High-pressure Cation-exchange Chromatography Research Articles

The Separation of Globin Chains by High Pressure Cation Exchange Chromatography

A high performance cation exchange system is described for isolating globin chains. The method is rapid and inexpensive, using total lysate, a 14 min gradient, and a 3 cm column. The running buffers contain 6 M urea to dissociate and unfold the globin, and a NaCl gradient is employed for elution. The procedure provides a convenient way of detecting mutant globins with an intrinsic alteration in their charge.

Nonenzymatic glycation of human lens crystallin. Effect of aging and diabetes mellitus.

We have examined the nonenzymatic glycation of human lens crystallin, an extremely long-lived protein, from 16 normal human ocular lenses 0.2-99 yr of age, and from 11 diabetic lenses 52-82-yr-old. The glucitol-lysine (Glc-Lys) content of soluble and insoluble crystallin was determined after reduction with H-borohydride followed by acid hydrolysis, boronic acid affinity chromatography, and high pressure cation exchange chromatography. Normal lens crystallin, soluble and insoluble, had 0.028 +/- 0.011 nanomoles Glc-Lys per nanomole crystallin monomer. Soluble and insoluble crystallins had equivalent levels of glycation. The content of Glc-Lys in normal lens crystallin increased with age in a linear fashion. Thus, the nonenzymatic glycation of nondiabetic lens crystallin may be regarded as a biological clock. The diabetic lens crystallin samples (n = 11) had a higher content of Glc-Lys (0.070 +/- 0.034 nmol/nmol monomer). Over an age range comparable to that of the control samples, the diabetic crystallin samples contained about twice as much Glc-Lys. The Glc-Lys content of the diabetic lens crystallin samples did not increase with lens age.

Variables in the high-pressure cation-exchange chromatography of proteins

The use of cation-exchange high-pressure liquid chromatography for the separation of proteins has been investigated. Several factors, including solvent composition, pH, flow rate, and temperature, were examined for their effects on the resolution of protein standards (insulin, β-lactoglobulin, and carbonic anhydrase B; molecular weight range, 6000 to 30,000 and p I range, 5.3 to 6.5). An initial comparison was made of the recovery of these proteins from three commercially available columns (Whatman Partisil SCX, Separation Industry CM silica, and MCB Reagents Lichrosorb KAT). In general, under the conditions employed, the SCX column gave the highest recovery of applied protein. Based on this recovery data, the Partisil SCX column was chosen for subsequent examination of chromatographic parameters that would optimize protein resolution. An increase in temperature decreased retention and resolution but increased recovery, with some proteins being affected more than others. A decrease in pH in the final eluant or an increase in pH in the initial eluant caused an increase in retention times. For some proteins, the decrease in pH resulted in a greater total recovery of protein. This information has been applied to the purification by cation-exchange high-pressure liquid chromatography of transforming growth factors from a human tumor cell line.

Studies on polyamine biosynthesis in Euglena gracilis

Euglene gracilis (strain Z) was found to contain five polyamines which could be separated by high-pressure cation-exchange chromatography. 1,3-Diaminopropane, putrescine, norspermidine ( N-(3- aminopropyl)-1,3- diaminopropane ), spermidine and norspermine ( N,N′- bis(aminopropyl)-1,3- diaminopropane ) were identified. Biosynthesis of putrescine in E. gracilis proceeds through decarboxylation of l-ornithine, no arginine decarboxylase (EC 4.1.1.19) activity could be detected. The properties of the enzymes ornithine decarboxylase (EC 4.1.1.17) and S- adenosylmethionine decarboxylase (EC 4.1.1.50) in this alga were found to be similar to those of the enzymes isolated from animal tissues or yeast cells. A biosynthetic scheme is proposed which relates the different polyamines occurring in E. gracilis.

Disposition of theophylline after a single intravenous infusion of aminophylline.

Theophylline serum concentration and disposition after a single 30-min intravenous infusion of 7.4 ± 1 mg of anhydrous theophylline per kg of body weight were determined in 20 otherwise healthy adult asthmatic volunteers using high-pressure cation-exchange chromatography. This dose resulted in a mean peak serum concentration of 18 ± 3.3 μg per ml, which remained > 10 μg per ml for 6 hours and > 5 μg per ml for the entire 12-hour period of observation. The rate of theophylline elimination in these subjects was generally slower than previously reported. Among the 4 cigarette smokers, however, theophylline plasma clearance was significantly (P < 0.001) more rapid than that observed in the 16 nonsmokers. Theophylline clearance was highly correlated with elimination half-life (r = 0.83, P < 0.001). These data support previous recommendations for an intravenous loading dose of 5 mg of theophylline per kg of body weight (6 mg of aminophylline per kg) when a mean increase in serum concentration of approximately 1...

