Novel Double-Isotope Technique for Enzymatic Assay of Catecholamines, Permitting High Precision, Sensitivity and Plasma Sample Capacity

1981 ◽  
Vol 61 (5) ◽  
pp. 591-598 ◽  
Author(s):  
M. J. Brown ◽  
D. A. Jenner
Keyword(s):  
Plasma Sample ◽  
Specific Radioactivity ◽  
High Specific Radioactivity ◽  
Coefficients Of Variation ◽  
Isotope Technique ◽  
Noradrenaline And Adrenaline ◽  
Radioenzymatic Assays ◽  
Double Isotope ◽  
Catecholamine Concentration

1. A novel use of a double-isotope method is described which allows radioenzymatic assays to combine precision and sensitivity. 2. In the catechol O-methyltransferase assay separate portions of each plasma sample are incubated with either S-[3H]- or S-[14C]-adenosyl-l-methionine. Standards of noradrenaline and adrenaline are added to the latter portions and are thus converted into standards of [14C]metadrenalines. These are added to the 3H-labelled portions after the incubation, where they function as tracers. 3. The final recovery of 14C radioactivity corrects for (a) the efficiency of methylation in the plasma sample concerned and (b) the recovery of metadrenalines during the extraction procedures. 4. The 3H/14C ratio is constant in each assay for a given catecholamine concentration and is determined for samples to which standards of noradrenaline and adrenaline are added to the 3H- (as well as the I4C-) labelled portions before the initial incubation. 5. The sensitivity of the assay is increased by using high specific radioactivity S-[3H]adenosyl-l-methionine (60-85 Ci/mmol), and low backgrounds are maintained by catecholamine depletion in vivo in the rats used for enzyme preparation. 6. Both catecholamines (1.5 pg/ml; 10 pmol/l) may be detected; the coefficients of variation are 3.0 and 3.2% for noradrenaline and adrenaline respectively (intra-assay) and 4.6 and 5.0% (inter-assay).

scholarly journals Reverse cholesterol transport in the isolated perfused rat spleen

1990 ◽  
Vol 268 (2) ◽  
pp. 499-505 ◽  
Author(s):  
M A Mindham ◽  
P A Mayes ◽  
N E Miller
Keyword(s):  
Whole Blood ◽  
Chemical Potential ◽  
Specific Radioactivity ◽  
Density Lipoprotein ◽  
Potential Gradient ◽  
Cholesterol Transport ◽  
Unesterified Cholesterol ◽  
Chemical Potential Gradient ◽  
High Specific Radioactivity

1. A method has been developed which enables the rat spleen to be loaded in vivo with [3H]cholesterol to a high specific radioactivity using cholesterol-labelled erythrocytes. The erythrocytes were shown to be rapidly degraded by the spleen and not released intact during subsequent perfusion. 2. When labelled spleens were perfused with whole blood or serum, lipoproteins in the high-density lipoprotein (HDL) range were shown to be the principal lipoprotein vehicles for the removal of cholesterol, the specific radioactivity of cholesterol being much greater in the HDL fractions than in other lipoproteins, particularly in the d 1.175-1.210 fraction. 3. The formation of [3H]cholesteryl ester was restricted to the major HDL fractions. 4. Experiments utilizing individual HDL fractions added to a basal perfusate indicated that HDL1 (d 1.050-1.085) was of less importance in the removal of cholesterol from the spleen than HDL subfractions of higher density. Also, a decrease in density of the lipoproteins was observed during perfusion, concurrent with uptake of cholesterol, especially in the d 1.085-1.125 subfraction. 5. When [3H]cholesterol-labelled spleens were perfused with whole blood, about half of the radioactivity released was detected in erythrocytes, indicating a rapid exchange or transport of cholesterol. Thus erythrocytes could play an important role in the transfer of unesterified cholesterol when the chemical potential gradient is favourable.

