Small Plasma Samples Research Articles

Plasma Levels of Aldosterone, Corticosterone, 11-Deoxycorticosterone, Progesterone, 17-Hydroxyprogesterone, Cortisol, and Cortisone During Infancy and Childhood

Summary: Plasma aldosterone (A), corticosterone (B), deoxycorticosterone (DOC), progesterone (P), 17-hydroxyprogesterone (17-OHP), cortisol (F), and cortisone (E) were measured simultaneously by specific radioimmunoassays in small plasma samples obtained from 174 normal infants and children between 2 hr and 15 yr of age. The significantly elevated neonatal mean levels (ng/ml) of 2.5 (A), 4.1 (DOC), 53.0 (P), and 6.6 (17-OHP) dropped significantly during infancy reaching prepubertal levels between 3 months and 3 yr of age, with a transient, significant DOC increase between 1-7 yr. The glucocorticoids F and B declined significantly from means of 68 and 4.4 to 11.4 and 0.28 ng/ml, respectively, during the first weeks of life, then increased significantly reaching adult levels between 1-3 yr of age. Mean E fell progressively from 74 ng/ml after birth to 10 ng/ml during 1-5 yr (P << 0.0001), then slightly increased to adult levels. After age 7 yr, P and 17-OHP, in contrast to the other steroids, rose significantly in both boys and girls relative to pubertal development. The observed changes are thought to be due to (1) adaptation of the adrenal neocortex to extrauterine life after disruption of the fetoplacental unit, (2) a physiologic lack of corticosteroid binding globulin (CBG) during infancy due to maturation of hepatic CBG biosynthesis, (3) the functional immaturity of the infant kidney compensated by an increased activity of the renin-angiotensin-aldosterone system, and (4) gradually increasing gonadal secretion of progestins during puberty. Speculation: From birth to adulthood, marked evolutional changes were observed in the basal plasma concentrations of all physiologically important unconjugated corticosteroids and progestins in normal children. Detailed knowledge of the age-dependent normal plasma steroid pattern reflecting maturational processes of both the hypothalamo-adrenocortical and the hypothalamo-gonadal axis, of the renin-angiotensin-aldosterone system, and of hepatic and renal function, therefore, is a prerequisite for understanding pathologic conditions in pediatric endocrinology.

Increased plasma and urinary normetanephrine in young patients with primary hypertension.

1. Normetanephrine was measured in small samples of plasma and urine of hypertensive patients and normal volunteers (age 20-60 years) by a specific radioenzymatic assay with bovine adrenal phenylethanolamine N-methyltransferase and tritiated S-adenosylmethionine. 2. Noradrenaline was measured simultaneously in plasma and urine. 3. Plasma normetanephrine and noradrenaline concentrations varied in direct proportion to activation or suppression of sympathetic nerve function. 4. Both plasma and urinary normetanephrine concentrations were elevated in patients with phaeochromocytoma. 5. Plasma normetanephrine concentrations were related to plasma noradrenaline concentrations of hypertensive subjects. 6. Plasma normetanephrine and noradrenaline concentrations and urinary normetanephrine excretion rates were increased in some young patients with primary hypertension, suggesting that sympathetic nerve hyperactivity is a pathogenic factor in these patients.

Improved detectability of barbiturates in high-performance liquid chromatography by pre-column labelling and ultraviolet detection

2-Naphthacyl derivatives of barbiturates are formed in acetone at 30° within 30 min, in an essentially quantitative manner, using caesium carbonate as a catalyst. These derivatives absorb UV radiation strongly at 254 nm. Using a variable-wavelength detector set at 249 nm, 1 ng of N,N-dinaphthacylphenobarbital could be detected after chromatography using a high-performance liquid chromatograph equipped with a microparticulate reversed-phase column. This derivatization technique has the potential of allowing the determination of barbiturates, in small plasma or serum samples, in concentrations well below the therapeutic range.

High‐Pressure Liquid Chromatographic Analysis of Antipyrine in Small Plasma Samples

A high‐pressure liquid chromatographic (HPLC) method was developed for the assay of antipyrine in small (0.1‐ml) plasma samples using aminopyrine as the internal standard and a reversed‐phase microparticulate column. The assay sensitivity (1 μ/ml) permits development of a plasma level‐time curve using a single rat. The mean (±SE) plasma elimination half‐life in rats was 1.28 ± 0.14hr. A comparison of the spectrophotometric method with the HPLC method yielded a correlation coefficient of 0.98. The HPLC assay for antipyrine is rapid and precise and can be used for hepatic drug metabolism study in a single animal.

