Donald C. Wigfield

Tissue porphyrin pattern determination by high-speed high-performance liquid chromatography

A rapid and sensitive method for the determination of porphyrin carboxylic acids in liver, kidney, and spleen by high-speed high-performance liquid chromatography is described. Porphyrins were extracted with recoveries greater than or equal to 98%, concentrated on disposable octadecylsilyl cartridges, and analyzed with a liquid chromatograph equipped with a 3 microns X 3 cm octadecylsilyl column and a fluorescence detector. Separation of di-, tetra-, penta-, hexa-, hepta-, and octacarboxylic acids was achieved within 5 min. The detection limits for uro, copro, and protoporphyrin were 20, 10, and 20 fmol, respectively.

Dose-response relationships in hexachlorobenzene-induced porphyria.

The rate of development of hexachlorobenzene (HCB)-induced porphyria in female Wistar rats was determined using HCB dosage and porphyrin analysis protocols designed to determine factors which contribute to the delay commonly observed between initial exposure to HCB and the detection of porphyria. Measurements were made of HCB and porphyrin concentrations in the livers, kidneys, and spleens of female Wistar rats exposed continuously (up to 56 days) or for 1 day to HCB (at dietary concentrations of 1000 ppm and 100 ppm). The experiments showed that when a corn oil solution of HCB was added to the diet at a concentration of 1000 ppm, HCB accumulated rapidly in all organs, and the delay in appearance of elevated liver highly carboxylated porphyrins (HCPs) was at most 4 days (approximately 8-fold elevation of HCPs on day 4). One day of exposure to this diet was sufficient to cause elevated liver HCPs, thus showing that continuous exposure to HCB was not required to cause porphyria in this species. Solid HCB added directly to the diet (1000 ppm) resulted in less rapid HCB accumulation and less rapid development of porphyria. The experiments demonstrated that the appearance of a delay in HCB-induced porphyria in the Wistar rat is caused by the rate at which HCB is absorbed, and by using total hepatic porphyrins (rather than HCPs) as the indicator of the disorder. The experiments also showed that HCB-induced liver enlargement and neurotoxicity are not necessarily associated with the severity of porphyria.

The delay in polyhalogenated aromatic hydrocarbon-induced porphyria: Mechanistic reality or methodological artefact?

Hepatic porphyria was induced in female Wistar rats exposed to dietary hexachlorobenzene (HCB) for 56 days. The well-documented several-week delay before liver total porphyrins became elevated was observed using conventional methods. However, a newly developed high-performance liquid chromatography (HPLC) technique revealed a much earlier response. Highly carboxylated porphyrins were found to increase soon after exposure to the toxicant. The long delay observed by total porphyrin analysis is shown to be due to the relatively small contribution of highly carboxylated porphyrins to the total porphyrin pool. It is concluded that the concept of a latent period is largely a methodological artefact which has confused the search for a fundamental understanding of chemically induced porphyria.

Investigations on the question of multiple mechanisms in the cope rearrangement

Abstract The possible occurrence of the ionic Cope rearrangement, and other non-concerted mechanisms is discussed. The synthesis of 2 - (1 - ethyl - 1 - propenyl) -2- (3 - p - methoxyphenylallyl)malononitrile (1b) and its clean thermal 1,3 rearrangement to (1 - ethyl - 5 - p - methoxyphenyl - 2 - methyl - 4 - pentenylidene)malononitrile (4) are reported. This result contrasts with the rearrangement of 2 - (1,1 - dideuterioallyl) - 2 -(1 - ethyl - 1 - propenyl)malononitrile (1c) which isomerizes cleanly in a 3,3 rearrangement. Rearrangement of 2 - (1 - cyclohexenyl) - 2 - (3 - p - methoxyphenylallyl)malononitrile (11), however, leads sluggishly to [2 - (p - methoxy - α - vinylbenzyl)cyclohexylidene]malononitrile (19) (3,3 shift) and rearrangement of 2 - (1 - isopropyl - 2 - methyl - 1 - propenyl) - 2 -(3 - p - methoxyphenylallyl)malononitrile (12) leads, also slowly, to (1 - isopropyl - 5-p- methoxyphenyl - 2,2 - dimethyl - 4 - pentenylidene)malononitrile (14) (1,3 shift). Rearrangement of 1b in the presence of sodium borohydride allows interception of the proposed ionic intermediates and isolation of 2 - (1 - ethylpropylidene)malononitrile (5) and anethole (21c). Ion trapping experiments also gave positive results in the 3,3 rearrangement of 11. These results are discussed in terms of the ionic Cope rearrangement.

