Pamela Angwin

Simultaneous determination of indole- and catecholamines in tissues using a weak cation-exchange resin☆☆☆

During the past decade, a number of procedures have been used for routine assay of catecholamines and the indoleamines, in various tissues. Many of these procedures do not allow simultaneous assay of the amines, or, if they do, as in the case of some solvent extraction procedures, do not allow for separation of amines from their precursor amino acids (l-6). The successful application of the weak cation-exchange resins by some authors (7-13) for one or two amines made it possible for us to adapt and improve those methods and, at the same time, to study various parameters of the procedure which have not been previously reported in order to increase the versatility of the assay method. The procedure can be used for simultaneous determination of norepinephrine (NE), epinephrine (E) , dopamine (DA), serotonin, (5-HT) and 5-methoxytryptamine (5-MT). Basically, the methods involve the deproteinieation of the tissue with either perchloric acid or ethanol, adsorption of the amines on the weak cation-exchange resin Bio-Rex 70, elution, and spectrofluorometric determination of the amines. The advantages of the method described in this paper over other published ones are: (a) the purification steps of the amines are greatly reduced, requiring only 2.5 hr to run up to 50 samples through the columns, (b) the recovery of the amines is higher, (c) one can determine quantitatively 20 ng of any of the four amines in one tissue sample, and (d) the method is highly reproducible.

Simultaneous determination of indole- and catecholamines in small brain regions in the rat using a weak cation exchange resin

Abstract A fluorometric procedure is described which allows the measurement of nanogram amounts of serotonin, horepinephrine, and dopamine in small brain areas (20–350 mg) from individual rats. The amines are separated from their precursor amino acids and acid metabolites by a single-column chromotography step using a weak cation exchange resin. It is possible quantitatively to determine 10–30 ng of each amine when all three are measured simultaneously. When either serotonin or the catecholamines are assayed in a tissue sample, as little as 5–15 ng may be detected. Recoveries of the amines range from 85 to 93% as measured by addition of 14 C-labeled amines to tissue supernatants.

Isolation and primary structure of human peptide YY

The isolation, primary structure and chemical synthesis of human peptide YY (PYY) are described. The peptide was purified from human colonic extracts using a chemical method which detected the C-terminal tyrosine amide structure of PYY. Human PYY consists of 36 amino acid residues and the complete amino acid sequence is: Tyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu- Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu- Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH2. The differences between the structures of porcine and human PYY are at positions 3 (Ala/Ile replacement) and 18 (Ser/Asn). Synthetic human PYY prepared using a solid-phase synthetic technique was found to be structurally identical to the natural peptide.

Isolation and characterization of bovine pancreastatin

Bovine pancreastatin, a 47 amino acid residue peptide, was isolated from the pancreas and the pituitary gland using a chemical method which detects its C-terminal glycine amide structure. The complete amino acid sequence of the pancreatic peptide is 74% homologous to that of porcine pancreastatin and is identical to bovine chromogranin A-(248-294), as deduced from its cDNA sequence. The sequence of the first 28 amino-terminal residues of the pituitary peptide was determined to be identical to the corresponding sequence of the pancreatic peptide. Since the pituitary peptide also contains the C-terminal glycine amide, it is therefore likely to be identical in structure to the pancreatic peptide. Thus, we conclude that bovine chromogranin A is the precursor of bovine pancreastatin. Synthetic bovine pancreastatin inhibited pancreatic exocrine secretion in a similar manner to porcine pancreastatin.

α‐N‐Acetyl β‐Endorphins in the Pituitary: Immunohistochemical Localization Using Antibodies Raised Against Dynorphin(1‐13)

Abstract: Intense immunohistochemical staining of the intermediate lobe of the pituitary was observed by using an antiserum raised against synthetic dynorphin(1‐13) treated with a water‐soluble carbodiimide (CDI). Subsequent studies showed that the immunostaining was blocked by preincubation of the antiserum with acetylated derivatives of both β‐endorphin and dynorphin(1‐13) as well as by CDI‐treated dynorphin(1‐13), but only weakly by authentic dynorphin(1‐13). Neither nonacetylated β‐endorphin nor any other fragments of the ACTH/endorphin precursor blocked the immunostaining of the intermediate lobe. Analysis of the CDI‐treated dynorphin(1‐13) used as an antigen showed that most of the peptide was acetylated at primary amino groups. CDI treatment of dynorphin(1‐13) results in the formation of an acetyl derivative because the commercially available peptide is supplied as the acetate salt. The antibodies responsible for the intermediate lobe staining were isolated by affinity chromatography, using a column containing partially purified intermediate lobe extract linked to an affinity resin and a radioimmunoassay (RIA) was developed with CDI‐treated dynorphin(1‐13) used as a trace and as a standard. Competition studies showed 0.5‐1% cross‐reactivity with α‐N‐acetyl β‐endorphin(1‐31), α‐N‐acetyl β‐endorphin(1‐27), and totally acetylated β‐endorphin(1‐31). Nonacetylated β‐endorphins did not cross‐react. Posterior‐intermediate lobe extracts from rat and beef were fractionated by gel filtration. Rat posterior‐intermediate lobe extracts were also fractionated by cation‐exchange chromatography. Fractionated extracts were analyzed by RIAs for β‐endorphin, CDI‐treated dynorphin(1‐13), and authentic dynorphin(1‐13). The results suggested that the peptides responsible for the intermediate lobe staining were mainly four different derivatives of β‐endorphin bearing an acetyl group at the amino terminus. No immunostaining was seen in the posterior and anterior lobes of the pituitary. This suggests that the intermediate lobe is the main source of acetylated β‐endorphins in the pituitary.

5-Hydroxytryptophan: A method for monitoring human plasma content during clinical administration.

A method is described for the assay of 5-hydroxytryptophan (5-HTP) in human blood plasma following administration of the compound. The procedure involves extracting the amino acid into butanol, returning the 5-HTP to an aqueous phase, and separating it from other interfering 5-hydroxyindoles on a column of Dowex 50W-X4, a strong cation-exchange resin. The concentration of 5-HTP in the buffer eluate is determined spectrofluorometrically in 3 N HCl; 25 ng/ml plasma can be detected. Using this method, it was possible to determine the time-course of plasma 5-HTP concentrations during the 24-h period after a single 300-mg dose. 5-HTP is being utilized in clinical trials for several psychiatric and neurological disorders.