Xenobiotic-metabolizing cytochromes P450 convert prostaglandin endoperoxide to hydroxyheptadecatrienoic acid and the mutagen, malondialdehyde.

Cyclooxygenases catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides. Cyclooxygenase-2- and the xenobiotic-metabolizing cytochrome P450s 1A and 3A are all aberrantly expressed during colorectal carcinogenesis. To probe for a role of P450s in prostaglandin endoperoxide metabolism, we studied the 12-hydroxyheptadecatrienoate (HHT)/malondialdehyde (MDA) synthase activity of human liver microsomes and purified P450s. We found that human liver microsomes have HHT/MDA synthase activity that is concentration-dependent and inhibited by the P450 inhibitors, ketoconazole and clotrimazole with IC(50) values of 1 and 0.4 microM, respectively. This activity does not require P450 reductase. HHT/MDA synthase activity was present in purified P450s but not in heme alone or other heme proteins. The catalytic activities of various purified P450s were determined by measuring rates of MDA production from prostaglandin endoperoxide. At 50 microM substrate, the catalytic activities of purified human P450s varied from 10 +/- 1 to 0.62 +/- 0.02 min(-1), 3A4 >> 2E1 > 1A2. Oxabicycloheptane analogs of prostaglandin endoperoxide, U-44069 and U-46619, induced spectral changes in human P450 3A4 with K(s) values of 240 +/- 20 and 130 +/- 10 microM, respectively. These results suggest that co-expression of cyclooxygenase-2 and P450s in developing cancers may contribute to genomic instability due to production of the endogenous mutagen, MDA.

Modulation of endoperoxide product levels and cyclophosphamide-induced injury by glutathione repletion.

Glutathione is a tripeptide important in a number of diverse cellular functions including enzymatic reactions involved in prostaglandin endoperoxide metabolism. We have previously reported that cyclophosphamide administration to rats results in acute lung injury manifested by increased bronchoalveolar lavage albumin concentrations. In the current study we examine whether cyclophosphamide treatment affects pulmonary glutathione stores or bronchoalveolar endoperoxide metabolic product levels and whether these effects may be related to acute lung injury caused by the drug. We show that cyclophosphamide treatment causes a dose-dependent reduction in pulmonary glutathione stores 4 h after drug administration. In addition, acute lung injury as the result of cyclophosphamide can be abrogated by coadministration of oxothiazolidine carboxylate, an intracellular cysteine delivery system that also reverses pulmonary glutathione depletion induced by cyclophosphamide in our study. Finally, cyclophosphamide treatment reduces prostaglandin E2 concentrations in bronchoalveolar lavage and alveolar macrophage culture supernatant in a dose-dependent fashion and increases bronchoalveolar thromboxane concentrations in low dose-treated animals. These effects are reversed to a variable degree by coadministration of oxothiazolidine carboxylate. Our study suggests in vivo pulmonary arachidonic acid metabolism and cyclophosphamide-induced acute lung injury are modulated by cellular glutathione stores. These findings may have important implications for the treatment of acute lung injury.

Activation of aromatic amines by prostaglandin H synthase.

Prostaglandin H synthase catalyzes the first step in the synthesis of prostaglandins from arachidonic acid. The peroxidase activity of this enzyme can support the oxidation of xenobiotics, particularly aromatic amines. This pathway of metabolism may contribute to the activation of carcinogenic aromatic amines in target tissues such as the skin, lung, and bladder. In this review, recent work on this subject is summarized. I emphasize the elucidation of the structures of aromatic amine oxidation products, and their interactions with biological macromolecules. Prostaglandin H synthase supports the activation of benzidine to a mutagenic species in the Ames (Salmonella typhimurium) test, and our studies of the mechanism of this activation are described.

Dihydropyridine agonist Bay K 8644 inhibits platelet activation by competitive antagonism of thromboxane A2-prostaglandin H2 receptor.

