Direct Chiral Supercritical Fluid Chromatography-Mass Spectrometry Analysis of Monoacylglycerol and Diacylglycerol Isomers for the Study of Lipase-Catalyzed Hydrolysis of Triacylglycerols.

The fast and selective separation method of intact monoacylglycerol (MG) and diacylglycerol (DG) isomers using chiral supercritical fluid chromatography-mass spectrometry (SFC-MS) was developed and employed to study lipase selectivity in the hydrolysis of triacylglycerols (TGs). The synthesis of 28 enantiomerically pure MG and DG isomers was performed in the first stage using the most commonly occurring fatty acids in biological samples such as palmitic, stearic, oleic, linoleic, linolenic, arachidonic, and docosahexaenoic acids. To develop the SFC separation method, different chromatographic conditions such as column chemistry, mobile phase composition and gradient, flow rate, backpressure, and temperature were carefully assessed. Our SFC-MS method used a chiral column based on a tris(3,5-dimethylphenylcarbamate) derivative of amylose and neat methanol as a mobile phase modifier, which provides baseline separation of all the tested enantiomers in 5 min. This method was used to evaluate hydrolysis selectivity of lipases from porcine pancreas (PPL) and Pseudomonas fluorescens (PFL) using nine TGs differing in acyl chain length (14-22 carbon atoms) and number of double bonds (0-6) and three DG regioisomer/enantiomers as hydrolysis intermediate products. PFL exhibited preference of the fatty acyl hydrolysis from the sn-1 position of TG more pronounced for the substrates with long polyunsaturated acyls, while PPL did not show considerable stereoselectivity to TGs. Conversely, PPL preferred hydrolysis from the sn-1 position of prochiral sn-1,3-DG regioisomer, whereas PFL exhibited no preference. Both lipases showed selectivity for the hydrolysis of outer positions of DG enantiomers. The results show complex reaction kinetics of lipase-catalyzed hydrolysis given by different stereoselectivities for substrates.

Complement mediates the binding of HIV to erythrocytes.

A fraction of HIV is associated with erythrocytes even when the virus becomes undetectable in plasma under antiretroviral therapy. The aim of the present work was to further characterize this association in vitro. We developed an in vitro model to study the factors involved in the adherence of HIV-1 to erythrocytes. Radiolabeled HIV-1 (HIV) and preformed HIV-1/anti-HIV immune complexes (HIV-IC) were opsonized in various human sera, purified using sucrose density gradient ultracentrifugation, and incubated with human erythrocytes. We observed that, when opsonized in normal human serum, not only HIV-IC, but also HIV, bound to erythrocytes, although the adherence of HIV was lower than that of HIV-IC. The adherence was abolished when the complement system was blocked, but was maintained in hypogammaglobulinemic sera. Complement-deficient sera indicated that both pathways of complement were important for optimal adherence. No adherence was seen in C1q-deficient serum, and the adherence of HIV was reduced when the alternative pathway was blocked using anti-factor D Abs. The adherence could be inhibited by an mAb against complement receptor 1. At supraphysiological concentrations, purified C1q mediated the binding of a small fraction of HIV and HIV-IC to erythrocytes. In conclusion, HIV-IC bound to erythrocytes as other types of IC do when exposed to complement. Of particular interest was that HIV alone bound also to erythrocytes in a complement/complement receptor 1-dependent manner. Thus, erythrocytes may not only deliver HIV-IC to organs susceptible to infection, but free HIV as well. This may play a crucial role in the progression of the primary infection.

Association of a pool of HIV-1 with erythrocytes in vivo: a cohort study.

BACKGROUND: Treatment of HIV-1-infected individuals with antiretrovirals can result in sustained suppression of plasma viral RNA at concentrations below the detection limit of available assays. However, continuing virus replication has been detected in patients with viral RNA in plasma suppressed for months to years, and many cell types are known to act as reservoirs or carriers for the virus. In vitro, erythrocytes bind HIV-1 immune complexes, so we tested for a circulating pool of HIV-1 associated with erythrocytes in people with HIV-1 infection. METHODS: We investigated 82 chronically HIV-1-infected individuals. Plasma, white cells, and erythrocytes were tested for HIV-1 RNA by RT-PCR. FINDINGS: Erythrocyte-associated HIV-1 RNA was detected in 80 of 82 individuals. In 23, plasma HIV-1 RNA had been undetectable (<20 copies/mL) for up to 32 months; in corresponding erythrocyte samples, there were up to 82878 HIV-1 RNA copies per mL whole blood. HIV-1 associated with erythrocytes in vivo was shown to be infectious. Within the subgroup of patients with undetectable plasma viral load, higher numbers of HIV-1 associated with erythrocytes were correlated with a history of advanced clinical stages of HIV-1 infection (p=0.014). INTERPRETATION: A pool of HIV-1 is associated with erythrocytes even after long-term suppression of viral RNA in plasma. This finding is direct evidence for continuing virus replication or release in these individuals. Quantification of this viral pool may help to judge suppression of HIV-1 replication in individuals with undetectable plasma HIV-1 RNA.