Determination of sulthiame, tetrahydro-2- p-sulphamoyl-phenyl-2H-1,2-thiazine-1,1-dioxide, using high-performance liquid chromatography

A specific and sensitive liquid chromatographic method for the determination of sulthiame in small (0.5 ml) plasma samples is described. After adding an internal standard, a direct extract of the sample is examined by reversed-phase liquid chromatography with ultraviolet spectrophotometric detection. The method is rapid, simple and capable of determining plasma levels after therapeutic ingestio of sulthiame.

Gas—liquid chromatography of isobutyl ester, N(O)-heptafluorobutyrate derivatives of amino acids on a glass capillary column for quantitative separation in clinical biology

A gas chromatographic method adapted to routine analysis has been developed for quantitative separation on glass capillary columns for free proteic and other known amino acids normally or abnormally found in physiological fluids. The procedure involves ion-exchange chromatography and isobutyl ester, N(O)-heptafluorobutyrate derivatization of free plasma and urine amino acid samples. Derivatized components were ascertained by combined gas chromatography—mass spectrometry. The use of glass for the capillary column is mandatory to achieve qualitative and quantitative analysis of the known occurring amino acids in urine and small plasma samples. Quantitative analysis of several types of human amino acid disorders are presented.

Caffeine determination in rat plasma : A comparative study of micromethods

Confronted with the need for a sensitive (1 mg/l), specific and reproducible (<5%) method for the determination of caffeine in small plasma samples (10–50 μl) of small laboratory animals, two existing methods, radioactive labelling and gas chromatography, were adapted. The first step, common to both methods, is chloroform extraction, followed by either gas-chromatographic analysis or radioactivity measurement. These methods were compared by using a common internal standard, labelled caffeine, measured before and after the extraction step. The initial requirements were fulfilled and the correlation of the results proved to be excellent. These methods can be extended to animals other than the rat, and to organs or biological fluids other than plasma. The very small amount of plasma needed for the determination allows pharmacokinetic studies to be conducted in small laboratory animals. Further, by combining the two methods it is possible to perform rather complex investigations, such as evaluating the extent of caffeine metabolism and, at the same time, determining levels in the case of chronic administration.

Determination of Caffeine in Small Plasma Samples by Gas-Liquid Chromatography with Thin-Layer Chromatographic Sample Clean-Up

Abstract A sensitive method for the determination of caffeine in small plasma samples (50 μ1) is described. The samples are extracted with chloroform and the extrects are freed from interfering compounds by thin-layer chronatography. The spots of caffeine and the internal standard, 7-ethyl theophylline, are scraped off the plates and both compounds are eluted from the scrapings by means of a glass capillary collector. The caffeine in the eluate is quantitated by gas-

Simple sensitive microbioassay for adenine arabinoside and hypoxanthine arabinoside in human plasma.

Previous methods using low- or high-pressure liquid chromatography and UV absorbance for quantitation of arabinosides in plasma can practically detect only >/=200 ng of adenine arabinoside and >/=100 ng of hypoxanthine arabinoside per ml, and they require expensive equipment and expert technical assistance. We describe in this report a simple quantitative microbioassay for arabinosides in human plasma based on their ability to inhibit the cytopathic effect of vaccinia virus in an adenosine deaminase-free cell culture system. Using prior separation of nucleosides in plasma by thin-layer chromatography, followed by the microbioassay, we quantitated adenine arabinoside with a sensitivity of approximately 4 ng/ml and hypoxanthine arabinoside at approximately 625 ng/ml. This assay method is simple, sensitive, and reproducible, requires small plasma samples, measures biological activity, and is adaptable to routine use. It is an important tool for evaluating the pharmacology of adenine arabinoside and Ara-AMP in patients in current clinical trials.

Radioenzymatic estimation of noradrenalin in small plasma samples without prior extraction

We have developed a radioenzymatic method for the estimation of nor-adrenalin in small plasma samples by utilizing partly purified phenylethanol-amine- N-ethyltransferase. Noradrenalin can be estimated in deproteinised plasma without prior extraction. The sensitivity of the assay is better than 2.5 pg. Inclusion of an internal standard with each sample is necessary as the enzyme reaction is slightly influenced by some components of the plasma. Prior extraction with alumina severely decreases the sensitivity of the assay.

