Sodium ascorbate improves yield of urinary steroids during hydrolysis with Helix pomatia juice

TY - JOUR

T1 - Sodium ascorbate improves yield of urinary steroids during hydrolysis with Helix pomatia juice

AU - Christakoudi, S

AU - Cowan, D A

AU - Taylor, N E

PY - 2008/3

Y1 - 2008/3

N2 - Urinary steroid profile analysis requires enzymatic hydrolysis of glucuronide and sulfate conjugates and this is achieved simultaneously using Helix pomatia juice (HPJ), but steroids with 3 beta-hydroxy-5-ene structure undergo transformation and yield of 5 alpha-reduced corticosteroid glucuronides is poor. We describe the use of sodium ascorbate to solve these problems and provide a basis for its mode of action. Steroid conjugates were extracted from urine, hydrolyzed in acetate buffer with HPJ and sodium ascorbate and analyzed as methyloxime-trimethylsilylether derivatives by gas chromatography-mass spectrometry. Ranges of temperature, pH and ascorbate, substrate and HPJ concentrations were compared for urine and pure standards. Activity of other antioxidants and that of bacterial cholesterol oxidase were examined. Helix pomatia enzyme preparations from different commercial sources were compared. Loss of 3 beta-hydroxy-5-ene steroids was enzyme-dependant, since it required HPJ, was saturable, subject to substrate competition and heat-inactivated. Products were 3-oxo-4-ene steroids and 4,6-diene and 6-oxy derivatives of these but the latter were not formed from 3-oxo-4-ene precursors. Ascorbate, other antioxidants or oxygen exclusion diminished activity. These characteristics were shared by cholesterol oxidase. Yield of 5 alpha-reduced steroids was diminished by pre-incubation of HPJ before ascorbate addition and this was reversed if ascorbate was added to the pre-incubation mixture. We conclude that transformation of 3 beta-hydroxy-5-ene steroids by HPJ is due to cholesterol oxidase and is diminished by antioxidants or oxygen denial. Yield of 5 alpha-reduced steroids is low due to oxidative damage of P-glucuronidase during hydrolysis, prevented by ascorbate. These features are shared by most commercial Helix pomatia enzyme preparations tested. (C) 2007 Elsevier Inc. All rights reserved

AB - Urinary steroid profile analysis requires enzymatic hydrolysis of glucuronide and sulfate conjugates and this is achieved simultaneously using Helix pomatia juice (HPJ), but steroids with 3 beta-hydroxy-5-ene structure undergo transformation and yield of 5 alpha-reduced corticosteroid glucuronides is poor. We describe the use of sodium ascorbate to solve these problems and provide a basis for its mode of action. Steroid conjugates were extracted from urine, hydrolyzed in acetate buffer with HPJ and sodium ascorbate and analyzed as methyloxime-trimethylsilylether derivatives by gas chromatography-mass spectrometry. Ranges of temperature, pH and ascorbate, substrate and HPJ concentrations were compared for urine and pure standards. Activity of other antioxidants and that of bacterial cholesterol oxidase were examined. Helix pomatia enzyme preparations from different commercial sources were compared. Loss of 3 beta-hydroxy-5-ene steroids was enzyme-dependant, since it required HPJ, was saturable, subject to substrate competition and heat-inactivated. Products were 3-oxo-4-ene steroids and 4,6-diene and 6-oxy derivatives of these but the latter were not formed from 3-oxo-4-ene precursors. Ascorbate, other antioxidants or oxygen exclusion diminished activity. These characteristics were shared by cholesterol oxidase. Yield of 5 alpha-reduced steroids was diminished by pre-incubation of HPJ before ascorbate addition and this was reversed if ascorbate was added to the pre-incubation mixture. We conclude that transformation of 3 beta-hydroxy-5-ene steroids by HPJ is due to cholesterol oxidase and is diminished by antioxidants or oxygen denial. Yield of 5 alpha-reduced steroids is low due to oxidative damage of P-glucuronidase during hydrolysis, prevented by ascorbate. These features are shared by most commercial Helix pomatia enzyme preparations tested. (C) 2007 Elsevier Inc. All rights reserved

U2 - 10.1016/j.steroids.2007.11.001

DO - 10.1016/j.steroids.2007.11.001

M3 - Article

VL - 73

SP - 309

EP - 319

JO - Steroids

JF - Steroids

IS - 3

ER -