scholarly journals The separation of intracellular serum albumin from rat liver

1971 ◽  
Vol 123 (4) ◽  
pp. 643-648 ◽  
Author(s):  
J. D. Judah ◽  
Marion R. Nicholls
Keyword(s):  
Serum Albumin ◽  
Rat Liver ◽  
Specific Radioactivity ◽  
Exchange Chromatography ◽  
Rat Serum ◽  
Radioactive Impurity ◽  
Simple Method ◽  
High Specific Radioactivity ◽  
Chromatographic Procedure

1. Antibody precipitation of serum albumin from rat liver extracts yields impure preparations of the protein. 2. When rat liver is labelled with l-[1-14C]leucine, antibody precipitation of albumin leads to material that is contaminated with a protein or proteins of very high specific radioactivity. Only 10–25% of the radioactivity of the antibody precipitate is associated with serum albumin. 3. A chromatographic procedure is described that can be used to separate radiochemically pure serum albumin from antibody precipitates obtained from extracts of rat liver. 4. Extracellular albumin secreted by liver slices yields a precipitate with antibody which contains much less radioactive impurity. About 70–90% of the radioactivity is associated with serum albumin. Serum albumin separated by antibody precipitation from rat serum labelled in vivo was not contaminated with the radiochemical impurities associated with intracellular albumin. 5. A simple method is described of obtaining the content of serum albumin in rat liver extracts by the technique of isotope dilution and ion-exchange chromatography.

Comparison of double- and single-isotope enzymatic derivative methods for measuring catecholamines in human plasma.

1978 ◽  
Vol 24 (4) ◽  
pp. 567-570 ◽  
Author(s):  
M I Evans ◽  
J B Halter ◽  
D Porte
Keyword(s):  
Plasma Catecholamines ◽  
Assay Method ◽  
Plasma Catecholamine ◽  
Isotope Method ◽  
Coefficients Of Variation ◽  
Basal Plasma ◽  
Isotope Technique ◽  
Assay Time ◽  
Plasma Catecholamine Concentrations ◽  
Double Isotope

Abstract We directly compared the reliability of a single-isotope enzymatic derivative technique for measurement of plasma catecholamines with that of the well-established double-isotope method. A significant (p less than 0.001) correlation was observed between measurements (n = 52) in the two assays, both for norepinephrine (r = 0.97) and epinephrine (r = 0.80). Means and coefficients of variation for the two analytes in a pooled specimen of plasma, measured repeatedly during six months, were virtually identical by each assay method. Basal plasma catecholamine concentrations in two different groups of apparently healthy subjects were also similar by each method. Dopamine concentrations in plasma were consistently below the limits measurable by either technique. The single-isotope assay requires half the assay time and 1/200th the sample as the double-isotope method. We conclude that this assay is just as reliable as the double-isotope technique and gives virtually identical values for norepinephrine and epinephrine concentrations in the physiological range.

scholarly journals Dietary ascorbic acid raises iron absorption in anaemic rats through enhancing mucosal iron uptake independent of iron solubility in the digesta

1997 ◽  
Vol 77 (1) ◽  
pp. 123-131 ◽  
Author(s):  
K. J. H. Wienk ◽  
J. J. M. Marx ◽  
M. Santos ◽  
A. G. Lemmens ◽  
E. J. Brink ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Iron Uptake ◽  
Iron Absorption ◽  
Iron Solubility ◽  
Fe Uptake ◽  
Isotope Technique ◽  
Proximal Intestine ◽  
Liquid And Solid Phases ◽  
Double Isotope

We studied Fe absorption from FeSO4 in rats with Fe deficiency-induced anaemia that were given an Fe-sufficient purified diet without or with ascorbic acid (10·4 g/kg diet). Attention was focused on mucosal Fe uptake as measured in vivo by a double-isotope technique. Haemoglobin repletion and liver Fe levels were not affected when the ascorbic acid-supplemented diet was given, but apparent Fe absorption and retention of orally administered 59Fe were significantly enhanced. The distribution of Fe between liquid and solid phases of contents of both the stomach and the proximal intestine was not affected by the feeding of the ascorbic acid, but ascorbic acid significantly enhanced mucosal Fe uptake. It is concluded that ascorbic acid in the diet raises mucosal Fe uptake through a mechanism independent of the intestinal Fe solubility.

