High-pressure Anion-exchange Chromatography Research Articles

3] Separation, purification and analysis of cyclic nucleotides by high pressure ion exchange chromatography

High-pressure anion exchange chromatography has been used to measure cyclic AMP (cAMP) produced by adenylate cyclase and to measure cAMP in tissues and in patient urine. In addition, it has been shown useful in the measurement of cyclic GMP (eGMP) and cAMP analogs. The anion exchange resin used in this method is that originally described by Horvath. Anion exchange resin is polymerized upon small glass beads and has been termed “pellicular” resin. The glass bead matrix eliminates changes in column volume when the ionic strength is altered and markedly reduces the bleed of ultraviolet absorbing materials from the resin. The ability to obtain good results at the picomole level is dependent upon optimal performance of the mechanical and electronic systems.

Deficient renal cyclic adenosine 3'-5' monophosphate production in nephrogenic diabetes insipidus.

Vasopressin (ADH) (10 mU/min) was infused for 2–4 hr in 6 water loaded normal subjects, 5 patients with primary diabetes insipidus, and 2 with nephrogenic diabetes insipidus. Cyclic AMP in hourly urine samples prior to and during ADH administration was measured by high pressure anion exchange chromatography. In normals, urinary cyclic AMP was 2.7 ± 0.5 μm/g Cr (1 sd) during H2O loads increasing to 4.8 ± 1.0 with ADH, and in diabetes insipidus, urinary cyclic AMP was 2.5 ± 0.3 on no therapy increasing to 3.9 ± 1.0 μm/g Cr with ADH as urine osmolality rose from less than 100 to more than 330 mOsm/kg. Urinary cyclic AMP was significantly (p < 0.001) greater, 1.7 ± 0.4 times control, with ADH. However, in 2 patients with nephrogenic diabetes insipidus, urinary cyclic AMP was 2.0 ± 0.1 and 2.7 ± 0.2 μm/g Cr prior to and remained less than or equal to control values during ADH infusion. Tolbutamide (1 g iv) increased urine osmolality from 101 to 579 mOsm/kg in a glucose loaded diabetes insipidus patient while urinary cyclic AMP rose to 1.6 times baseline; while in nephrogenic diabetes insipidus tolbutamide produced no increase in urine osmolality or urinary cyclic AMP. Parathyroid hormone (200 U iv) increased urinary cyclic AMP 71–121-fold in 6 normals, while urinary cyclic AMP increased only 10- and 30-fold in the 2 nephrogenic diabetes insipidus patients. Glucagon (1 mg iv) increased urinary cyclic AMP 7–25-fold in 6 normals, and 6 times in nephrogenic diabetes insipidus. These findings show that in nephrogenic diabetes insipidus, urinary cyclic AMP fails to rise with ADH or tolbutamide in contrast to normal and diabetes insipidus patients. Nephrogenic diabetes insipidus patients appear to have a defect in ADH and parathyroid hormone stimulated urinary cyclic AMP excretion.

Analysis of DNA derivatives : II. High-pressure liquid anion-exchange chromatography of deoxyribonucleotides

A procedure for assaying deoxyribomononucleotides released by diesterase digestion of oligomers is reported. The method is based on high-pressure anion-exchange chromatography on Zipax SAX at ambient temperature using a single buffer of low ultraviolet absorption. A simple procedure for determining peak overlap is included.

Determination of picomole amounts of enzymatically formed adenosine 3',5'-cyclic monophosphate by high pressure anion exchange chromatography.

Determination of picomole amounts of enzymatically formed adenosine 3',5'-cyclic monophosphate by high pressure anion exchange chromatography.

Ultraviolet-Absorbing Compounds in Urine of Normal Newborns and Young Children

Abstract Ultraviolet-absorbing compounds excreted in the urine of normal newborns, infants, and children have been measured by high-pressure anion-exchange chromatography. The chromatograms increased in complexity with increasing age of the subject. Preliminary data indicate that the excretion of pseudouridine and uridine was significantly higher in newborns and infants than in older children and adults. Excretion of hippuric acid was low in the newborn. Excretion of ergothioneine was lower in older children than in newborns, infants, or adults. However, hypoxanthine excretion was constant in all groups.

Analysis of Urine for Its Ultraviolet-Absorbing Constituents by High-Pressure Anion-Exchange Chromatography

Abstract An automatic, high-resolution analytic system for the quantitative determination of the ultraviolet-absorbing molecular constituents of urine and other body fluids is being developed. This system is comprised of a heated, high-pressure anion-exchange column for the separation step and a continuous-flow ultraviolet spectrophotometer for the detection step. Separation is achieved by elution with an acetate buffer of gradually increasing concentration. The total analysis time is 40 hr. Chromatograms showing the absorbance of the column effluent as a function of time are recorded by a strip-chart recorder and by a data acquisition system containing a digital voltmeter and paper tape punch. The output from the latter is then used as the input to a digital computer for analysis of the chromatogram. More than 140 chromatographic peaks have been resolved from a 2-ml. urine sample. Normal urine samples yield similar chromatograms. A variation in the diurnal cycle has been noted.