Comparison of glycated albumin and hemoglobin A(1c) levels in diabetic subjects on hemodialysis.

Glycated albumin is thought to more accurately reflect glycemic control in diabetic hemodialysis patients than hemoglobin A(1c) because of shortened red cell survival. To test this, glycated hemoglobin and albumin levels were measured in blood samples collected from 307 diabetic subjects of whom 258 were on hemodialysis and 49 were without overt renal disease. In diabetic subjects with renal disease, relative to those without, the mean serum glucose and glycated albumin concentrations were significantly higher while hemoglobin A(1c) tended to be lower. The glycated albumin to hemoglobin A(1c) ratio was significantly increased in dialysis patients compared with the controls. Hemoglobin A(1c) was positively associated with hemoglobin and negatively associated with the erythropoietin dose in hemodialysis patients, whereas these factors and serum albumin did not significantly impact glycated albumin levels. Using best-fit multivariate models, dialysis status significantly impacted hemoglobin A(1c) levels without a significant effect on glycated albumin. Our results show that in diabetic hemodialysis patients, hemoglobin A(1c) levels significantly underestimate glycemic control while those of glycated albumin more accurately reflect this control.

Is a left ventricular vent necessary for coronary artery bypass operations performed with cardioplegic arrest?

The need for ventricular venting with hypothermic cardioplegic arrest is controversial. We report an evaluation of the need for left ventricular venting in a canine model that closely simulates conditions during routine coronary artery bypass grafting (CABG). Thirty-five dogs were placed on cardiopulmonary bypass for 60 minutes of hypothermic cardioplegic arrest (18 vented, 17 nonvented) and then reperfused for 30 minutes. Myocardial temperature and left atrial pressure (LAP) were recorded continuously. Before and 30 minutes after hypothermic cardioplegic arrest, left ventricular function curves were generated (six vented, six nonvented), and biopsy specimens of the left ventricle were taken for adenosine triphosphate (ATP) determinations (11 vented, 10 nonvented) and semiquantitative grading of mitochondrial ultrastructure (six vented, six nonvented). LAP in nonvented dogs was 7.4 mm Hg during hypothermic cardioplegic arrest and 5.0 mm Hg during reperfusion. Temperature during hypothermic cardioplegic arrest was 12.3 degrees C in vented dogs and 11.3 degrees C in nonvented dogs (p = 0.5). There were no differences in left ventricular function or preservation of mitochondrial ultrastructure between vented and nonvented dogs. ATP after hypothermic cardioplegic arrest was 96.6% of control (4.30 microM/gm) in vented dogs and 94.6% (4.37 microM/gm) in nonvented dogs (p = 0.7). The absence of left ventricular venting did not lead to ventricular distention or more rapid rewarming. These data in vented dogs and nonvented dogs strongly support the belief that left ventricular venting is not necessary during routine CABG.

Colorimetric assay of urinary 5-hydroxy-3-indoleacetic acid.

We describe a method for 5-hydroxy-3-indoleacetic acid in urine based on its reaction with nitrosonaphthol in the presence of mercaptoethanolamine, an odorless compound. Mercaptoethanolamine shifts the color maximum from 540 nm to 640 nm, intensifies the color and discharges the color of interfering substances. The method is rapid and free from interferences.

The taurine and hypotaurine content of human semen.

Taurine and hypotaurine levels were measured in human sperm and seminal fluid. Sperm taurine ranged from 17 nmol/mg DNA to 348 nmol/mg DNA, and hypotaurine from 0 nmol/mg DNA to 251 nmol/mg DNA. Seminal fluid contained 319 mumol/L to 1590 mumol/L of taurine, but no detectable hypotaurine. The coefficient of variation in multiple ejaculates from a single man for these components ranged from 12% for hypotaurine to 24% for seminal fluid taurine, indicating a relative constancy in their concentrations. Sperm hypotaurine content was significantly correlated with sperm morphology, sperm relative forward progression, the percentage of motile sperm, and the total number of sperm in the ejaculate. By contrast, sperm taurine content was negatively correlated with these parameters. The mean hypotaurine content of sperm from 8 fertile men was 149 +/- 92 nmol/mg DNA, four times higher than that of sperm from 9 infertile men, which was 35 +/- 19 nmol/mg DNA (P = 0.011). In contrast, the mean sperm taurine content of the fertile men was lower than that of the infertile men (83 +/- 33 nmol/mg DNA versus 168 +/- 119 nmol/mg DNA, respectively; P = 0.07). Seminal fluid taurine concentrations, however, were similar for both groups. Hypotaurine, an antioxidant, may play an important role in protecting sperm from reactive oxygen species. Higher concentrations of taurine in the sperm of infertile men suggest that accelerated oxidation of hypotaurine to taurine may accompany the observed decline in other sperm parameters.

Liquid chromatographic assay of urinary free cortisol.