scholarly journals The realiability of rates of glucose appearance in vivo calculated from constant tracer infusions

1978 ◽  
Vol 172 (3) ◽  
pp. 407-416 ◽  
Author(s):  
J R Allsop ◽  
R R Wolfe ◽  
J F Burke
Keyword(s):  
Specific Radioactivity ◽  
Glucose Production ◽  
Volume Of Distribution ◽  
Endogenous Glucose Production ◽  
Single Step ◽  
Actual Rate ◽  
Steady State Rate ◽  
Isotope Technique ◽  
State Rate

The rate of appearance of unlabelled glucose was calculated from tracer data and compared with the actual rate of infusion of unlabelled glucose into a anaesthetized dog with all sources of endogenous glucose production surgically removed. The mean steady-state rate of appearance of unlabelled glucose calculated from the equilibrium specific radioactivity was insignificantly higher (0.3%) than the actual rate of infusion of unlabelled glucose (n = 6). During non-steady states, a time-variable volume of distribution of glucose (V) was necessary to predict the rate of appearance of unlabelled glucose correctly from the pool-dependent equation described by Steele [(1959) Ann. N.Y. Acad. Sci. 82, 420–430]. Rapid fluctuations in the rate of appearance of glucose could be predicted reasonably well by using a fixed value of V for 40ml/kg, but by using larger fixed values for V (100–160ml/kg) the rates were inaccurate. The pool-dependent two-radiactive-isotope technique described by Issekutz, Issekutz & Elahi [(1974) Can. J. Physiol. Pharmacol. 52, 215–224] predicted single-step increases in the rate of infusion of glucose reasonably accurately, but the Steele (1959) equation was better at predicting sequential changes in the rate of infusion of unlabelled glucose.

Glycosylation of plasma proteins with [U-14C]gIucose in vivo

1982 ◽  
Vol 60 (10) ◽  
pp. 942-951
Author(s):  
Rudolf Vrba ◽  
Stephen P. Adams
Keyword(s):  
Molecular Weight ◽  
Body Weight ◽  
Sialic Acid ◽  
Plasma Proteins ◽  
Gel Filtration ◽  
Specific Radioactivity ◽  
Apparent Molecular Weight ◽  
Covalent Bonds ◽  
High Specific Radioactivity

Within 1–12 h after subcutaneous injection of [U-14C]glucose, significant amounts of 14C attached by stable (probably covalent) bonds were found in plasma proteins of rats. More than 30% of 14C of [U-14C]glucose injected per 1 g body weight was incorporated into proteins contained in 1 mL of plasma. Less than 5% of 14C of the [14C]glucose-labelled plasma proteins was soluble in cold dilute perchloric acid, whereas more than 80% of 14C in the [14C]glucose-labelled plasma proteins was soluble in 50% saturated solutions of ammonium sulfate. From [U-14C]glucose-labelled plasma proteins, approximately 50% of the incorporated 14C was recovered in carbohydrate moieties (sialic acid, 8–12%; mannose and galactose, 15–31%; hexosamines, 8–14%) and the rest of the 14C (42–64%) was recovered from protein residue. Gel-filtration and electrophoresis profiles of distribution of 14C in [U-14C]leucine-labelled plasma proteins were very similar to those of [U-14C]glucose-labelled plasma proteins; a relatively high specific radioactivity was observed in fractions having an apparent molecular weight of 105 × 103 or its multiples (220 × 103 and 520 × 103). However, about 99% of the incorporated 14C was recovered from the protein residue of [14C]leucine-labelled plasma proteins, whereas only traces of 14C were found in the carbohydrate moieties. [U-14C]Alanine is also a relatively poor precursor of 14C for incorporation into carbohydrate moieties of plasma proteins as compared with [U-14C]glucose.

scholarly journals An alternative procedure for incorporating radiolabelled cholesteryl ester into human plasma lipoproteins in vitro