Activated Charcoal Adsorption of Theophylline in Vitro

This study was initiated to evaluate the in vitro efficacy of activated charcoal as a potential agent in the treatment of acute theophylline overdoses. Two, ten, and twenty mg/ml solutions were analyzed for theophylline concentration by high-pressure cation-exchange chromatography before and after shaking with 10 g of activated charcoal. The results indicate that 1 g of activated charcoal may adsorb in vitro as much as 300 mg of theophylline. Controlled studies in humans are now indicated.A wide variety of written forms of prescription drug information for patients are catalogued and discussed. These materials are designed to be delivered by the pharmacist, the physician or the nurse, or to serve as general reference material provided directly to consumers. Materials that describe specific prescription drugs come in the form of stickers or folded sheets directly attached to the medicine container, checklists, wallet or index card size materials, one page sheets, leaflets, brochures and booklets. The best form of patient oriented prescription drug printed material must be evaluated in the context of the goal that it is desired to achieve. Whereas shorter materials are more apt to be read, they cannot provide complete information, the rationale or explanations for the cause or prevention of therapeutic or adverse drug effects.The importance of providing written prescription drug information to patients is indicated by the fact that many health care professionals produce or distribute these materials to augment and reinforce verbal consultation. In addition, consumer surveys indicate that written communication is a preferred source for this additional information. The examination of existing forms of written prescription drug information for patients is particularly relevant to the Food and Drug Administration (FDA). Senior officials in the FDA have announced support for the concept of written reinforcement of verbal consultation and have said that plans will be proposed for the expanded requirement of patient oriented prescription drug labeling via patient package inserts.

Absolute bioavailability of oral theophylline

The absolute bioavailability of theophylline was investigated by comparing the areas under concentration-time curves for intravenous theophylline with a plain uncoated anhydrous theophylline tablet and a theophylline solution. Twenty asthmatic adults received approximately 7.5 mg/kg theophylline intravenously over 30 minutes; either seven days before or after the i.v. dose, 10 of these patients received tablets and the remainder solution in a similar dose. Blood samples were obtained at 0, 10, 20, 30, 45, 60, 90, 120, 180 and 240 minutes and then every two hours for at least 12 hours. Theophylline concentration was measured in serum by high-pressure cation exchange chromatography. The fraction of the dose absorbed averaged 0.96 +/- 0.03 for the tablet while the value for the solution was 0.99 +/- 0.02. The time of peak absorption averaged 2 +/- 0.3 hours for the tablets and 1.4 +/- 0.3 hours for the solution. The maximum serum concentration attained was 15.3 +/- 0.7 microng/ml after a dose of 7.6 +/- 0.4 mg/kg of the tablet, and 14.6 +/- 0.6 microng/ml after a dose of 7.3 +/- 0.2 mg/kg of the solution. Absorption of the tested theophylline tablets and solution approached 100% of the available drug.

Polyamine levels in normal human serum. Comparison of analytical methods

A comparison of levels of the polyamines, putrescine, spermidine and spermine, in individual and pooled normal human serum, as determined by three independent methods, high pressure cation-exchange chromatography (HPCC), radioimmunoassay (RIA) and gas chromatography-mass spectrometry (GC-MS), has been made. Generally good correlations were found for values derived by the three different methods.

High pressure cation exchange chromatography of biogenic amines

Fourteen selected neuroamines and related compounds (phenethylamines, β-hydroxyphenethylamines, and indoleamines) were chromatographed by high pressure cation exchange chromatography using ammonium phosphate solutions as elutants. The capacity factor ( k′) values for all compounds studied exhibited linear relationships with respect to both elutant concentration and pH. Values of k′ were strongly dependent on the degree of hydrophilicity or hydrophobicity of the individual bases. The method proved capable of quantitation of 1–5 nmol of most biogenic amines. Sensitivity to serotonin was even greater, with detectability extending into the picomole range.

Rapid Analysis for Theophylline in Serum by Use of High-Pressure Cation-Exchange Chromatography

We have developed a quantitative assay for serum theophylline by high-pressure cation-exchange chromatography. As little as 0.1-ml aliquots of serum were prepared for analysis simply by diluting them with a solution of the internal standard (8-chlorotheophylline). Theophylline and the internal standard were eluted in 17 and 27.5 min, respectively, and the peaks for them were distinct from those of other xanthines and uric acids. We encountered no interference when we used: (a) plasma obtained from blood anticoagulated with citrate; (b) hemolyzed, lipemic, or icteric serum; or (c) 52 samples from patients who were receiving various other medications. An analysis of calibration suggested that errors greater than plus or minus 2.6 mg/liter, a clinically acceptable range, were highly unlikely. Sensitivity was sufficient to identify less than 2.5 mg/liter. This assay was compared with the usual method involving solvent extraction and absorbance measurement in the ultraviolet and found to have similar accuracy, although it is easier, faster, requires less sample, and potentially is more specific.

Separation of some chloramphenicol intermediates by high-pressure ion-exchange chromatography

High-pressure cation-exchange chromatography was used to separate three of the by-product compounds formed in the chloramphenicol production process. Baseline separation can be achieved within 8.5 min employing a 1-m column packed with Zipax SCX pellicular cation exchanger and 0.05 M sodium sulphate solution as eluent at pH 1.1 in a Varian Aerograph LC 4020 liquid chromatograph.

The determination of the base composition of rna by high-pressure cation-exchange chromatography

A method has been developed to determine the base composition of micro- and nanogram quantities of RNA in 4 min. The RNA's were hydrolyzed (either chemically or enzymatically) to their nucleoside level and separated by cation-exchange chromatography. The nanogram quantities of ribonucleosides were separated in less than 4 min by eluting with formate buffer at a flow rate and pressure of 50 ml/h and 3000 p.s.i., respectively. The separation time is reduced to 2 min by eluting at 80 ml/h and 4600 p.s.i. The column was monitored with a sensitive UV detector which permitted absorbance to be recorded linearly and allowed analysis of nanomole to picomole quantities of nucleosides with a relative standard error of 2–3%. Data from four RNA samples are presented. The method is also applicable to the determination of the base composition of DNA since the deoxynucleosides are also separated by the procedure.