Characterization of dynamically prepared phospholipid-modified reversed-phase columns.

We have modified a reversed-phase (RP8) column by passing through it an aqueous solution of phosphatidylcholine-based liposomes. The phospholipids from the liposomes adsorb onto the octyl chain of the stationary phase, thus altering the nature of the stationary phase and of the chromatographic interactions. The properties of the phospholipid-modified column were investigated using solutes belonging to several chemical classes. We found that the retention factors of negatively and positively charged solutes decreased as the amount of phospholipid in the column was increased. For the solutes studied here the extent of the decrease was smaller for the positive solutes. With neutral solutes, the retention factors of some (benzenediols) increased markedly while with others (ketones) the retention factors decreased. The selectivities between the various solutes on the phospholipid-modified column were different than on the original reversed-phase column. The retention behavior of the solutes can be explained in terms of (1) effective shielding of the hydrophobic part of the stationary phase by the polar head groups of the phospholipids and (2) hydrogen bond formation between the solutes and the carbonyl oxygens as well as the non-ester phosphate oxygens in the polar head groups of the phospholipids.

Optimization by isochronal analysis. II. Changes in mobile phase velocity and temperature, and in mobile phase composition and temperature.

The theory of resolution optimization by isochronal analysis is further extended to the cases involving simultaneous changes in mobile phase velocity and column temperature, and in mobile phase composition and temperature. In each case, the resolution equation, when written in terms of isochronal conditions, shows that improvements can be obtained without sacrificing analysis time. Several subcases, which relate to the dependence of the selectivity factors on the changed parameters, are described. For each system, the contributions of the various parameters to the resolution are examined. The results of these discussions are plotted as resolution surfaces, on which lines of constant analysis time are indicated. The experimental routes that chromatographers must take in order to apply isochronal analysis are described.

Separation and determination of saturated very-long-chain free fatty acids in plasma of patients with adrenoleukodystrophy using solid-phase extraction and high-performance liquid chromatographic analysis.

An improved method was developed for the isolation of very-long-chain free fatty acids (VLCFFAs) in plasma and their separation and determination by high-performance liquid chromatography (HPLC). The method includes sample clean-up using solid-phase extraction, fluorophoric labelling of the FFAs and reversed-phase HPLC separation. The solid-phase extraction was carried out with aminopropyl-bonded phase columns. The FFAs were then derivatized with 9-anthryldiazomethane (fluorescent) reagent and separated by HPLC on an RP-18 column with methanol as the mobile phase. Using this method, the concentrations of C20:0, C22:0, C24:0 and C26:0 were determined in the plasma of five adrenoleukodystrophy (ALD) patients, one obligatory heterozygote, four healthy male volunteers and one child with cerebral leukodystrophy but without any other ALD symptoms. Statistically significant differences were found in the levels of C24 and C26 and in the ratios C24/C22 and C26/C22 in ALD patients and in normal controls. The values were higher in patients with X-ALD. This method therefore provides a rapid and accurate procedure for the laboratory confirmation of X-ALD.

High-performance liquid chromatographic analysis of free palmitic and stearic acids in cerebrospinal fluid.

A relatively simple method for extraction of free fatty acids from cerebrospinal fluid with aminopropyl bonded-phase columns, and the estimation of palmitic acid (C16:0) and stearic acid (C18:0) concentrations by high-performance liquid chromatographic analysis is described. The values of C16:0 and C18:0 in patients with non-neurological disorders lie within a narrow range, with a mean (+/- S.D.) of 4.02 +/- 0.33 micrograms/ml for C16:0 and 2.72 +/- 0.39 micrograms/ml for C18:0.

Numerical solution of the complete mass balance equation in chromatography.

A new approach to simulate the movement of bands through a chromatographic column is presented. Similar to the Craig distribution model, the mass balance equation is divided into two equations describing two successive processes. The first equation includes two effects: solute diffusion in the mobile phase and migration of the solute band with the mobile phase. The second equation deals with the distribution of the solute between phases, i.e., the adsorption isotherm. The partial differential equations are integrated numerically over time and space using two methods. The first approach is a finite difference method. In the second approach, the propagation operator is expanded in a Chebyshev series, where large time steps can be used. The rate of adsorption and desorption is determined by the size of the time increment. By varying the size of the time step, it is possible to study kinetic effects. The influences of sample size, injection width, rate of mass transfer, and mobile phase velocity on the elution profile were studied. Simulations using the modified Craig approach with either of the two numerical procedures showed that the solutes behaved in the chromatographically expected manner. Moreover, with linear adsorption isotherms, direct relationships between HETP, as well as retention times, and experimental parameters could be established.

High-pressure liquid chromatography of amino acids and dipeptides on a tripeptide bonded stationary phase.

