Dissection autoradiography: a screening technique using storage phosphor autoradiography to detect the biodistribution of radiolabelled compounds.

INTRODUCTION: This study reports an alternative, rapid, whole body autoradiography technique which utilises storage-phosphor imaging technology. Conventionally, tissue or whole body sections have been used to examine the distribution of radiolabelled test compounds. However, the information acquired relates only to the sections examined, and the amount of radioactivity within the whole organ cannot be quantified. We have developed a rapid semi-quantitative technique that produces a concise visual representation of the distribution of the isotope throughout the entire animal: dissection autoradiography (DAR). METHODS: By dissecting a mouse which has been administered 14C-labelled methotrexate (MTX) and drying the tissues on a gel dryer, whole organs and aliquots of body fluids can be exposed to a phosphor imaging plate. The data obtained was analysed with the software associated with the phosphor imaging system and, by using 14C standards, the amount of 14C per total organ or tissue was quantified relative to other samples. Another widely used method to detect radiolabelled material in vivo is tissue solubilisation (TS) followed by liquid scintillation counting (LSC). This conventional method was compared with DAR. RESULTS: The new technique described in this communication was found to have a high level of reproducibility (R(2)= 88-95%). Whilst DAR was less sensitive than TS and LSC, trends over time in the biodistribution of 14C-MTX throughout most tissues were consistent between techniques. DISCUSSION: Whilst TS and LSC was a more sensitive technique, it was labour intensive and expensive in terms of consumables and time when compared with DAR. Dissection autoradiography has the potential to be used to screen quickly large numbers of samples in the biodistribution studies of various conjugates, isomers, derivatives or formulations of a parent compound, following a variety of routes of administration.

Role of the liver and kidney in the desulphation of heparin in vivo.

Heparin radiolabelled with 125I was given intravenously to intact, bilaterally nephrectomised or completely hepatectomised rats in the presence and absence of 1 mg unlabelled heparin/kg. Plasma samples were collected and analysed by gel filtration chromatography and affinity chromatography on Polybrene-Sepharose, which binds sulphated glycosaminoglycans. Radiolabel in the plasma was associated with both intact heparin and fully desulphated macromolecular carbohydrate chains. Levels of intact heparin in plasma from control rats decreased with time at both doses with a concomitant increase in desulphated material. Livers accumulated the greatest amounts of radiolabel on a per organ basis. Hepatectomy both increased levels of radiolabel in plasma and decreased amounts of desulphated heparin produced, indicating that the liver was the major site of desulphation. Even after removal of the liver, the desulphated metabolite comprised approximately 15% of radiolabelled material at all times and doses, suggesting an additional site of GAG desulphation which may be the vascular endothelium. The kidneys contributed significantly to in vivo desulphation of heparin only at the high dose.

Detection and preliminary characterization of two enzymes involved in biosynthesis of platelet-activating factor in mouse oocytes, zygotes and preimplantation embryos: dithiothreitol-insensitive cytidinediphosphocholine: 1-O-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase and acetyl-coenzyme A:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase.

The aim of this study was to determine whether the final enzymes in the two biosynthetic pathways for platelet-activating factor (PAF) (the 'de novo' and the 'membrane remodelling' pathways) are present in mouse embryos, zygotes and oocytes. The enzymes are dithiothreitol-insensitive cytidinediphosphocholine: 1-O-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (cholinephosphotransferase) in the de novo pathway and acetyl-coenzyme A:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase (acetyltransferase) in the membrane remodelling pathway. Activity of both enzymes was detected in the unfertilized oocyte, the zygote and also in the preimplantation embryo (48, 72 and 96 h after the ovulatory injection of hCG). In both cases the activity was destroyed by boiling and increased linearly with incubation time and the concentration of embryo homogenate present, indicating that the reactions were catalysed by enzymes. The product of the reactions was confirmed as PAF using HPLC and structural analyses by enzymatic digestion. Cholinephosphotransferase required Mg2+ and was inhibited by Ca2+, while acetyltransferase required the presence of NaF (a phosphatase inhibitor). The activity of cholinephosphotransferase was similar in unfertilized oocytes and zygotes, and did not change significantly with advancing developmental stage in preimplantation embryos. Acetyltransferase had a significantly lower specific activity (0.078 +/- 0.044 fmol PAF per oocyte per min, mean +/- SEM) in unfertilized oocytes than in zygotes of corresponding age (0.358 +/- 0.097 fmol PAF per zygote per min) (P < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis of platelet-activating factor by two-cell mouse embryos.