The dihydropyridine calcium channel antagonists, such as nifedipine, inhibit platelet aggregation in vitro and ex vivo, but the mechanism by which this occurs is uncertain. Bay K 8644 (BAY) is a substituted dihydropyridine that has effects on voltage-dependent calcium channels in cardiac and smooth muscle that are opposite the effects of nifedipine. To evaluate the mechanism responsible for dihydropyridine-induced inhibition of platelet function, we studied the in vitro effects of BAY on human platelet aggregation and secretion plus several related biochemical parameters, including cytoplasmic ionized calcium ([Ca2+]i). BAY exerted concentration-dependent effects on platelet aggregation and secretion of [14C]serotonin. BAY (1-10 microns) inhibited the second wave of platelet aggregation and secretion stimulated by adenosine diphosphate or epinephrine and blocked shape change, aggregation, and secretion induced by the thromboxane A2 (TXA2) mimic, U46619. BAY also inhibited U46619-induced phosphorylation of the approximately 40,000-dalton cytoplasmic protein substrate of protein kinase C (40K protein), formation of TXA2, and rise in [Ca2+]i, all biochemical consequences of platelet activation. The (+)-(R) enantiomer of BAY [BAY(+)] was predominantly responsible for the inhibitory effects of racemic BAY. Nifedipine had the same inhibitory effects on platelet function and biochemistry, except it was approximately 10 times less potent than BAY. Since these results suggested inhibition of the TXA2-prostaglandin H2 (PGH2) receptor, we measured binding of [3H]U46619 to intact platelets. BAY, BAY(+), and nifedipine all functioned as competitive antagonists of [3H]U46619 binding (BAY Ki = 1.47 microM). They did not inhibit binding of [3H]yohimbine to platelet alpha 2-adrenergic receptors. At 1-10 nM BAY, BAY(+) and the (-)-(S) enantiomer of BAY [BAY(-)] all resulted in slight stimulation of platelet function and biochemical events. No significant increase in [3H]U46619 binding was demonstrable, however. Therefore, dihydropyridines that function as either calcium channel agonists or antagonists in cardiac or smooth muscle exert concentration-dependent effects on platelet function. In nanomolar concentrations, they augment, and in micromolar concentrations, they inhibit platelet activation induced by TXA2 or U46619. These data indicate that dihydropyridines do not inhibit TXA2-induced platelet activation by an effect on voltage-dependent calcium channels; they define the mechanism of inhibition as competitive antagonism of the TXA2-PGH2 receptor. The mechanism responsible for augmentation of platelet activation is uncertain.(ABSTRACT TRUNCATED AT 400 WORDS)

Formation of 6,15-diketoprostaglandin F1 alpha from prostaglandin G2 by bovine aortic endothelial cells.

Besides 6-ketoprostaglandin F1 alpha, bovine aortic endothelial cells also produced considerable amounts of 6,15-diketoprostaglandin F1 alpha from arachidonic acid, either exogenously added or released from cellular phospholipids. Incubations of particulate fractions of endothelial cells with the cyclic endoperoxides prostaglandin G2 and prostaglandin H2 showed that 6,15-diketoprostaglandin F1 alpha is formed by the action of prostaglandin I2 synthetase on prostaglandin G2. The labile metabolite 15-hydroperoxyprostaglandin I2 is then converted nonenzymatically to the 15-keto derivative. In the presence of reduced glutathione, quantitative analysis of both metabolites by gas chromatography-mass spectrometry showed a significant decrease of 6,15-diketoprostaglandin F1 alpha formation, whereas prostaglandin I2 synthesis was markedly increased. This shift seems to be due to a stimulation of peroxidase by GSH, a well known cofactor of this enzyme. Thus, it seems that a decreased endothelial prostaglandin I2 formation may occur when cellular glutathione levels are reduced as a consequence of oxidant injury and lipid peroxidation. Additionally, ferrous ions seems to be involved in the regulation of endothelial prostaglandin I2 synthesis, since Desferal, a specific ferrous ion chelator that might have antimetastatic properties, produced a pronounced shift from 6,15-diketoprostaglandin F1 alpha to the 6-keto derivative, i.e., prostaglandin I2.

Macrophage eicosanoid formation is stimulated by platelet arachidonic acid and prostaglandin endoperoxide transfer.

