Zakariya K. Shihabi

Manganese accumulates in iron-deficient rat brain regions in a heterogeneous fashion and is associated with neurochemical alterations

Previous studies have shown that iron deficiency (ID) increases brain manganese (Mn), but specific regional changes have not been addressed. Weanling rats were fed one of three semipurified diets: control (CN), iron deficient (ID), or iron deficient/manganese fortified (IDMn+). Seven brain regions were analyzed for Mn concentration and amino acid (glutamate, glutamine, taurine, γ-aminobutyric acid) concentrations. Both ID and IDMn+ diets caused significant (p<0.05) increases in Mn concentration across brain regions compared to CN. The hippocampus was the only brain region in which the IDMn+ group accumulated significantly more Mn than both the CN and ID groups. ID significantly decreased GABA concentration in hippocampus, caudate putamen, and globus pallidus compared to CN rats. Taurine was significantly increased in the substantia nigra of the IDMn+ group compared to both ID and CN. ID also altered glutamate and glutamine concentrations in cortex, caudate putamen, and thalamus compared to CN. In the substantia nigra, Mn concentration positively correlated with increased taurine concentration, whereas in caudate putamen, Mn concentration negatively correlated with decreased GABA. These data show that ID is a significant risk factor for central nervous system Mn accumulation and that some of the neurochemical alterations associated with ID are specifically attributable to Mn accumulation.

Transient pseudo‐isotachophoresis for sample concentration in capillary electrophoresis

We show that many water miscible organic solvents such as acetonitrile, acetone and small alcohols can function as a terminating ion in transient isotachophoresis, which leads to sample concentration on the capillary. It is suggested that this method could be termed transient “pseudo‐isotachophoresis” (pseudo‐ITP). Because of their low conductivity, these water miscible organic solvents provide the high field strength necessary for band sharpening similar to that provided by the terminating ion. Salts, when present in such samples act briefly as leading ions, migrating rapidly in the organic solvent until they are slowed at the interface of the separation buffer. When the organic solvents are added to the sample, both the migrations as well as the stacking of the analytes are affected by the concentration of salts (leading ions) in the sample, similar to that observed in isotachophoresis. Our results show that this type of stacking offers good reproducibility and reliability for practical analysis. In practice, pseudo‐ITP stacking is much easier to perform compared to that of true ITP with several added practical advantages as discussed.

Sample matrix effects in capillary electrophoresis: II. Acetonitrile deproteinization

Abstract Acetonitrile deproteinization [acetonitrile-serum (3:2, v/v)] was found to be a suitable method for removal of serum proteins for the analysis of drugs and small molecules by capillary electrophoresis. Many compounds exhibited higher plate numbers and peak heights in the presence of 60% acetonitrile. This effect is due to a special stacking brought along by the low resistivity of the acetonitrile in the sample. In addition to removing serum proteins, acetonitrile improves the solubility of some compounds and increases the sample volume which can be introduced into the capillary. Serum theophylline analysis was used here as an example for the analysis of drugs by capillary electrophoresis using acetonitrile deproteinization.

Angiotensin II Type 1 Receptor Blockade Prevents Alcoholic Cardiomyopathy

Background— Activation of the renin-angiotensin system (RAS) may contribute to the development of alcoholic cardiomyopathy. We evaluated the effect of angiotensin II (Ang II) type 1 receptor (AT1) blockade on the development of alcoholic cardiomyopathy. Methods and Results— We serially evaluated left ventricular (LV) and cardiomyocyte function and the RAS over 6 months in 3 groups of instrumented dogs. Eight animals received alcohol (once per day orally, providing 33% of total daily caloric intake); 6 received alcohol and irbesartan (5 mg · kg−1 · d−1 PO); and 8 were controls. Compared with controls, alcohol ingestion caused sustained RAS activation with progressive increases in plasma levels of Ang II, renin activity, LV angiotensin-converting enzyme activity, and LV myocyte Ang II AT1 receptor expression. The RAS activation was followed by a progressive fall in LV contractility (EES, alcohol-fed dogs 3.9±0.8 versus control dogs 8.1±1.0 mm Hg/mL); reductions in the peak velocity of myocyte shortening (78.9±5.1 versus 153.9±6.2 &mgr;m/s) and relengthening; and decreased peak systolic Ca2+ transient ([Ca2+]iT) and L-type Ca2+ current (ICa,L; P<0.05). Irbesartan prevented the alcohol-induced decreases in LV and myocyte contraction, relaxation, peak [Ca2+]iT, and ICa,L. With alcohol plus irbesartan, plasma Ang II, cardiac angiotensin-converting enzyme activity, and AT1 remained close to control values. Conclusions— Chronic alcohol consumption produces RAS activation followed by progressive cardiac dysfunction. The cardiac dysfunction is prevented by AT1 receptor blockade.

