Daniel Couri

Toxic Polyneuropathy Produced by Methyl N-Butyl Ketone

A polyneuropathy affecting a large number of workers was recently observed at a plant producing plastic-coated and color-printed fabrics. Epidemiological data suggested strongly that methyl N-butyl ketone (MBK) was responsible for the outbreak. This hypothesis is now supported by the development of a peripheral neuropathy in chickens, rats, and cats exposed to MBK at atmospheric concentrations of 200 to 600 parts per million, 24 hours per day, 7 days per week. Although the animals were exposed continuously and the affected workers were exposed intermittently, the averages of the total number of hours of exposure for development of the peripheral neutropathy in the animals and workers were remarkably close.

Toxicity and metabolism of methyl n-butyl ketone.

MEK (2-butanone) when combined with MBK (2-hexanone) markedly enhanced MBK neurotoxicity. MBK in rat plasma after exposure to MBK/MEK increased with time. Metabolites of MBK identified in blood and urine of rats and guinea pigs were 2-hexanol and 2,5-hexanedione.

Alterations of fast axoplasmic transport in experimental methyl n-butyl ketone neuropathy

Methyl n-butyl ketone (MBK) is known to produce a giant axonal neuropathy in man and experimental animals characterized pathologically by a gradual increase in the number of neurofilaments which become associated with focal areas of axonal swelling and thinning of the myelin sheath. Fast axoplasmic transport was studied in rats exposed to MBK. In 10 severely paralyzed rats exposed to MBK there was a significant impediment of fast axoplasmic transport following dorsal root ganglion injections (x +/- S.D. = 283.2 +/- 20.34 mm/day) compared to normal controls (417.6 +/- 23.78 mm/day). In rats undergoing injections into the ventral horn of the spinal cord there was a gradual impairment of the mean down flow rate for transport of [3H]leucine which correlated with the severity of the MBK induced neuropathy. Quantitative morphological determinations showed that the total number of neurotubules per unit cross-sectional myelin area and the number of neurotubules associated with mitochondria in swollen axons was unchanged from normal. The total number of mitochondria in randomly sampled axons varied significantly from controls but the absolute number of mitochondria associated with neurotubules was unchanged from normal. The results of these studies suggest that the impediment of fast axoplasmic transport may be related to the increased neurofilaments producing focal areas of axonal blockage.

Biotransformation of n-hexane and methyl n-butyl ketone in guinea pigs and mice

Peripheral neuropathies caused by exposures to the industrial solvents n-hexane and MBK exhibit strinkingly similar characteristics. In in vivo studies, the metabolites of MBK and n-hexane identified in blood and urine of guinea pigs were 2-hexanol (partly as glucuronide in urine); and 2,5-hexanedione which was detected only in MBK treated groups. Phenobarbital pretreatment increased 2-hexanol urinary excretion in both solvent treatment groups. In in vitro studies, hepatic reduction of MBK required the cytosol fraction to form 2-hexanol; whereas the oxidation of MBK and n-hexane required the microsomal fraction to form 2,5-hexanedione and 2-hexanol, respectively. The in vivo and in vitro biotransformation of MBK and n-hexane to a common metabolite (2-hexanol) suggests that the neurotoxic action of these solvents may be metabolite related.

Analysis of enkephalins, β-endorphins and small peptides in their sequences by highly sensitive high-performance liquid chromatography with electrochemical detection: implications in opioid peptide metabolism

Sensitivity in the 10-100 pg range for enkephalins, beta-endorphin, tyrosine (T), 12 tyrosylglycine (T-G) and tyrosylglycylglycine (T-G-G) was attained by using a high-performance liquid chromatographic (HPLC) method with electrochemical detection which is at least 100 times more sensitive than HPLC with UV detection. The chromatographic conditions on a reversed-phase C18 silica column were 50 mM sodium phosphate buffer (pH 2.1) (A) in acetonitrile-methanol (1:1) (B), isocratic mixture, flow-rate 0.6-1 ml/min, UV detection at 205 nm, electrochemical oxidation potential + 1.25 V. The separation of T, T-G and T-G-G was obtained by using 10% B while the separation of the pentapeptide, enkephalins required 40% B. Separation of enkephalins from beta-endorphin was attained at a shorter retention times did not exceed 15 min. This method can be used to determine tissue levels and pharmacodynamics of enkephalins and beta-endorphin. A highly specific measurement of the different enzymes involved in the metabolism of enkephalin has been achieved.