Measurement of 1α-hydroxycorticosterone and other corticosteroids in elasmobranch plasma by radioimmunoassay

A simple radioimmunoassay method for the simultaneous determination of 1α-hydroxycorticosterone, 1-dehydrocorticosterone, corticosterone, and cortisol in small samples of elasmobranch plasma has been developed. Chromatographic separation of these steroids, followed by acid dehydration of 1α-hydroxycorticosterone and chromatography and subsequent radioimmunoassay of the resultant 1-dehydro derivative using an antibody raised for corticosterone, gave a highly specific and sensitive method for the determination of 1α-hydroxycorticosterone. 1α-Hydroxycorticosterone concentrations in the ray Raja clavata ranged from less than 0.08 to 13.9 μg/100 ml of plasma, and in the dogfish Scyliorhinus caniculus from 0.1 to 0.54 μg/100 ml of plasma. This compound was not detected in the rainbow trout Salmo gairdneri. Corticosterone and 1-dehydrocorticosterone were either undetectable or were present in extremely low concentrations in both species of elasmobranch. Cortisol, the major corticosteroid in the rainbow trout (8 μg/100 ml), was not detectable in either of the elasmobranch species.

A new method for deproteinization of small samples of blood plasma for amino acid determination

In this work is described a simplified plasma deproteinization procedure using acetone and very small amounts of plasma. The precipitation is carried out in small-bore capillary tubes, and acetone is used as deproteinizing agent. This method can be advantageously compared with heat, Somogyl's, perchloric, and even trichloroacetic deproteinization procedures for its good amino acid preservation and protein removal capabilities.

AN HOMOLOGOUS RADIOIMMUNOASSAY FOR CHICKEN FOLLICLE-STIMULATING HORMONE: OBSERVATIONS ON THE OVULATORY CYCLE

A highly purified FSH preparation has been used to develop a specific homologous radioimmunoassay for chicken FSH which is sufficiently sensitive and precise to measure the hormone in small sample (10-100 microliter) of plasma. The assay was used to measure plasma FSH in the chicken and turkey. The FSH concentration was higher in sexually mature chickens than in juvenile birds and further elevated after castration or ovariectomy. In turkeys, it was lower in birds held on a short daily photoperiod than in birds held on a long daily photoperiod. FSH rose in sexually quiescent female turkeys after injection of synthetic LH-releasing hormone and was increased in laying hens after injection of progesterone. No major changes were observed in FSH concentration during the chicken ovulatory cycle, although there was a small increase 15 and 14 h before ovulation.

Adsorption of polypeptide hormones to silicate powders: Development of reproducible methods for extracting ACTH and MSH from plasma

Several rapid and easy methods have been compared to extract human ACTH. α-MSH, and human β-MSH from small plasma samples (0.5 ml). The extraction methods were based on the adsorption of these polypeptide hormones to various silicate powders and subsequent elution by different eluants. The best extraction procedures were achieved under the following conditions: The ACTH was adsorbed to 40 mg of silicic acid and eluted with 2 ml of an acetic acid:acetone:water mixture (1:40:59); α-MSH was adsorbed to 50 mg of talcum powder and eluted with 2 ml of 75% aqueous ethanol: β-MSH was adsorbed to 40 mg of silicic acid and eluted with 2 ml of 0.1 n HCl. These extraction techniques did not affect the immunoactivity of the hormones and will prove useful for radioimmunoassays.

Sephadex LH-20 multiple-column chromatography for the simultaneous separation of progesterone, deoxycorticosterone and 17α-hydroxyprogesterone from small plasma samples

A simple and convenient chromatographic method is described for the simultaneous and complete separation of the unconjugated steroids progesterone, deoxycorticosterone and 17α-hydroxyprogesterone on 40-cm columns of Sephadex LH-20 using a water-saturated solvent system containing n-heptane-chloroform (1:1) plus 0.25% of ethanol. Manual operation of up to ten columns run in parallel is facilitated by the use of graduated, cylindrical, solvent reservoirs on top of the columns. Thus, negligible column-to-column, and only limited day-to-day, variations occur in the elution patterns, and constant high recoveries are obtained. When combined with a previously described 60-cm LH-20 chromatography, eight important corticosteroids can be isolated individually from 16 to 20 small plasma samples per day.