Peptide stacking by acetonitrile-salt mixtures for capillary zone electrophoresis

Many small natural and synthetic peptides can be stacked for capillary zone electrophoresis by dissolving the peptides in a mixture containing acetonitrile and high concentrations of inorganic salts. In many instances one third of the capillary can be loaded with peptides dissolved in a mixture of 2 volumes acetonitrile and 1 volume of 1% sodium chloride leading to about 20-fold enhanced detection. This stacking is dependent on the presence of both salts and acetonitrile. Natural peptides such as enkephalins, angiotensin and insulin chain B in addition to peptides released from the action of proteolytic enzymes on proteins were concentrated by this method. From a practical point of view, the stacking in acetonitrile is more useful since it removes proteins, counteracts the deleterious effects of high concentrations of inorganic ions present in the sample and stops the enzymatic reaction. Furthermore, it allows a larger volume of the sample to be loaded on the capillary increasing the sensitivity of the CE. This stacking produces a greater sample concentration and better resolution than the traditional stacking obtained in aqueous low ionic strength buffers. The mechanism is also different since it is improved by a high concentration of ions in the sample. Furthermore, since proteins are eliminated, the electropherograms are cleaner and the capillary does not require thorough washings between samples, speeding up the analysis and extending the capillary life.

Sample matrix effects in capillary electrophoresis: I. Basic considerations

Abstract Serum samples contain high concentrations of proteins and ions which interfere in the analysis of small molecules such as theophylline. These effects are minimized by employing an electrophoresis buffer with high ionic strength. Although such buffers slow the speed of the analysis, they minimize the effects of proteins and ions, improve the separation and enable a larger volume of sample to be introduced into the capillary leading to enhanced signals. Therefore, serum samples can be diluted in weak buffers and analyzed directly rendering capillary zone electrophoresis an attractive technique in the clinical laboratories.

Analysis of isoflavones by capillary electrophoresis

A simple capillary electrophoresis method is described for the assay of several isoflavones and coumestrol isolated from plant extracts. The method has good reproducibility; it compares well to HPLC, and it can be performed in less than 10 min.

Capillary electrophoresis for the determination of glomerular filtration rate using nonradioactive iohexol

High-performance liquid chromatography (HPLC) has been used as an alternative to the isotopic method to calculate glomerular filtration rate (GFR). With the HPLC method, serum iohexol or iothalamate levels are measured, and the plasma clearance rate of the compound is used as a surrogate for GFR. However, HPLC is a labor-intensive procedure, which limits its usefulness in the clinical setting. Capillary electrophoresis, a newer technique in which electrophoretic separations are performed in capillary tubes, is easier and faster than HPLC. We used capillary electrophoresis for the determination of serum iohexol levels and the calculation of GFR. Patients underwent a simultaneous 125I-iothalamate clearance test and a plasma iohexol clearance test to determine GFR. Mean GFR (+/-SD) was 70.9 +/- 29.9 mL/min (range, 14.5 to 131 mL/min) in 52 patients as determined by standard iothalamate clearance methods. For iohexol clearance, the correlation coefficient and standard error were 0.93 and 10.9 mL/min, respectively, using capillary electrophoresis compared with the iothalamate method. Capillary electrophoresis is a simple, rapid method that can be used to calculate GFR and provides results at least as accurate as those obtained by HPLC and x-ray fluorescence.

Stacking and discontinuous buffers in capillary zone electrophoresis.

Discontinuous buffers for capillary zone electrophoresis (CZE) can be used under less rigid conditions compard to those for isotachophoresis for stacking. They can be prepared simply by modifying the sample itself, either by addition of small inorganic ions, low conductivity diluents, or both, and also by adjusting its pH, meanwhile injecting a large volume on the capillary. Zwitterionic and organic‐based buffers such as triethanolamine and tris(hyroxmethyl)aminomethane (Tris) are well suited for stacking due to their low conductivity, provided the buffer is discontinuous as demonstrated here. A simple mechanism based on discontinuous buffers is described to explain many of the observed stacking types in CZE, pointing out the many similarities to transient isotachophoresis.

Measurement of Glomerular Filtration Rate Using Nonradioactive lohexol: Comparison of Two One-Compartment Models

Radioisotopic methods for the determination of the glomerular filtration rate (GFR) are highly accurate but require the collection of multiple blood and urine samples and are costly to perform due to personnel, material, and analysis costs. Nonradioactive methods of GFR determination have the potential of minimizing procedure costs while preserving accuracy. We determined the GFR simultaneously by 125I-iothalamate and nonradioactive iohexol clearance methods in 41 adults. The study group consisted of 54% males, with a mean age of 50.7 (range 28-79) years and a mean GFR by 125I-iothalamate clearance of 66.5 +/- 28.3 (range 10-118) ml/min. The iohexol concentrations were measured by a simplified high-performance liquid chromatography method that did not require sample preparation. The iohexol plasma clearance was calculated by both a new one-compartment model as well as by Jacobssons one-compartment model. Using Jacobssons single-sample model and data from the 240-min point, there was an excellent correlation between 125 I-iothalamate and nonradioactive iohexol clearance values: r2 = 0.95, standard error of the estimate = 11.4 ml/min, and intrapatient coefficient of variation = 16.9%. However, this formula tended to overestimate GFRs < 30 ml/min and to underestimate GFRs > 80 ml/min. The new one-compartment model is a modification of Bubecks model, originally used for the determination of renal plasma blood flow. Using this modified model, there was an excellent correlation between 125I-iothalamate and nonradioactive iohexol clearance values at all levels of GFR tested: r2 = 0.95, standard error of the estimate = 9.2 ml/min, and intrapatient coefficient of variation = 13.7%. In conclusion, the determination of the plasma clearance of iohexol by a nonradioactive technique and a monoexponential model is a simple and accurate method of determining the GFR in patients with varying degrees of renal impairment.