A bonded tripeptide (L-valyl-L-phenylalanyl-L-valine) phase has been used as a stationary phase in liquid chromatography. Unique retention orders and retention variations are shown for phenylthiohydantoin (PTH) derivatives of amino acids and isomeric dipeptides using acidic and basic mobile phases. It is shown that the 25 PTH-amino acids studied here have different capacity ratios when 1% citric acid in water (ca. pH 2.5) is used as the mobile phase. The analysis time for these amino acid derivatives could be shortened without a loss in the resolution by adding 5% methanol to the above mobile phase. Of particular importance is the fact that the basic amino acids are eluted first. Isomeric dipeptides have been resolved by using deionized water (pH 5.5), and 1% sodium citrate in water (pH 7.9), as mobile phases. The data show that the nature of the mobile phases, the pH, and the ionic strength are the important factors affecting the selectivity and efficiency of the separations of amino acids and dipeptides.

Bonded peptide stationary phases for the separation of amino acids and peptides using liquid chromatography.

Bonded optically active tripeptides have been applied as stationary phases for liquid chromatography. Significant retention variations are shown for some UV absorbing amino acids when compared to a silica gel column using the same mobile phase. The separation of certain isomeric dipeptides has been accomplished using the bonded optically active tripeptide stationary phases. It is shown that these separations are superior to those possible on silica gel, although the efficiencies of the column are low. Phenylthiohydatoin (PTH)-amino acids have also been analyzed using the bonded tripeptide L-Val-L-Ala-L-Ser on silica gel CT and 1% citric acid-water as the mobile phase. No two PTH-amino acids showed the same capacity ratio out of 25 tested and the separation of 15 in one isocratic run is presented. The implications and future for the use of bonded opitcally active peptides as stationary phases for liquid chromatography are discussed.

Use of gold nanoparticles to enhance capillary electrophoresis.

We describe here the use of gold nanoparticles to manipulate the selectivity between solutes in capillary electrophoresis. Two different gold-based nanoparticles were added to the run buffer. In one case, the nanoparticles were stabilized with citrate ions, but in another study, the gold nanoparticles were capped with mercaptopropionate ions (thiol-stablized). Citrate-stabilized gold nanoparticles were used in conjunction with capillaries treated with poly(diallyldimethylammonium chloride) (PDADMAC). The positively charged PDADMAC layer on the capillary walls adsorbs the negatively charged gold nanoparticles. The model solutes that were used to study the effect of the presence of the citrate-stabilized gold nanoparticles are structural isomers of aromatic acids and bases. The presence of the PDADMAC layer and the PDADMAC plus the gold nanoparticles changes both the electroosmotic mobility and the observed mobility of the solutes. These changes in the mobilities influence the observed selectivities and the separations of the system. Thiol-stabilized gold nanoparticles were used without PDADMAC in the capillary. The model solutes studied in this part are various aromatic amines. In this case as well, the presence of the gold nanoparticles modifies the electroosmotic mobility and the observed mobility of the solutes. These changes in the mobilities are manifested in selectivity alterations. The largest change in the selectivities occurs at low concentrations of the gold nanoparticles in the run buffer. The presence of nanoparticles improves the precision of the analysis and increases the separation efficiency.

Liquid chromatographic separation and detection of nanogram quantities of biologically important dicarboxylic acids.

Phenacyl and benzyl derivatives of some biologically significant dicarboxylic acids have been prepared in a quantitative manner using a crown ether catalyst. They were separated using reversed-phase chromatography. The column used was packed with Corasil II to which a C9 phase (nonyl) was bonded. Water-methanol (68:32) was used as the mobile phase. Detection limits in the range of 5--15 ng have been obtained for a non-optimized system. The ease of preparation of these derivatives, coupled with their excellent chromatographic properties, makes this a very attractive procedure for the investigation of mixtures containing biologically significant acids. The implications and future of this technique are discussed.

Reversed-phase liquid chromatographic retention and selectivity surfaces. II. Deoxyribonucleosides.

By plotting capacity factors and selectivity factors as functions of temperature and pH or methanol concentration of the mobile phase, retention and selectivity surfaces of four deoxyribonucleosides were generated. Although changes in the methanol content had little effect on selectivity, changes in pH and temperature could be used to improve selectivity. However, changes in pH or temperature can be coupled with changes in methanol concentration to optimize the analysis time. In addition, by using lines of constant analysis time (isochrons), it was found that several sets of conditions will result in nearly identical retention times. These isochrons can be used to optimize the resolution of the solutes.

Gold nanoparticle-enhanced microchip capillary electrophoresis.

We describe here the use of gold nanoparticles in conjunction with chip-based capillary electrophoresis to improve the selectivities between solutes and to increase the efficiency of the separation. We coated the microchannel wall of a microfluidic device with a layer of poly(diallyldimethylammonium chloride) (PDADMAC) and then collected on it citrate-stabilized gold nanoparticles. The resolutions and the plate numbers of the solutes were doubled in the presence of the gold nanoparticles. Such selectivity improvements reflect changes in the observed mobility accrued from interactions of solutes with the particle surface. The electrochemical detection and the quantitation of the solutes were not effected by the PDADMAC and the gold nanoparticles.

Cardiac arrhythmia in chronic renal failure: Holter monitoring during hemodialysis and intermittent peritoneal dialysis.