1985 ◽  
Vol 226 (1) ◽  
pp. 319-322 ◽  
Author(s):  
D C K Roberts ◽  
N E Miller ◽  
S G L Price ◽  
D Crook ◽  
C Cortese ◽  
...  
Keyword(s):  
Cholesteryl Ester ◽  
Human Plasma ◽  
Specific Radioactivity ◽  
Density Lipoprotein ◽  
Plasma Lipoproteins ◽  
Cholesteryl Esters ◽  
Simple Method ◽  
High Specific Radioactivity

A simple method has been developed for labelling human plasma lipoproteins to high specific radioactivity with radioactive cholesteryl esters in vitro. After isolation by preparative ultracentrifugation, the selected lipoprotein was incubated for 30 min at 4 degrees C in human serum (d greater than 1.215) that had been prelabelled with [4-14C]cholesteryl oleate or [1,2-3H]cholesteryl linoleate, and was then re-isolated by ultracentrifugation. All major lipoprotein classes were labelled by the procedure. Specific radioactivities of up to 18 d.p.m. pmol-1 (46 d.p.m. ng-1) were achieved. When radiolabelled high-density lipoprotein was infused intravenously, the radioactive cholesteryl ester behaved in vivo indistinguishably from endogenous cholesteryl esters produced by the lecithin (phosphatidylcholine): cholesterol acyltransferase reaction.

scholarly journals 125I-Labelling of Erythropoietin without Loss of Biological Activity

1976 ◽  
Vol 159 (2) ◽  
pp. 287-292 ◽  
Author(s):  
M J Murphy
Keyword(s):  
Biological Activity ◽  
Glucose Oxidase ◽  
Catalytic Properties ◽  
Specific Radioactivity ◽  
Biological Integrity ◽  
High Specific Radioactivity

Erythropoietin, the hormone that regulates erythropoiesis in mammals, was 125I-labelled by using the catalytic properties of lactoperoxidase and the H2O2-generating properties of glucose oxidase. This methodology, both rapid and simple, not only produced hormone preparations with high specific radioactivity but also did not substantially alter the biological integrity of erythropoietin when it was assayed in vivo.

scholarly journals A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo

1983 ◽  
Vol 212 (3) ◽  
pp. 791-800 ◽  
Author(s):  
R C Pittman ◽  
T E Carew ◽  
C K Glass ◽  
S R Green ◽  
C A Taylor ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Specific Radioactivity ◽  
Apolipoprotein A1 ◽  
Intact Protein ◽  
Gut Contents ◽  
Tissue Samples ◽  
Cultured Fibroblasts ◽  
High Specific Radioactivity

We recently developed a general method for determining tissue sites of degradation of plasma proteins in vivo that made use of covalently attached radioactive sucrose. On degradation of the protein, the sucrose remained trapped in the cells as a cumulative marker of protein degradation. The method described here depends on the same principles, but uses an adduct of cellobiose and tyramine that is radioiodinated to high specific radioactivity and then covalently attached to protein. Use of the radioiodinated ligand increases the sensitivity of the method at least 100-fold and allows simplified tissue analysis. Proteins derivatized with the radioiodinated ligand were recognized as underivatized proteins both in vitro and in vivo. On degradation of derivatized low-density lipoprotein, the rate of leakage from cultured fibroblasts was only 5% during 24 h. Similarly, on injection of labelled proteins into rats and rabbits, urinary excretion of the label was in all cases less than 10% of total labelled catabolic products recovered 24 h after injection. Examination of the tissue contents of label at two times after injection of labelled asialofetuin or apolipoprotein A1 in rats, and asialotransferrin in rabbits showed that the label did not detectably redistribute between tissues after initial uptake and catabolism; a significant leakage from liver was quantitatively accounted for by label appearing in gut contents and faeces. A simple double-label method was devised to provide a correction for intact protein in trapped plasma, the extravascular spaces, and within cells. By using this method it becomes unnecessary to fractionate tissue samples.

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