The present study was designed to determine whether platelets transfer arachidonic acid or prostaglandin endoperoxide intermediates to macrophages which may be further metabolized into cyclooxygenase products. Adherent peritoneal macrophages were prepared from rats fed either a control diet or an essential fatty acid-deficient diet, and incubated with a suspension of washed rat platelets. Macrophage cyclooxygenase metabolism was inhibited by aspirin. In the presence of a thromboxane synthetase inhibitor, 7-(1-imidazolyl)heptanoic acid, immunoreactive 6-ketoprostaglandin F1 alpha formation was significantly increased 3-fold. Since this increase was greater (P less than 0.01) than that seen with either 7-(1-imidazolyl)heptanoic acid-treated platelets or aspirin-treated macrophages alone, these results indicate that shunting of endoperoxide from platelets to macrophages may have occurred. In further experiments, macrophages from essential fatty acid-deficient rats were substituted for normal macrophages. Essential fatty acid-deficient macrophages, depleted of arachidonic acid, produced only 2% of the amount of eicosanoids compared to macrophages from control rats. When platelets were exposed to aspirin, stimulated with thrombin, and added to essential fatty acid-deficient macrophages, significantly more immunoreactive 6-ketoprostaglandin F1 alpha was formed than in the absence of platelets. This increased macrophage immunoreactive 6-ketoprostaglandin F1 alpha synthesis, therefore, must have occurred from platelet-derived arachidonic acid. These data indicate that in vitro, in the presence of an inhibition of thromboxane synthetase, prostaglandin endoperoxides, as well as arachidonic acid, may be transferred between these two cell types.

Rat lens prostaglandin generation proceeds by the non-enzymatic degradation of PGH2 endoperoxide.

Experiments were performed using [1-14C]PGH2 endoperoxide as substrate to assess which, if any, of the PGH2 utilizing enzymes were present in rat lens microsomes. After a 15 min incubation, prostaglandins (PGs) were extracted, separated by thin-layer chromatography and labeled PGs located by autoradiography. The bands were cut and counted and product formation expressed as a percentage of the total recovered label. Prostaglandin PGE2 formation accounted for 24% of the label and was not increased (22%) when 1 mM reduced glutathione (GSH), the obligate cofactor of PGH2----PGE2 isomerase, was added. However, the addition of the GSH significantly increased PGF2 alpha generation from 25% to 37%. Pretreatment of the microsomes with p-hydroxymercuribenzoate, a sulfhydryl-blocking agent and an inhibitor of PGH2----PGE2 isomerase, did not limit lens microsomal PGE2 formation (24% vs. 39%) but dramatically inhibited PGF2 alpha production (25% vs. 6%). Boiling the microsomes did not alter PGF2 alpha or PGE2 generation suggesting that these PGs were non-enzymatically produced. No thromboxane B2 or 6-keto-PGF1 alpha (the stable breakdown products of thromboxane A2 and prostacyclin, respectively) were found indicating that their respective synthetases were absent in the lens microsomes. These data suggest that the lens is a unique tissue, generating the primary PGs in the absence of all known PGH2 endoperoxide-utilizing enzymes. It appears that the PGH2 formed by lens cyclo-oxygenase spontaneously breaks down in the lens microsomes into PGE2, PGD2 and PGF2 alpha.

Overview of physiological and pathophysiological effects of thromboxane A2.

Thromboxane (Tx) A2 is a biologically potent and chemically unstable metabolite of prostaglandin endoperoxides. Recent developments in measurement techniques and the availability of both selective inhibitors of Tx synthetase and TxA2 receptor antagonists have facilitated the implication of TxA2 as a physiological modulator and as a mediator in thrombotic, vasospastic, and bronchospastic conditions. TxA2 is synthesized by platelets and contributes to platelet activation and irreversible platelet aggregation in physiological hemostasis and in thrombosis (e.g., unstable angina, stroke). TxA2 is also synthesized in intestinal, pulmonary, and renal tissues by cells other than platelets. Particularly in these tissues, TxA2 appears to act as a physiological modulator of changes in blood flow distribution and airway caliber. Strong stimuli for TxA2 release from these tissues may initiate ulcer, pulmonary hypertension, bronchoconstriction, and renal vasoconstriction. Evidence supports participation of TxA2 and/or TxA2 receptors in modulation of natural cytotoxic cell cytotoxicity, in tumor growth and metastasis, in complications of pregnancy (e.g., preeclampsia), and in the progression of ischemic injury after coronary artery occlusion. This evidence supports pivotal involvement of TxA2 in pathophysiology and provides a strong rationale for pursuing TxA2-blocking strategies in drug development.

