Aldo Roda

Structure-activity relationship studies on natural and synthetic bile acid analogs

The objective of our research was to develop ursodiol analogs that are structurally modified to modulate hepatic side-chain amidation and prevent 7-dehydroxylation by intestinal bacteria while at the same time maintaining the critical micellar concentration (CMC) and hydrophilicity of ursodiol. More than 20 naturally occurring bile acids were screened for physicochemical properties. Then, two generations of analogs were studied, and those with physicochemical properties similar to ursodiols were analyzed for physiologic properties. The first generation of analogs included molecules with steric and/or electronic hindrance on the side chain; the second group consisted of the same molecules conjugated with glycine or taurine and also “pseudoconjugated” analogs (23-hydroxylated, esterified, and amidated with other amino acids). Of the first-generation analogs, only cyclopropane D derivative and trans-olefin were useful to our purposes, being conjugated by the liver and almost completely recovered in bile. These two analogs were deconjugated and 7-dehydroxylated but with slower kinetics. The hydrophilicity of the molecules could be augmented by increasing the polarity of the steroid ring. Among the pseudoconjugated analogs, the CMC values were similar to those of the natural analogs, although hydrophobicity differed among the group. The analogs that were not deconjugated were not 7-dehydroxylated either. All of the pseudoconjugated bile acids were efficiently taken up by the liver, and their recovery in bile was similar to that of glycine and taurine ursodiol. From these studies we now know that side chain configuration and conformation are important in the conjugation and deconjugation processes. Mild modification of the side chain can prevent 7-dehydroxylation and thus yield a bile acid more resistant to intestinal bacteria and more bioavailable. Prevention of hepatic conjugation improves biliary secretion and recovery of many analogs.

Bioluminescent flow sensor for D-(-)-lactate

Abstract Previously, a bioluminescent flow sensor was developed for the determination of the content of l -lactate in biological fluids (serum, plasma, ventricular fluid) by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH), produced by immobilized lactate dehydrogenase (LDH), with bacterial bioluminescent enzymes immobilized on a separate nylon coil. Based on a similar scheme, a sensor has now been developed for d -(−)-lactate. The co-immobilization of alanine aminotransferase (ALT) with d -LDH improved the lactate transformation to 40–60%. The response was linear from 1 to 100 μmol 1 −1 at 25 °C for the LDH-ALT reactor. The intra- and inter-assay relative standard deviations were less than 75% and the recoveries ranged from 93 to 106%. The results agreed well with those obtained with a spectrophotometric method and applications to d -(−)-lactate determination in serum, milk and microorganism extracts are reported together with those for l -(+)-lactate.

A theoretical model for the critical micelle concentration of bile salts

Critical micelle concentration (CMC) is a fundamental parameter in the evaluation of the biological activity of natural and synthetic bile salts. The CMC is logarithmically related to the free energy of solute micellization in aqueous solution. Hydrophobic and hydrogen bonding interaction energies were identified as the primary contributors to this free energy and the logarithm of the CMC was modeled as a linear function of relevant chemical group contributions to the solvent accessible molecular surface area of the solute. The structures (three-dimensional atomic coordinates) of 23 mono-, di-, and tri-hydroxyl bile acids were generated and optimized by energy minimization. The accessible surface area for each structure was computed and partitioned according to calculated charge distribution and polar group orientation. Experimental CMC values were fitted to these computed quantities by least squares multiple linear regression. Two regression equations, based on slightly different surface area partition schemes, were derived and compared. Their significance in explaining the aggregation process and in predicting the CMC of new bile salts is discussed.

Microdialysis and luminescent probe: analytical and clinical aspects☆

Immobilized enzymes are widely used in the clinical laboratory to assay several analytes and enzymes. The use of immobilized enzymes makes these reagents recoverable, disposable and in most cases increases their stability and catalytic activity. In conjunction with bioluminescent enzymes (firefly and bacterial luciferases) and chemiluminescent catalyst (peroxidase) we set up high-sensitive flow sensors based on the use of nylon tube coil or epoxy methacrylate column as solid support. For in-vivo determination a suitable microdialysis probe inserted directly into brain or blood allows continuous measurement of extracellular lactate levels by means of a bioluminescent flow detector system. This procedure performs more measurements in the same time interval than other systems (HPLC), e.g. to give a detailed description of the effects of ischemia, or other pathological events, on lactate concentration in the brain.