ENDOCRINE RESPONSES TO “STRESS” IN CHILDREN

It is known that growth hormone, ACTH and cortisol are secreted in response to stress. Although the physiological role of pancreatic glucagon is not yet fully unterstood, it has recently been shown, that “stress” also leads to an increase in plasma glucagon concentration. dAnesthesia, as well as minor or major surgical procedures should provoke a “stress”-response. The development of sensitive radio-immunological methods allows the assay of a series of hormones in small plasma samples. Using these methods the presumed stress response could therefore be investigated in pediatric patients. The plasma concentrations of HGH, ACTH, cortisol and glucagon were determined before, during and after anesthesia pneumencephalography, cardiac catheterisation and minor or major surgery in children of different ages.

Rapid Separation of Plasma Creatine Kinase Isoenzymes by Batch Adsorption on Glass Beads

To separate the MM and MB creatine kinase (EC 2.7.3.2) isoenzymes in small plasma samples, we developed a simple, rapid ion-exchange batch-adsorption procedure with "DEAE Glycophase" glass beads (Corning). All separative steps are performed in a single test tube and can be completed within a few minutes. Results of measurements of isoenzymes in plasma samples from patients with acute myocardial infarction were compared to those obtained with an independent quantitative assay method previously reported. Additional measurements were performed on standard plasma samples containing mixtures of MM and MB isoenzyme purified from human myocardium. Results by the two independent assay procedures agreed well, as did measured isoenzyme activities and activities predicted from the amounts of isoenzyme added. Activities in the fractions containing separated isoenzymes were measured by a direct kinetic fluorometric assay. MB activities in normal plasma averaged 1.6 plus and minus 0.28 U/liter (mean plus and minus SD).

Automation of multiple sephadex LH-20 column chromatography for the simultaneous separation of plasma corticosteroids

An automated method of Sephadex LH-20 chromatography has been developed for the simultaneous and complete separation of corticosterone, 11-deoxy-cortisol, aldosterone, cortisone and cortisol from a small plasma sample prior to radioimmunoassay. Using a controlled elution flow-rate of 40 ml/h and automated programme tape-controlled collection of the eluates from six columns eluted simultaneously, excellent reproducibility of elution patterns with minimal day-to-day and column-to-column variations could be achieved. The method can be easily extended and permits the reliable, rapid and easy routine separation of individual steroids from a single plasma or tissue extract on a multi-column scale.

Constancy and linearity of the metabolic clearance of adrenocorticotropin in dogs.

The study of the turnover characteristics of adrenocorticotropin (ACTH) has been facilitated by (1) a sensitive bioassay for ACTH in plasma based upon the response of suspended adrenal cells to ACTH and (2) a technique for preserving the ACTH in small plasma samples. The metabolic clearance rate (MCR) of ACTH was determined by intravenous step infusions of ACTH into dogs lightly anesthetized with Nembutal, without blockade of ACTH secretion; in these dogs the endogenous plasma ACTH level was negligible. The infusion rates ranged from 0.25 to 3.0 mU/kg∙min, yielding plasma ACTH plateau concentrations of 1.8 to 37 mU/100 ml. In no dog did the MCR correlate significantly with plasma concentration, nor did the MCR differ significantly from dog to dog. The 34 successful plateaux yielded a mean MCR of 9.54 ± 0.24 ml/kg∙min. Linear regression of MCR versus concentration yielded a correlation coefficient of −0.00026. The findings showed that the disappearance of ACTH from blood is not dose-dependent and varies negligibly with time or among animals. Hence the measured MCR may be used to convert concentrations of ACTH in plasma into turnover rates.

Determination of barbiturates and their metabolites in small plasma samples by gas chromatography-mass spectrometry

Abstract Techniques for the measurement of amylobarbitone and its pricipal metabolite, 3′-hydroxyamylobarbitone in 100-μl plasma samples are described. The methods which employ a mass spectrometer as a gas chromatographic detector may be used to determine 0.01 μ/ml of the drug and 0.0 μ/ml of the metabolite and were developed initially to permit pharmacodynamic studies in human newborn. With minor modification, they are applicable to other barbiturates including butobarbitone, pentobarbitone and the metabolites 3′-hydroxybutobarbitone and 3′-hydroxypentobarbitone providing scope for more general pharmacodynamic studies with barbiturates where sample size prrecludes conventional assay.