A nonisotopic method for determination of the in vivo activities of tyrosine hydroxylase in the rat adrenal gland.
A rapid and reliable method for determination of in vivo activities of tyrosine hydroxylase in the rat adrenal gland is presented. This method involves determining the rate of accumulation of 3,4-dihydroxyphenylalanine (Dopa) in the adrenal gland after decarboxylase inhibition by NSD 1015, using HPLC with electrochemical detection after purification of the acid-deproteinized tissue extract with Bio-Rex 70 columns followed by alumina batch method. Purification of the sample with alumina adsorption alone, a method usually used for purification of catecholamines and Dopa, was ineffective: epinephrine and norepinephrine, which are present in high concentrations, interfered with an accurate determination of Dopa, and dopamine, which is retained strongly on the reverse-phase column, interfered with a rapid analysis. Purification with Sephadex G-10 columns followed by alumina adsorption was also ineffective. After purification with columns of weak cation-exchange resins such as Bio-Rex 70 or Amberlite CG-50 followed by alumina adsorption, most of the epinephrine and norepinephrine was removed and dopamine was eliminated. Thus a rapid and accurate determination of Dopa could be made. Of the two cation exchangers, Bio-Rex 70 was more effective. Accumulation of Dopa in the adrenal gland was linear up to 30 min after administration of NSD 1015 and a plateau was reached with doses over 10 mg/kg. Using this method, we investigated the effects of immobilization stress, reserpine, and hypoxia on in vivo activities of tyrosine hydroxylase in the adrenal gland.
D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004295:Dihydroxyphenylalanine / Q000032:analysis / Q000378:metabolism; D004296:Dopa Decarboxylase / Q000378:metabolism; D006834:Hydrazines / Q000494:pharmacology; D000860:Hypoxia / Q000201:enzymology; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012110:Reserpine / Q000494:pharmacology; D013312:Stress, Physiological / Q000201:enzymology; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D006834:Hydrazines; D004295:Dihydroxyphenylalanine; D012110:Reserpine; C004297:3-hydroxybenzylhydrazine; D014446:Tyrosine 3-Monooxygenase; D004296:Dopa Decarboxylase