Twenty patients undergoing intermittent peritoneal dialysis (IPD) and 20 patients on hemodialysis (HD) for end-stage kidney disease underwent Holter ECG monitoring for a continuous 24-h period, starting prior to the onset of dialysis and continuing until several hours after it. Clinically significant ventricular arrhythmias were found in only 2 of 20 patients in the HD group; no such ventricular arrhythmias were observed in the IPD group. Supraventricular arrhythmias were equally prevalent in both groups. Thus, the differences between IPD and HD are not great and it seems that HD is, at most, a mild arrhythmogenic procedure. However, in patients with proven heart disease or signs of left ventricular hypertrophy, peritoneal dialysis (intermittent or continuous ambulatory) appears to be preferable to HD.

Atrial natriuretic peptide and renal cGMP in rats with experimental heart failure.

Rats with chronic aortocaval (AV) fistula, an experimental model of congestive heart failure, display high plasma levels of atrial natriuretic factor (ANF) and a blunted natriuretic response to ANF infusion. We previously reported that rats with AV fistula either develop progressive sodium retention (urinary sodium excretion, UNaV less than 100 microeq/24 h) or compensate (UNaV greater than 1,200 microeq/24 h). To gain further insight into the mechanism of renal hyporesponsiveness to ANF, we evaluated the effect of ANF on renal guanosine 3',5'-cyclic monophosphate (cGMP) production in sham-operated control rats and in the two groups of rats with AV fistula. Infusion of synthetic ANF-(99-126) (at either 10 or 50 micrograms.kg-1.h-1) resulted in a reduced fractional sodium excretion (P less than 0.05) in both compensated rats (0.7 +/- 0.2 and 7.9 +/- 1.6%) and sodium-retaining rats (0.3 +/- 0.1 and 0.5 +/- 0.1%) compared with controls (8.5 +/- 1.2 and 13.7 +/- 2.3% for low and high doses, respectively). Similarly, urinary cGMP excretion corrected by glomerular filtration rate (UcGMPV/GFR) during low-dose ANF infusion was significantly reduced (P less than 0.05) in both groups with AV fistula (compensated: 39 +/- 10 pmol/ml; sodium-retaining: 55 +/- 13 pmol/ml) compared with controls (115 +/- 16 pmol/ml). During high-dose ANF infusion, compensated rats, but not sodium-retaining rats, displayed a significant increase in UcGMPV/GFR. The differences in UcGMPV/GFR are probably not due to variations in urine flow because furosemide infusion to a separate group of rats with AV fistula increased urine flow approximately eightfold but did not increase UcGMPV/GFR.(ABSTRACT TRUNCATED AT 250 WORDS)

Copper and zinc levels after external damage by laser photocoagulation to the retinal pigment epithelium. Preliminary reports.

Several reports have appeared in the literature linking abnormalities in the Retinal Pigment Epithelium (RPE) related with changes in the metabolism of copper and zinc. It has not been yet elucidated if the changes in the metabolism of these metals are secondary to a progressive disease that affects the RPE, or the changes found in this retinal layer are a manifestation of a basic metabolic disease. In the present study a group of 22 diabetic patients with different degree of retinopathy was evaluated. Eleven of these patients underwent laser treatment, and blood and urine copper and zinc were assessed prior to the treatment and at intervals of one day, one week and one month post treatment. All these patients were compared with a control group. We found a rise in the mean concentration of copper and zinc in serum, and zinc in the urine, in those patients who underwent more than 600 laser applications. The significance of these findings and their relationship to the RPE is discussed.

Copper and zinc metabolism in high myopic patients with retinal detachment evaluation of the Cu/Zn ratio.

In a previous work abnormalities in the copper and zinc metabolism in high myopic patients were found. Primary Retinal Detachment is a common complication of High Myopia. In order to establish further their relationship, a study of the metabolism of these metals was performed in myopic patients with Retinal Detachment. A significant high copper and zinc concentration in the urine of these patients was found. An inverse ratio of copper and zinc between their concentration in the subretinal fluid and their concentration in serum was present as well. The significance of these findings and their connection to the Retinal Pigment Epithelium is discussed.

Auditory brain-stem evoked potentials in patients undergoing dialysis.

Auditory brain-stem evoked potentials (ABEPs) and pure-tone audiograms were obtained for 38 patients with renal failure, undergoing dialysis, before and after a dialysis session, and for 40 healthy normal subjects. Blood chemistry was also evaluated for each patient before and after dialysis. ABEP abnormality (using 10/sec click rate) was observed for 24% of the patients, rising to 44% when 55/sec measures were included. Abnormalities included prolongations of peak latencies for both click rates, and prolongation of interpeak latency differences. Pre-dialysis calcium was significantly different between patients with or without ABEP abnormality. In addition to this chronic effect on ABEPs, an acute effect of the dialysis session was found. Blood chemistry data, ABEP latencies and I-III interpeak latency differences were significantly different before, as compared to after dialysis. The acute effect of dialysis on blood calcium levels correlated with its effect on latencies of peaks III and V at 10/sec click rate, and on peaks I and V latency at 55/sec. These results may indicate the types of dysfunction revealed by increased stimulus rate measures.