Liquid scintillation counting and sample adsorption. Structural factors in organic molecules controlling likelihood of adsorption

The Adsorption Shift method of detecting adsorption (previously described(4)) has been utilized to examine counting characteristics of 44 specifically labelled 14C-labelled compounds. 11 compounds showed adsorption and 33 were not seriously adsorbed. The relationships between structural features in organic molecules and their likelihood of adsorption to the walls of liquid scintillation counting vials are discussed, and the conclusions summarized.

The determination of the radioactivity of 14C samples adsorbed on glass vials by direct liquid scintillation counting

Abstract A method for obtaining the true activity and counting efficiency of a 14 C sample partially or completely adsorbed on the walls of a counting vial by liquid scintillation counting is presented.

Gas-phase adsorption losses of elemental mercury in cold-vapor atomic absorption spectrometry

Abstract The experimental application of a normalized-absorbance for the purpose of standardless calibration in cold-vapor atomic absorption spectrometry for mercury has been hindered by adsorptional losses of the trace analyte from the carrier gas. Surface adsorption has been evaluated for fourteen types of tubing using syringe injection of mercury vapor. For a flow-rate of 100 ml min−1 through 92-cm lenghts of tubing, losses varied between undetectable levels for nickel tubing and 100% for Silastic.

Interaction of divalent metal lons with normal and lead-inhibited human erythrocytic porphobilinogen synthase in vitro

The effects of Zn2+, Hg2+, Cd2+, and Cu2+ on normal and lead-inhibited erythrocytic porphobilinogen synthase (PBG-S) were studied in vitro using human whole blood hemolysate. The results demonstrate that each of the divalent ions tested has a characteristic effect on the pH-activity relationship of PBG-S. The effects for a given ion are concentration- and pH-dependent. For Zn2+ these effects are also time-dependent. The results obtained provide an explanation for the contradictory reports of the action of some of the metals in vitro and indicate that future investigations of the effects of metals on an enzyme such as PBG-S are best performed over a judiciously selected pH range rather than at a single pH value. It is also shown that the metals studied will only have a significant effect on the proposed PBG-S activity ratio test for lead intoxication in instances of gross contamination of blood collection devices. The activation and/or inhibition of PBG-S and associate pH-activity profile changes resulting from interaction with the four metal ions tested were attributed to their respective affinity for the thiol and other groups at the active sites. The occurrence of a relatively specific pH optimum for PBG-S after interaction with each ion investigated remains unexplained.

The transition state in the reduction of ketones by complex metal hydrides. Reassessment and limited reincarnation of the concept of product development control.

Abstract Using the conclusion of the previous communication, and other data, it is concluded that NaBH 4 reductions involve and product-like transition state and LiAlH 4 reductions involve a reactant-like transition state.

The delay in polyhalogenated aromatic hydrocarbon-induced porphyria: the effect of diet preparation

Porphyria development in female Wistar rats has been followed by dosing the animals with hexachlorobenzene (HCB) either dissolved in corn oil or as a solid mixed with the diet. It was found that the corn oil preparation resulted in much faster uptake of HCB into the liver, and much faster accumulation of liver porphyrins. Diet preparation is thus shown to be a major factor in determining whether the development of porphyria is associated with the delay phenomenon. Evidence is also presented suggesting that the neurological symptoms of porphyria are not caused by high porphyrin levels.