Cortisol in concentrated sulfuric acid forms two major fluorescing compounds which are separated by chromatography on a reversed-phase column. Based on this reaction, urine free cortisol, after double extraction was assayed by high-performance liquid chromatography with fluorescent detection. The method is sensitive, rapid, and free from interferences. The values by this method are 30% lower than radioimmunoassay. In addition to Cushing's syndrome, patients with acute pancreatitis had elevated levels of urinary free cortisol which parallel urinary amylase levels while patients with myocardial infarction had normal levels. Urinary free cortisol showed diurnal variation with elevated values early in the morning.

Stacking in capillary zone electrophoresis.

Due to the short light path of the capillaries, the CE detection limit based on concentration, is far less than that of HPLC and not sufficient for many practical applications. Several methods, based on different electrophoretic maneuvers, can concentrate the sample (stack) easily on the capillary before the separation step of capillary zone electrophoresis (CZE). These methods incorporate different types of discontinuous buffers as the means for invoking different velocities to the same analyte molecules to produce a sharpening of the band (stacking). In CZE, these buffers can be often very simple such as sample dilution or adding to the sample a high concentration of a fast mobility ion. However, in other applications these buffers can be as complicated as those required for isotachophoresis. Stacking can often yield a concentration factor of 5-30-fold, which can improve greatly in CZE the detection limits bringing them very close to those of HPLC. Different methods of stacking, the importance of discontinuous buffers and the different mechanism for concentration on the capillary are reviewed here. As there is a need for more practical applications, there will be more methods devised for stacking in CZE.

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.

Field amplified injection in the presence of salts for capillary electrophoresis.

Salts in the sample are detrimental to the stacking by the field-amplified injection. However, physiological samples often contain salts at levels of about 1% which can diminish the peak height or cause band spreading instead of stacking. Using different analytes which contain salts, we demonstrate that the presence of acetonitrile at 66% in the sample reverses the deleterious effect of salts and favors the stacking by the electrokinetic injection. The advantage of this type of stacking is that it favors certain analytes over others and it can give, in some instances, better theoretical plate numbers.

Analysis of angiotension-converting enzyme by capillary electrophoresis.

A method is described for determination of serum angiotension-converting enzyme by capillary electrophoresis (CE) based on incubation of the substrate, a synthetic peptide, with the serum outside the capillary and cleaving hippuric acid and a dipeptide. The reaction is stopped by the addition of acetonitrile, followed by injection of the supernatant on the capillary. The acetonitrile allows injection of a large volume of sample on the capillary. Both the substrate and the reaction product (hippuric acid) can be monitored at the same time. The CE step is rapid and can be performed in about 6 min. The CE method compared well to a kinetic assay method (= 0.98).

Capillary electrophoresis of double-stranded DNA in an untreated capillary.

Using the zwitterionic buffer N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) in the presence of a high-molecular-mass hydroxypropylmethylcellulose (HPMC) as a sieving polymer and ethidium bromide double-stranded DNA (dsDNA) was separated in an untreated capillary. The HEPES buffer shielded the DNA against the capillary wall interaction and decreased the electroosmotic flow enabling a good separation of the DNA similar to that obtained in a commercially coated capillary. In addition to the low cost of the untreated capillary it can be washed after each run. Furthermore, stacking with hydrodynamic injection filling about half of the capillary volume is demonstrated.

Therapeutic drug monitoring by capillary electrophoresis.

Because of the ease of analysis and the high resolution, drug analysis is becoming the best example for the application of capillary electrophoresis. Therapeutic drug monitoring is a specialized area of drug analysis performed in clinical laboratories for patient care. CE offers high resolution and speed with the low operating costs needed in patient care. However, CE has a few limitations, mainly poor detection limits and precision. Simple methods of stacking, which enhance drug detection to overcome the poor sensitivity of CE are stressed. Serum has a unique matrix with a high content of proteins and salts which can have adverse effects on separation by CE. For successful analysis, special maneuvers are employed to decrease these matrix effects. Studies that have addressed the improvement of the precision of CE are summarized. CE offers the possibility of bringing chiral separations into the routine arena.

Insulin stacking for capillary electrophoresis.

Stacking methods are very important in overcoming the poor detection limits in capillary electrophoresis. Human insulin, a polypeptide, was concentrated on the capillary (stacked) based on three different and simple treatment methods to the sample: dilute buffers, high salt content, and acetonitrile (66%) were added to the sample to induce stacking. A dilute buffer in the sample caused a limited stacking, while acetonitrile treatment and high salt content in the sample caused much greater (approximately 20-fold) stacking. High salt concentration in the sample caused stacking presumably by a transient isotachophoretic method. In addition to stacking, the acetonitrile treatment removed the excess proteins in the sample. Insulin did not denature or precipitate in 66% acetonitrile as confirmed by high-performance liquid chromatography (HPLC) and immunoassays. Acetonitrile treatment enabled one-third of the capillary to be loaded with sample thus increasing the detection signal greatly. The insulin peak after acetonitrile treatment and separation by capillary electrophoresis (CE) was confirmed by HPLC and by CE fraction collection followed by immunoassay. Based on acetonitrile treatment, insulin detection in pancreatic tissue homogenates is shown to be feasible.