Chick retinal pigment epithelium exhibits glutathione requiring prostaglandin D2 synthetase activity.

Prostaglandin synthetic activity of neural retina and retinal pigment epithelium in chick retina was studied using arachidonic acid and prostaglandin H2 (intermediate prostaglandin endoperoxide) as substrates. To obtain sufficient enzyme activity in retinal pigment epithelium, cultured chick retinal pigment epithelial cells were used. Little arachidonic acid was converted into prostaglandins, and details of prostaglandin synthetic activity were not clear. The assay from prostaglandin H2 showed that: 1) preparations consisting of both neural retina and retinal pigment epithelium dissected from 7-day-old chicks have activity of glutathione-requiring prostaglandin D2 synthetase; 2) preparations consisting of only neural retina dissected from 7-day-old chicks have no activity of prostaglandin synthetase; and 3) cultured chick retinal pigment epithelial cells have high activity of glutathione-requiring prostaglandin D2 synthetase.

Prostaglandins and human platelet aggregation. Implications for the anti-aggregating activity of thromboxane-synthase inhibitors.

Selective pharmacological blockade of thromboxane-synthase in human platelets by dazoxiben resulted in the reorientation of cyclic-endoperoxides towards PGE2, PGD2 and PGF2 alpha. At concentrations which can be reached when thromboxane-synthase is inhibited, PGE2 (100-500 nM) exerted a marked, concentration-dependent pro-aggregatory effect. This required the formation of endogenous or the addition of exogenous endoperoxides and was prevented by PGD2 or 13-aza-prostanoic acid, a selective antagonist of PGH2/TxA2 receptors. The anti-aggregating effect of PGD2 was evident at concentrations lower than those obtained in dazoxiben-treated platelets. It is proposed that in the absence of TxA2 generation, a combination of endoperoxides and PGE2 may result in normal aggregation. The latter may be inhibited by PGD2. No interference of PGF2 alpha on platelet function could be shown.

Prostaglandin H synthase-catalyzed activation of benzidine: a model to assess pharmacologic intervention of the initiation of chemical carcinogenesis.

Carcinogens which cause cancers in tissues distal to their entry are thought to require metabolic activation before covalent binding to macromolecules. The hydroperoxidase component of prostaglandin H synthase (PHS) activates certain carcinogens and a model describing this process is presented. The procarcinogen benzidine was used to identify sites at which microsomal PHS-catalyzed binding might be inhibited by pharmacologic agents. Activation of benzidine was determined by assessing free radical cation formation and covalent binding to protein. Reduction of benzidine diimine to diamine was also assessed. This study provides the first demonstration of inhibition of PHS-activated benzidine binding by propylthiouracil, methimazole, MK447, vitamin C and phenidone. The agents tested identified the following sites at which PHS-catalyzed binding of benzidine can be prevented: 1) inhibition of generation of the peroxide cosubstrate for benzidine oxidation; 2) inhibition of prostaglandin hydroperoxidase; 3) reduction of oxidized intermediate(s) to the parent compound; and 4) conjugation of the activated intermediate(s). This study provides a basis for further investigations of the pharmacologic intervention of chemical carcinogenesis.

Comparative effects of prostaglandin H synthase-catalyzed binding of two 5-nitrofuran urinary bladder carcinogens.

