Adriana M. Almará

Inhibitory effect of bilirubin on complement-mediated hemolysis

We investigated the in vitro action of the bile pigments, unconjugated bilirubin (UB) and bilirubin monoglucuronide (BMG) on complement (C) cascade reaction. Both UB and BMG inhibited hemolysis in the classical pathway (CP) in a dose-dependent manner at low micromolar concentrations, UB showing a stronger effect than BMG. The analysis of the action of UB on the hemolytic activity of the C1, C4, C2 and C-EDTA components of the C cascade revealed that the C1 step was the most inhibited. An enzyme immunoassay was developed to evaluate the effect of UB on the binding of C1q, one of the subcomponents of C1, to human IgM and IgG. The study demonstrated that the unconjugated pigment interferes both the C1q-IgM and -IgG interactions, thus tentatively explaining the inhibitory action of UB on hemolytic activity of C1. We conclude that the anti-complement effect of UB is mainly exerted on the C1 component, the recognition unit of CP. The potential clinical implication of the reported effects in hyperbilirubinemia is discussed.

In vivo anti-complement effect of bilirubin-IXα

The effect of the IXalpha isomer of unconjugated bilirubin (UB) on complement-mediated intravascular hemolysis was evaluated in rats carrying naturally occurring heteroantibodies against sheep erythrocytes. Several doses of UB were administered i.v. to these animals in order to induce different levels of hyperbilirubinemia. Intravascular hemolysis was promoted by transfusion with a sheep red cell suspension. Hemoglobin in urine was assessed as a marker of intravascular hemolysis. The urinary excretion of hemoglobin was attenuated by UB in a dose-dependent manner. To establish whether complement was involved in the hemolytic reaction, we evaluated the hemolytic activity of complement in these same animals, before and after sheep erythrocyte transfusion. The significant consumption of complement, which was partially prevented by UB, corroborated its participation in the intravascular hemolytic reaction in the current experimental conditions. The data suggest an inhibitory action of UB on complement-mediated hemolysis in vivo.

Evaluation of urinary N-acetyl-beta-D-glucosaminidase as a marker of early renal damage in patients with type 2 diabetes mellitus

OBJECTIVE To evaluate the clinical usefulness of urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion for the detection of early tubular damage in type 2 diabetes mellitus (T2DM). SUBJECTS AND METHODS Thirty six patients with T2DM were divided into two groups based on urinary albumin to creatinine ratio (ACR): normoalbuminuria (ACR <30 mg/g; n=19) and microalbuminuria (ACR =30-300 mg/g; n=17). The following parameters were determined in both groups: urinary NAG and albumin, serum and urine creatinine, fasting plasma glucose and glycated hemoglobin (HbA1c). RESULTS Urinary NAG levels [Units/g creatinine; median (range)] were significantly increased in microalbuminuria group [17.0 (5.9 - 23.3)] compared to normoalbuminuria group [4.4 (1.5 - 9.2)] (P<0.001). No differences between groups were observed in fasting glucose, HbA1c, serum creatinine levels and estimated glomerular filtration rates (eGFR). Urinary NAG positively correlated with ACR (r=0.628; p<0.0001), while no significant association was observed between NAG and glycemia, HbA1c, serum creatinine and eGFR. CONCLUSIONS The increase of urinary NAG at the microalbuminuria stage of diabetic nephropathy (DN) suggests that tubular dysfunction is already present in this period. The significant positive association between urinary NAG excretion and ACR indicates the possible clinical application of urinary NAG as a complementary marker for early detection of DN in T2DM.

Unconjugated bilirubin inhibits C1 esterase activity

OBJECTIVE To evaluate if unconjugated bilirubin (UB) inhibits C1 esterase activity. DESIGN AND METHODS Esterase activity was evaluated by C1-mediated hydrolysis of N-acetyl-L-tyrosine ethyl ester, and binding of UB to C1r and C1s was assessed by dot-blot analysis. RESULTS UB inhibited C1 enzymatic activity. C1r, C1s and human serum albumin bound [(14)C]-UB to a similar extent. CONCLUSIONS UB inhibits C1 esterase activity, apparently due to a direct pigment-protein interaction. This could explain the inhibitory action of UB on complement activation.