages of PBC patients were found significantly higher than controls in agreement with a few previous studies.17, 19 Cirrhotic patients in our study were also found to have significantly increased levels of NOx, a fact that has also been described elsewhere.23, 39, 40 In this respect, it is possible that the high levels of NOx, found in PBC stages III and IV, are indeed related to the advanced liver injury. However, such an explanation cannot be applied to the increased levels found in early PBC. It could well be that this is due to an effort by the liver to overcome the effects of the increased ET-2, or alternatively it could be also a primary defect of endothelial cells.

The UDCA is the only drug currently approved for treatment of patients with PBC. Although data are conflicting regarding its efficacy on long-term survival, there are studies that report41–43 a significant delay of fibrosis progression in PBC and an improved survival44 after UDCA treatment, especially if given early in the disease. In our study, UDCA caused a significant decrease of ET-2 in all histological stages most pronounced in early stage PBC (Figure 3). At the same time a significant increase of NO in patients with early stage PBC was also noted. With these alterations, UDCA should reduce vasoconstriction and promote vasodilation. We hypothesize, therefore, that UDCA exerts its beneficial action in PBC through dilatation of vessels in the PVP and its action may be more beneficial if given early in the disease process.

Hepatic vein ET-2 is the only ET that is increased compared with the levels in hepatic vein of the cirrhotics. Although this increase was not significant it should be taken into account that ET hepatic vein levels in PBC were assessed after UDCA treatment, which strongly decreases peripheral levels. It seems plausible therefore that this difference is even higher. It is equally interesting that peripheral and hepatic vein NOx levels in late-PBC are similar in contrast to viral cirrhosis where levels are higher in the periphery. This finding further supports the idea of a more generalized endothelial dysfunction in PBC, which requires further studies. Alternatively, an increased uptake of ETs might occur in the liver of PBC patients.

In conclusion, we found that serum levels of ET-2 are increased in patients with both early and late-PBC. Additionally, UDCA treatment significantly reduces the ET-2 concentrations in PBC patients. Furthermore, comparisons of the ET levels between the hepatic vein and peripheral veins may indicate that the increased ET-2 levels may result from a more generalized endothelial cell dysfunction in PBC. Further studies are required to investigate the possibility that endothelial abnormalities are a primary event possibly contributing to the disease pathogenesis.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Patient population
  6. Materials
  7. Determination of endothelins
  8. Determination of nitrite/nitrate concentration
  9. Statistical analysis
  10. Results
  11. Endothelins
  12. Nitrites/nitrates
  13. The effect of UDCA treatment
  14. Hepatic vein endothelins and NOx
  15. Correlations
  16. Discussion
  17. Acknowledgement
  18. References
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