Incubation of two-cell mouse embryos with a range of radiolabelled compounds resulted in the incorporation of label into platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in the culture media. The demonstration that known precursors ([1-14C]hexadecanol, [1-3H]hexadecanol, 1-O-[alkyl-1'2'-3H]lyso-PAF, 1-O-[alkyl-1'2'-3H]acetyl-glycerol and [methyl-3H]choline chloride) were incorporated into PAF showed that embryo-derived PAF biosynthesis occurred via pathways present in other PAF-producing cells. The enzyme responsible for the formation of the ether linkage of the PAF molecule, alkyl-dihydroxyacetone-phosphate synthase, was present in the preimplantation embryo as [1-3H]hexadecanol was incorporated into PAF. Incorporation of label from alkylacetyl-glycerol and choline chloride into lyso-PAF was also observed, suggesting a role for lyso-PAF in the metabolism of embryo-derived PAF. Incubation of embryos with each of three [14C]carbohydrate energy substrates resulted in the incorporation of label into PAF in culture media, indicating that the composition of embryo culture media is important in the synthesis of PAF precursors. Incorporation of label from [2-14C]pyruvate was greatest and is consistent with the suggestion that pyruvate is the major energy source at the two-cell stage of development. L-[U-14C]Lactate was also incorporated into embryo-derived PAF, but the mean amount incorporated relative to the concentration of labelled substrate in the medium was 40 times less. The incorporation of D-[U-14C]glucose into PAF was 2405 times less than that from pyruvate, relative to the concentration in the medium.

Structural heterogeneity of platelet-activating factor produced by murine preimplantation embryos.

Platelet-activating factor (PAF) is 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine with the alkyl moiety predominantly a mixture of saturated hexadecyl and octadecyl chains (C16:0 and C18:0 PAF, respectively). Previously, a PAF bioassay was compared with a radioimmunoassay for PAF (NEN Du Pont). Both assays were sensitive and quantitative, but the correlation between PAF measured by the bioassay compared to the radioimmunoassay was poor for murine embryo-derived PAF (r = 0.773, n = 88), while being completely adequate (r = 0.961) for a PAF standard which was an equimolar mixture of C16:0 and C18:0 PAF (C16:0/C18:0 PAF). This study compared a larger sample size of murine embryo-derived PAF (n = 154) and found that the poor correlation between the two assays persisted (r = 0.791). When dose-response curves were generated with C16:0, C16:0/C18:0 and C18:0 PAF (over a concentration range of 0.3-30 ng/ml), the concentrations which gave a 50% response were equivalent in the bioassay (i.e. 6 ng/ml), but differed in the radioimmunoassay (i.e. 1.5, 3 and 6 ng/ml, respectively). Following separation of murine embryo-derived PAF (from medium in which 30 two-cell embryos had been cultured for 24 h) into C16:0 and C18:0 PAF by reverse phase high performance liquid chromatography, 9/20 cultures produced 100% C16:0 PAF, 2/20 cultures produced 100% C18:0 PAF and the remaining 9/20 cultures produced varying proportions of both.(ABSTRACT TRUNCATED AT 250 WORDS)

Ureidoglycolate amidohydrolase from developing French bean fruits (Phaseolus vulgaris [L.].).

Ureidoglycolate is an intermediate of allantoin catabolism in ureide-transporting legumes. This report describes the first purification of ureidoglycolate degrading activity (UGDA) from plant tissue in which the enzyme has been separated from urease. The enzyme from developing fruits of Phaseolus vulgaris has been purified 48-fold to give a preparation free of allantoinase and urease activity. UGDA was inhibited by EDTA while the Vmax was increased in the presence of Mn2+. The Km values for ureidoglycolate in the presence and the absence of Mn2+ were 2.0 and 5.4 mM, respectively. In the absence of Mn2+ UGDA was heat labile at 40 degrees C, but in the presence of Mn2+ the activity was stable up to temperatures of 60 degrees C. The Mr of UGDA was determined to be 300,000 by gel filtration chromatography and the pH optimum ranged from pH 7.0 to 8.5. Ammonia was determined to be the nitrogen-containing product of UGDA by a microdiffusion assay. This enzyme should therefore be described as ureidoglycolate amidohydrolase. The activity was shown to be associated with peroxisomes by fractionation of a crude extract on a sucrose density gradient. The products of ureidoglycolate degradation are glyoxylate, ammonia, and presumably carbon dioxide, which can be readily utilized by pathways of metabolism that are known to be present in this organelle.