Conjugated bilirubin triggers anemia by inducing erythrocyte death

Abstract

Hepatic failure is commonly associated with anemia, which may result from gastrointestinal bleeding, vitamin deficiency, or liver-damaging diseases, such as infection and alcohol intoxication. At least in theory, anemia during hepatic failure may result from accelerated clearance of circulating erythrocytes. Here we show that bile duct ligation (BDL) in mice leads to severe anemia despite increased reticulocyte numbers. Bilirubin stimulated suicidal death of human erythrocytes. Mechanistically, bilirubin triggered rapid Ca(2+) influx, sphingomyelinase activation, formation of ceramide, and subsequent translocation of phosphatidylserine to the erythrocyte surface. Consistent with our in vitro and in vivo findings, incubation of erythrocytes in serum from patients with liver disease induced suicidal death of erythrocytes in relation to their plasma bilirubin concentration. Consistently, patients with hyperbilirubinemia had significantly lower erythrocyte and significantly higher reticulocyte counts compared to patients with low bilirubin levels.

Conclusion: Bilirubin triggers suicidal erythrocyte death, thus contributing to anemia during liver disease.

Fig. 1

Mice suffer from anemia following bile duct ligation. (A-D) Mice with BDL were compared to sham controls. (A) Serum bilirubin concentration was measured (each point indicates one animal, n = 3-11). Arithmetic means ± SEM of (B) red blood cells, which were quantified in the blood of BDL and sham-operated mice (n = 5-10). (C) Hematocrit was determined after BDL and compared with corresponding sham controls (n = 5-10). (D) Total hemoglobin levels after BDL and in corresponding sham controls were analyzed (n = 5-10). (E) Hematocrit is shown as a function of bilirubin concentrations of individual BDL mice and sham controls 21 days postoperation (n = 24). R² = 0.7095; P < 0.001 indicates slope significantly nonzero.

Fig. 2

Reticulocyte increases and enhanced erythrocyte turnover in mice following BDL. (A) Percentage of reticulocytes after BDL and in corresponding sham controls was measured over time (n = 6-14). (B) Absolute reticulocyte count in BDL animals and sham controls 7 (7D) and 21 (21D) days postoperation is illustrated (n = 12-14). (C) Absolute reticulocyte count is presented in relation to the bilirubin concentration in individual BDL mice and sham controls 21 days postoperation (n = 24) R² = 0.7733; P < 0.001 indicates slope significantly nonzero. (D) CFSE⁺-labeled red blood cells were injected into BDL animals and sham controls followed by monitoring of transferred erythrocytes. Data are presented as percent of CFSE⁺ erythrocytes 6 hours after transfer (n = 8-10).

Fig. 3

BDL animals exhibit increased suicidal erythrocyte death. (A,B) Arithmetic mean ± SEM of the percentage of annexin V bound red blood cells (RBC) in mice following BDL as compared to sham controls. (A) PS exposure on the outer leaflet of the cell membrane reflected by annexin V binding 1.5 and 16 hours after collecting blood samples 3 weeks post-BDL (n = 12-14). (B) Percentage of annexin V-bound RBC from BDL and sham-operated mice following incubation with 1 μg/mL ionomycin (1.5 hours, Iono), hypertonic solution (3 hours, HS), and solution containing 0 mM glucose is shown (16 hours, ED, n = 12-14).

Fig. 4

Bilirubin-mediated Ca²⁺ influx and ceramide production triggers PS translocation to the outer leaflet of the cell membrane. (A) Percentage of annexin V-bound human erythrocytes after 6 hours exposure to the indicated concentration of conjugated bilirubin is shown (n = 8). (B) Mean fluorescence intensity (MFI) of Fluo3-AM is displayed after 6 hours exposure of human erythrocytes to conjugated bilirubin at the indicated concentration (n = 8). (C) MFI of ceramide abundance in human erythrocytes following a 6-hour incubation with the indicated concentrations of conjugated bilirubin is shown (n = 8).

Fig. 5

Bilirubin-mediated suicidal erythrocyte death can be reduced by calcium removal, and urea or albumin addition. (A) Percentage of annexin V-bound human red blood cells (RBC) normalized to exposure of conjugated bilirubin (6 hours, 30 mg/dL) in control solution (C), in solution containing 600 mM urea (Urea), in solution without Ca²⁺ and with EDTA (1 mM, 0C), and in solution without Ca²⁺ and with EDTA (1 mM) and urea (600 mM, 0C/Urea) is illustrated (n = 8) ***P < 0.001; **P < 0.01 indicates significant difference between bilirubin containing solutions and corresponding controls; ###P < 0.001 indicates significant difference between bilirubin containing Ringer's solution and bilirubin containing solution with Urea, without calcium or both. (B) Percentage of annexin V-bound RBC after 6-hour incubation with (white bars) or without (black bars) bilirubin in Ringer's solution containing the indicated percentage of BSA (n = 6). ***P < 0.001; *P < 0.05 indicates statistically significant difference between bilirubin containing solutions and the corresponding controls; ##P < 0.01; ###P < 0.001 indicates statistically significant difference between bilirubin and bilirubin containing indicated concentrations of BSA, n.s. = nonsignificant.

Fig. 6

Eryptosis and anemia in patients with liver disease. P values indicate slopes significantly nonzero. (A) Percentage of annexin V-bound red blood cells (RBC) in blood samples collected from patients (n = 23 from cohort A in Table 1) suffering from liver disease following a 48-hour incubation in their own serum as a function of plasma bilirubin concentration R² = 0.5898. (B) The percentage of annexin V-bound 0 rhesus-neg erythrocytes from healthy volunteers following a 48-hour incubation in sera of patients suffering from liver disease is shown as a function of the plasma bilirubin concentration R² = 0.7729 (n = 16 from cohort A in Table 1). (C) Red blood cell number is shown as a function of plasma bilirubin concentration in 41 patients R² = 0.1796 (cohort B in Table 1). (D) Albumin levels are shown as a function of plasma bilirubin levels R² = 0.5027 (n = 20 from cohort B in Table 1). (E) Red blood cell count is presented in relation to the albumin concentration R² = 0.3999 (n = 20 from cohort B in Table 1).