Metabolism of [¹⁴C]cholesterol in Manduca sexta pupae: Isolation and identification of sterol sulfates, free ecdysteroids, and ecdysteroid acids

Abstract

[14C]Cholesterol was injected into fifth-instar larvae of Manduca sexta, and the metabolites were isolated and identified from 8-day-old male and female pupae. A major portion of the metabolized cholesterol was esterified either with a sulfate group or with fatty acids. The predominant ecdysteroid metabolites were 20-hydroxyecdysone, 20,26-dihydroxyecdysone, 20-hyd roxyecdysonoic acid, and 3-epi-20-hyd roxyecdysonoic acid. Smaller amounts of ecdysteroids were identified as conjugates of 26-hydroxyecdysone, 3-epi-20-hydroxyecdysone, 20,26-dihydroxyecdysone, and its 3a-epimer. The metabolic profiles were similar for both male and female pupae. The two ecdysteroid acids were identified by nuclear magnetic resonance spectroscopy and chemical ionization mass spectrometry and by mass spectral analyses of their methyl esters. Detection of 3-epi-20-hydroxyecdysonoic acid as a major metabolite is significant, as its occurrence has been scarcely reported. 3-Epiecdysteroid acid formation is discussed as a possible ecdysteroid-inactivating pathway that may be operating specifically in lepidopterous insects or in particular developmental stages such as eggs or pupae.

Profiling of ecdysteroids in complex biological samples using liquid chromatography/ion trap mass spectrometry

“…Manduca sexta (tobacco hornworm) were reared under a 16 h light/8 h dark photoperiod and staged early on day 5 of the fifth instar following the gut purge 23, 24. Hemolymph (blood) was collected from day 6–7 fifth instar larvae of M. sexta into ∼50 mL of methanol to precipitate proteins.…”

“…20E and E have been found in the hemolymph during the pupal‐adult development23 of M. sexta. [ 14 C]20E and [ 14 C]E were also detected in the whole body extract of pupae after injection of [ 14 C]cholesterol into fifth‐instar larvae 24, 27…”

Profiles of free and conjugated ecdysteroids and ecdysteroid acids during pupal‐adult development of Manduca sexta

“…Ecdysteroids were fractionated according to class (free, acid, or conjugate) and were then analyzed by HPLC. * Separation and purification procedures using Florisil, XAD-16, XAD-2, DEAE-Sephadex A-50, and C18 Sep-Pak column chromatography have been detailed elsewhere [8,9].…”

“…An aliquot of each conjugate was hydrolyzed with an enzyme mixture [8]; after purification, each deconjugated ecdysteroid was identified by TLC [9] and HPLC.…”

“…Our studies have now extended to the longterm labeling of ecdysteroids during the pupal-adult development of male M. sextu. Of particular significance, two ecdysteroid acids, 20-hydroxyecdysonoic acid and its 3a-epimer, were isolated and identified from insects 8 days following pupation as metabolites of ['4C]cholesterol administered to M. sextu as fifth-instar larvae [8,9]. Ecdysteroid acids and 3-epiecdysteroids are generally considered as inactivation products of the active molting hormone, 20-hydroxyecdysone [lo], resulting from C-26 oxidation and C-3 epimerization.…”