Corticosteroid Modulation and Stress-Induced Analgesia in Rats

Early evidence has indicated the presence and involvement of specific neural systems which can inhibit the responses to painful stimuli. More recently, further advances suggest that the opiate system may interact with other systems to modulate the analgesia produced by the opiates or various stressors. Since corticosteroids were found to be elevated under the conditions of different stress-induced analgesia (SIA), there may be interactions between the pain-inhibiting systems and the corticosteroids. Recently it was reported that acute stress or long-term adrenalectomy can result in release of beta-endorphin (beta E) and ACTH from the pituitary gland, which can be blocked by dexamethasone. In our early studies we have shown partial antagonism of the SIA by dexamethasone and complete antagonism after naloxone. In this report it was found that chronic treatment of the rats with 0.02% metyrapone in drinking water for 8 weeks resulted in minor hyperalgesia. The chronic pretreatment with metyrapone resulted in a significant potentiation of the analgesia induced via the cold swim stress model, which was reversed by 1 mg/kg (IP) naloxone. Also, hyperalgesia was noted 18 days after the bilateral adrenalectomy of the rats as measured in our laboratory by the hot plate method and as reported by Heybach and Vernikos-Danellis in 1978. These results suggest that the corticosteroid modulation (pituitary-adrenal axis) may have a role in regulating the SIA, and this may implicate the interactions of the corticosteroids with pain-inhibiting systems.

Studies on the site of action of dicumarol on prothrombin biosynthesis

Abstract 1. (1) Indirectly acting anticoagulants (including dicumarol) inhibit the synthesis of the glycoproteins of the prothrombin complex at a post-translation site; we investigated the possibility that the site of action of dicumarol is at the glycosylation of prothrombin. Dicumarol treatment in Wistar rats, but not in warfarin-resistant rats, reduced the plasma content (units and mg protein) of prothrombin and inhibited the incorporation of glucosamine into prothrombin. There was also a decrease in the specific activity of the prothrombin isolated from dicumarol-treated rats indicative of there being synthesis and release from the liver of an inactive ‘prothrombin’. Vitamin K 1 administered to dicumarol-treated rats caused a rapid increase in the plasma content of active prothrombin and greatly increased (3 times the untreated control levels) the rate of glucosamine incorporation into prothrombin, thus suggesting that the protein precursor for prothrombin present in the liver of dicumarol-treated rats contains much less glucosamine than prothrombin from untreated rats. 2. (2) Cycloheximide administered 30 min prior to vitamin K 1 in dicumarol-pretreated rats did not effect the early (within 1 h of the administration of the vitamin) and rapid rise in plasma prothrombin content. Although the concentration of plasma prothrombin protein rose rapidly after the administration of the vitamin in these experiments, cycloheximide inhibited the incorporation of leucine into the newly released prothrombin. The data support the conclusions (1) that most of the newly released prothrombin does not require protein synthesis de novo and (2) that the action of dicumarol and vitamin K 1 is prior to or at the incorporation of glucosamine into prothrombin.

Dexamethasone and stress-induced analgesia.

Analgesia induced in rats by cold-water swim stress and measured by the tail-flick and hot-plate methods was significantly antagonized after IP pretreatment for 3 days with 8 mg/kg dexamethasone. The analgesia developed by the cold-water swim stressor was also attenuated by 1 mg/kg naloxone. These results suggest that the corticosteroids may have a role in modulating stress-induced analgesia and that the adrenal-pituitary axis modulates the endogenous opiate system. These conclusions are based on recent reports that indicate the release of the opiate-like peptide β-endorphin and adrenocorticotropin (ACTH) from the pituitary are increased by acute stress and inhibited by administration of the synthetic glucocorticoid dexamethasone.

The influence of inhaled ketone solvent vapors on hepatic microsomal biotransformation activities.

Abstract In studies conducted to determine the effects of inhaled ketone (methyl n-butyl ketone, MBK; methyl ethyl ketone, MEK) solvent vapors on hepatic microsomal oxidative and reductive enzyme activities in rats, groups of young male Wistar rats were housed in environmental chambers and exposed to solvent vapors (MBK, 225 ppm; MEK, 750 ppm; MBK, 225 ppm/MEK, 750 ppm). Hexobarbital sleep times were significantly reduced following exposure to MBK/MEK or MEK, but MBK exposure did not alter sleep time measurements. Aniline hydroxylase, aminopyrine demethylase, Neoprontosil reductase, and p-nitrobenzoate reductase activities were significantly enhanced two- to threefold over control values.

A method for the detection of drugs at therapeutic dosages in human urine using adsorption column chromatography and thin-layer chromatography

Abstract This communication describes methods utilized in our laboratory for the rapid toxicological analysis of emergency room patient urine samples. The techniques include Amberlite XAD-2 resin column Chromatographic separation from biologic material, followed by thin-layer chromatography and chromogenic spraying for detection. These methods and techniques are relatively simple to perform and drugs can be detected and tentatively identified at therapeutic dose levels.