The development of assays for atractyloside and its localisation in rat tissue.
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Date
1991
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Abstract
An extract of the tuber of Callilepis laureola is regarded as the source of a powerful
therapeutic agent, known as Impila. Its use is associated with fatal hepatic and renal
necrosis, the renal toxin being atractyloside (ATR). The aims of this study were threefold.
Firstly, to generate a model set of biological specimens (urine, serum, liver and
kidney) from rats dosed with 5-25 mg ATR/kg bwt. Secondly, to develop a competitive
ELISA and HPLC method for the diagnosis of ATR poisoning employing the model
specimens as test samples. Thirdly, to localise the target organs, cells and organelles of
ATR, in vivo.
The HPLC method necessitated the systematic development of the derivatisation of ATR
with 9-anthryldiazomethane, sample clean up employing hexane, methanolic hydrochloric
acid and a silica minicolumn, as well as the chromatographic conditions.
Optimal resolution was obtained with a 3.9 x 150 mm NovaPak reverse phase column,
fluorescence detection (excitation = 365 nm, emission = 425 nm) and a solvent system of
MeOH:1M ammonium acetate:1M glacial acetic acid:water (38:2:2:58). This method has
a detection limit of 0.001 ng ATR, shows a mean recovery of 89% and detected
approximately 6.7 ug ATR/g wet weight of tuber tissue. The toxin was also detected in some
of the urine samples at levels of about 200 pg/ml, but not in the serum.
The production of antibodies to ATR for use in the ELISA and immunocytochemical
investigations required the investigation of the conjugation procedure, carrier type, host
species and immunization protocol. Optimal antibody yield, specificity and affinity was
obtained with an acid-treated Salmonella minnesota bacterial carrier conjugated to ATR
by carbodiimide, although there were indications of class switch inhibition and Tlymphocyte
suppression by ATR. The development of the ELISA yielded a protocol
involving the coating with a bovine serum albumin-ATR conjugate, blocking with bovine
serum albumin, incubating the primary antibody at 4°C and detection with a secondary
antibody-alkaline phosphate conjugate. This method detected ATR in both urine and
serum from ATR-dosed rats and shows a detection limit of 10 ng. Since the less sensitive
ELISA detected ATR in samples where the HPLC did not, this suggested that ATR is
biotransformed in vivo, such that its retention time on a reverse phase column is affected,
but not its epitope determinants.
The results of the organ function assays demonstrated that, when administered intra-peritoneally,
ATR is not hepatotoxic, but is a powerful nephrotoxin, targeting for the
microvilli of the brush border of the proximal tubules, and compromising glomerular permselectivity and distal tubular function. In addition, this toxin inhibits proline transport in the proximal tubule, and therefore probably affects protein biosynthesis. Renal regeneration is noted 3 days post-dosing, as demonstrated by calcium excretion.
Immunocytochemistry was optimised on tuber tissue and necessitated the intracellular
fixation of the toxin, using carbodiimide, to prevent leaching out of the ATR. The toxin
was encapsulated in vesicles in the tuber tissue. Atractyloside was also located in the
kidney of ATR-treated rats, up to 72 hours after exposure, targeting the microvilli of the
proximal tubule brush border, the mitochondrial cristae and specific sites on the Golgi
apparatus membrane. Microvilli disruption and mitochondrial swelling was noted
within 24 hours after exposure to the toxin while after 72 hours, loss of mitochondrial
integrity was observed.
The development of these diagnostic assays for ATR have provided the means to monitor
the levels of this toxin in plant extracts and mammalian body fluids. Future work should
include the identification of the hepatotoxin associated with Impila, the effects of the route
of administration on the toxicity of this remedy and furthermore, the identification of a
suitable antidote, which could include the use of duramycin and stevioside. The
association between compounds blocking the ADP/ATP antiporter in the c-state and Reye's
syndrome should also provide an interesting area of research.
Description
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1991.
Keywords
Biological assay., Atractyloside., Toxins., Theses--Biochemistry.