Analysis of the antiepileptic drug keppra by capillary electrophoresis.

A simple and rapid method for determination of the new antiepileptic drug keppra (levetiracetam) by capillary electrophoresis in borate buffer containing sodium dodecyl sulfate is described. The serum was injected without any treatment. The method compared well to high performance liquid chromatography. The mean of keppra in the serum of 35 patients was 25 mg/l (range 7-77 mg/l).

Analysis of nitrate in biological fluids by capillary electrophoresis.

Nitrite and nitrate represent the products of the final pathway of nitric oxide metabolism. These two ions were analyzed by capillary electrophoresis (CE) in serum, cerebrospinal fluid, urine and tissue homogenates by mixing the sample with acetonitrile containing NaBr as an internal standard, followed by centrifugation. The supernatant was injected hydrodynamically on a capillary 50 cm x 75 microns (I.D.) and electrophoresed at 6 kV (reversed polarity) in 1.4% sodium chloride in phosphate buffer for 13 min with detection at 214 nm. In addition to removal of the proteins, acetonitrile caused sample stacking. Urinary nitrate analysis by CE was compared to that by the enzymatic Aspergillus nitrate reductase method, with a correlation coefficient of 0.96.

Changes in diabetic urinary transferrin excretion after moderate exercise.

Microalbuminuria following submaximal exercise testing has been proposed for detecting renal abnormalities in diabetic patients. We compared urinary transferrin and albumin excretion between eight adults with insulin dependent diabetes mellitus and eight nondiabetic controls without microalbuminuria before and after a standardized exercise challenge of only moderate intensity for 20 min. Both groups were similar for age, sex, and METs expended during treadmill walking. Urinary excretion ratios of transferrin (UTER) and albumin (UAER) did not significantly increase for nondiabetic subjects. After exercise, UTER increased on average 207% in diabetic subjects (P = 0.009) and UAER increased 209% (P = 0.046). The percent increase in UTER appears to be a function of workload intensity, while the percent increase in UAER appears less dependent on the duration of exercise. A standardized treadmill challenge of moderate intensity easily differentiated changes in urinary transferrin excretion ratios between diabetic and nondiabetic subjects. Measuring transferrin excretion may be a more sensitive parameter than albumin in studies using urinary protein excretion as a response to a provocative exercise challenge.

Analysis and classification of serum cryoglobulins.

Cryoglobulins (CG) are immunoglobulins that reversibly precipitate from serum in cold temperatures. They are classified into three types, based on the monoclonality of the γ-globulins present (1): Type I contains only monoclonal bands; type II contains mixed polyclonal immunoglobulins with a monoclonal component; and type III contains mixed polyclonal immunoglobulins only.

Myoglobin analysis.

Myoglobin represents the stores of oxygen in muscle tissues. Because of its relatively small mol wt, myoglobin is often used in electrophoretic techniques as a mol wt marker, and also as a test for separation efficiency in capillary electrophoresis (CE). The separation of myoglobin can be accomplished based on mol wt (1), based on charge, or both together (as used here).

Enzyme analysis : cathepsin d as an example.

In enzyme analysis, capillary electrophoresis (CE) offers the ease of product separation from the substrate, with the ability to use expensive reagents in microvolumes. In CE, enzymes can be measured either as mass (when they are present in high concentration) by direct light absorbency, or by catalytic activity. For example, the protease enzyme, savinase, which is used as an ingredient in washing powder, was determined directly by its absorbency at 200 nm (1). For catalytic activity measurements, the substrate, the product, or both can be measured in CE without the need for coupling reactions. Because of the increased sensitivity, most CE methods measure enzymes by their catalytic activity on a substrate. To accomplish this by CE, several approaches have been used.

Serum lamotrigine analysis.

Antiepileptic drugs vary greatly in chemical structure. Analysis of these compounds by capillary electrophoresis (CE) requires different conditions for each one, or for each group. Acidic drugs can be analyzed easily by capillary zone electrophoresis in borate buffers (1); the neutral and the mixtures can be better analyzed by micellar electrokinetic chromatography (2-4). Chapter 17 describes how phenobarbital and a few other antiepileptic drugs could be analyzed by CE.

Acetonitrile stacking : serum phenobarbital as an example.

Capillary electrophoresis (CE) has several advantages, such as high resolution, speed, and low cost of operation. However, it suffers from two major drawbacks: poor detection limits and matrix effects. Several approaches have been used to overcome these two problems. Here, acetonitrile stacking (AS) is presented as a simple, easy, and practical approach that can solve these two problems for many analytes, with minimum sample preparation. This laboratory successfully used AS for the analysis of several compounds by CE (1).