Bioluminescence flow sensor for determination of creatine kinase activity in blood

Abstract A bioluminescent flow sensor was developed for the assay of creatine kinase (CK) using firefly luciferase immobilized on a nylon coil. The CK-catalysed reaction of creatine phosphate with ADP took place in a cuvette before the injection into the bioluminescent detector coil. The response was linear from 0.1 to 100 U l − at 25°C. An advantage of the flow sensor is a detection limit of less than 0.1 U l −1 , which, together with a high precision, allows determination of the CK activity in blood sera in about 5 min. The intra- and inter-assay reproducibilities (RSD) were less than 10% and the recovery range was 86–110%. The results agreed well with those obtained with a spectrophotometric method and with the normal reference values.

Hepatic uptake and biliary secretion of bile acids in the perfused rat liver.

Hepatic uptake and biliary secretion have been evaluated in the isolated perfused rat liver for cholic, chenodeoxycholic, ursodeoxycholic acid, both free and taurine-conjugated; the physicochemical properties of the bile acids have also been calculated and related to these experimental parameters. Cholic acid disappearance rate from the perfusate was the fastest, followed by that of ursodeoxycholic and chenodeoxycholic; it was also faster for taurine-conjugated bile acids than for their respective unconjugated forms. The recovery in bile was higher for conjugated than for unconjugated bile acids, and among each class, was higher for cholic than for chenodeoxycholic and ursodeoxycholic. The hepatic uptake correlated negatively (r = -0.99) with the bile acid lipophilicity, while the biliary secretion correlated with the solubility of the molecules. These results show the effect of the physicochemical properties of BA on their hepatic handling, at the physiological concentration of BA in the portal blood.

Chapter 15:Cell-based Bioluminescent Biosensors

Cell-based biosensors are designed to take advantage of the unique selectivity afforded by biological recognition elements in combination with a protein reporter that generates a measurable signal, i.e., fluorescence, chemiluminescence, bioluminescence, colorimetric or electrochemical. These biosensors are constructed, in general, by transforming a living bacterial cell with an engineered plasmid on which the expression of a reporter protein is under the regulatory control of a promoter/regulatory protein which recognizes an analyte of interest. As a result of this design, the biosensors are capable of rapid, specific/selective, and sensitive measurement in standard as well as miniaturized, rugged, portable platforms. A further benefit of cell-based biosensors is that they provide information regarding the bioavailablility of the analyte in a sample. Cell-based biosensors have been developed using a number of genes for luminescent proteins such as prokaryotic and eukaryotic luciferases, green fluorescent protein (GFP) and its mutants, aequorin, and β-galactosidase. The spectrum of analytes for which cell-based biosensors have been constructed is constantly expanding. Currently, there are biosensors designed to detect general toxicants as well as stress factors and specific analytes or groups of analytes such as metals, inorganic molecules, organic pollutants, antibiotics, sugars, and quorum sensing molecules. Additionally, the development of rugged biosensors, like those based on spore-forming microbes, the miniaturization of cell-based assay formats, and their incorporation onto microfluidics platforms will further expand the applications in on-site environmental/clinical analysis.

Chapter 7:Whole-cell Sensing Systems in Chemical and Biological Surveillance

With the increasing threat of biological and chemical warfare agents, developing innovative strategies for rapid, simple and precise detection of these harmful agents is critically important. In response to this demand, much effort has been focused on developing biosensors and biomimetic systems tha...

Methodological and Clinical Aspects of Bile Acid Analysis in Biological Fluids

The more relevant physicochemical properties of bile acids (BA) in aqueous solution are described in relation to their structure. The acquisition of such information is useful to define the possible BA species that exist in a particular biological fluid. Interaction with proteins, micelles formation, complexation and ionizzation can modulate the activity of BA in solution with consequent differences in their biological and physiological properties.

Immobilized Biospecific Proteins in Analytical Clinical Chemistry

Immobilized proteins are widely used in the clinical laboratory, e.g., immobilized enzymes in assay for analytes such as glucose, uric acid and immobilized antigen and antibodies in immunoassay. In the enzymatic analysis the use of immobilized enzymes make these reagents recoverable and thus reusable and in most cases increases their stability and catalytic activity. In conjuction with bioluminescent enzymes (firefly and bacterial luciferases) we set up high sensitive flow methods based on the use of nylon tube coil as solid support.