Biomechanical and biochemical investigation of erythrocytes in late stage human leptospirosis

Leptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira, which can cause lipid changes in the erythrocyte membrane. Optical tweezers were used to characterize rheological changes in erythrocytes from patients with leptospirosis in the late stage. Biochemical methods were also used for quantification of plasma lipid, erythrocyte membrane lipid, and evaluation of liver function. Our data showed that the mean elastic constant of erythrocytes from patients with leptospirosis was around 67% higher than the control (healthy individuals), indicating that patient's erythrocytes were less elastic. In individuals with leptospirosis, several alterations in relation to control were observed in the plasma lipids, however, in the erythrocyte membrane, only phosphatidylcholine showed a significant difference compared to control, increasing around 41%. With respect to the evaluation of liver function of individuals with leptospirosis, there was a significant increase in levels of alanine transaminase (154%) and aspartate transaminase (150%), whereas albumin was 43.8% lower than control (P<0.01). The lecithin-cholesterol acyltransferase fractional activity was 3.6 times lower in individuals with leptospirosis than in the healthy individuals (P<0.01). The decrease of the erythrocyte elasticity may be related to the changes of erythrocyte membrane phospholipids composition caused by disturbances that occur during human leptospirosis, with phosphatidylcholine being a strong candidate in the erythrocyte rheological changes.

Elevated levels of erythrocyte-conjugated dienes indicate increased lipid peroxidation in schistosomiasis mansoni patients

Schistosoma mansoni causes liver disease by inducing granulomatous inflammation. This favors formation of reactive oxygen species, including superoxide ions, hydrogen peroxide and hydroxyl radicals all of which may induce lipid peroxidation. We have evaluated lipid peroxidation in 18 patients with hepatosplenic schistosomiasis mansoni previously treated with oxamniquine followed by splenectomy, ligature of the left gastric vein and auto-implantation of spleen tissue, by measuring levels of erythrocyte-conjugated dienes and plasma malondialdehyde (MDA). Age-matched, healthy individuals (N = 18) formed the control group. Erythrocyte-conjugated dienes were extracted with dichloromethane/methanol and quantified by UV spectrophotometry, while plasma MDA was measured by reaction with thiobarbituric acid. Patient erythrocytes contained two times more conjugated dienes than control cells (584.5 ± 67.8 vs 271.7 ± 20.1 æmol/l, P < 0.001), whereas the increase in plasma MDA concentration (about 10 percent) was not statistically significant. These elevated conjugated dienes in patients infected by S. mansoni suggest increased lipid peroxidation in cell membranes, although this was not evident when a common marker of oxidative stress, plasma MDA, was measured. Nevertheless, these two markers of lipid peroxidation, circulating MDA and erythrocyte-conjugated dienes, correlated significantly in both patient (r = 0.62; P < 0.01) and control (r = 0.57; P < 0.05) groups. Our data show that patients with schistosomiasis have abnormal lipid peroxidation, with elevated erythrocyte-conjugated dienes implying dysfunctional cell membranes, and also imply that this may be attenuated by the redox capacity of antioxidant agents, which prevent accumulation of plasma MDA.

Characterization and potential uses of rabbit polyclonal antibodies against human plasma lecithin-cholesterol acyltransferase

Familial and secondary deficiency of plasma lecithin-cholesterol acyltransferase (LCAT) produce circulating lipoprotein particles with gross structural and compositional abnormalities; these have adverse effects on a variety of cellular functions. Factors affecting hepatic synthesis and secretion of this plasma enzyme are largely unknown but, potentially, some of them can be investigated with monospecific antibodies. In the present study, enzymically active LCAT was purified 40,000-fold from human plasma and then used to raise polyclonal antibodies in New Zealand White rabbits. Addition of this antiserum (1 mul) to human plasma (25 mul) completely inhibited LCAT activity, although it was less effective against plasma from other species. The antibodies appeared to be monospecific to plasma LCAT. They gave a single precipitin arc by crossed immunoelectrophoresis, while immunodiffusion established that there was no cross-reactivity with several apolipoproteins or with serum albumin. Moreover, the antiserum was successfully used to detect LCAT in normal human plasma by Laurell rocket immunoelectrophoresis. By contrast, Western blotting of plasma proteins using whole LCAT antiserum was largely unsuccessful because of high background staining, although this could be substantially reduced by use of an IgG fraction. However, the whole antiserum readily immunoprecipitated LCAT secreted into the culture medium of HepG2 cells, a human hepatoblastoma cell line, pre-labelled with [35S]methionine, the [(35)S]-labelled LCAT appearing as a narrow 65-kDa protein band by electrophoresis and fluorography. We conclude that polyclonal antibodies may be an important tool to investigate the characteristics and underlying mechanisms of secondary LCAT deficiencies, including those associated with hepatic cirrhosis and schistosomiasis.