Understanding the role of prostaglandin H synthase (PHS) in the carcinogenic process and the metabolic steps involved in the activation of carcinogens will facilitate experiments using pharmacological agents to prevent carcinogenesis. This study assesses the relative amounts of PHS-catalyzed binding of the urinary tract carcinogens [2-14C]-N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) and [2-14C]-2-amino-4-(5-nitro-2-furyl) thiazole (ANFT). Binding to protein and nucleic acid was assessed using PHS prepared from ram seminal vesicle, dog bladder transitional epithelium and rabbit renal inner medulla. PHS-catalyzed binding of ANFT was significantly greater than FANFT in each tissue. Substrate and inhibitor experiments were consistent with the prostaglandin hydroperoxidase activity of PHS catalyzing the binding of FANFT and ANFT. Oxygen was required for metabolism with arachidonic acid but not with peroxide as cosubstrate. The amount of PHS-catalyzed ANFT binding to protein was at least 4-fold greater than FANFT. Whereas a significant amount of FANFT was bound to protein, no FANFT binding to DNA could be detected. By contrast, PHS catalyzed the binding of ANFT to both protein and DNA. A PHS-catalyzed metabolite of ANFT was tentatively identified as the 4-keto analog by mass spectral analysis. The lower rate of PHS-catalyzed metabolism of FANFT compared to ANFT and the lack of detectable FANFT binding to DNA suggest that the metabolic steps involved in the initiation of FANFT-induced bladder cancer include 1) deformylation of FANFT to ANFT, 2) PHS-catalyzed activation of ANFT and 3) binding of an activated ANFT metabolite(s) to DNA.

Reorientation of prostaglandin endoperoxide metabolism by a thromboxane synthetase inhibitor: in vitro and clinical observations.

1 Work with dazoxiben in vitro and in vivo suggests that a diminished capacity of platelets to synthesize thromboxane A2 results in a reorientation of the metabolism of cyclic endoperoxides. 2 Platelets of patients with congenital thromboxane synthetase deficiency show the same phenomenon. 3 In the presence of endothelium or leukocytes which have prostacyclin synthetase capacity, significant amounts of prostacyclin can be generated if thromboxane synthesis is blocked. 4 The local generation of aggregation inhibiting prostaglandins in areas of vascular damage may be an interesting therapeutic concept. 5 Pilot clinical studies of thromboxane synthetase inhibitor dazoxiben (UK 37248) in a small number of patients with peripheral arterial disease did not reveal major or consistent haemodynamic changes. 6 Attention is directed towards optimizing pharmacologically the generation and efficacy of the inhibitory prostaglandins produced when thromboxane synthetase is inhibited.

Prostaglandin hydroperoxidase-mediated 2-amino-4-(5-nitro-2-furyl) [14C]thiazole metabolism and nucleic acid binding.

Prostaglandin hydroperoxide-mediated metabolism and binding of 2-amino-4-(5-nitro-2-furyl) [14C]thiazole ([14C]ANFT) metabolite to nucleic acids and proteins were investigated with rabbit bladder transitional epithelial and solubilized ram seminal vesicle microsomes. Metabolism was assessed by spectrophotometric and radiochemical techniques. Substrate and inhibitor studies are consistent with both metabolism and binding of [14C]ANFT occurring by the prostaglandin hydroperoxidase activity of prostaglandin endoperoxide synthetase. The ratio of the rates of [14C]ANFT product formation is approximately 3:7:10 (organic soluble:non-trichloroacetic acid precipitable: trichloroacetic acid precipitable) over a wide range of arachidonic acid concentrations. Approximately 2 and 1% of the total [14C]ANFT metabolized binds to transfer RNA and DNA, respectively. The metabolite isolated from the organic phase had a chromatographic profile and ultraviolet spectra different from authentic ANFT. If transfer RNA or DNA is added at the end of a 5-min incubation, no binding to nucleic acids was observed. The demonstration of prostaglandin hydroperoxidase-mediated covalent binding to nucleic acids is consistent with the involvement of this enzyme in 5-nitrofuran-induced bladder carcinogenesis.

Familial bleeding tendency with partial platelet thromboxane synthetase deficiency: reorientation of cyclic endoperoxide metabolism.

Three family members from three successive generations presented with a moderate bleeding tendency and a functional platelet defect. They had absent aggregation with arachidonic acid (0.6--3 microM), reversible aggregation with ADP (4 microgram) and cyclic endoperoxide analogues, single wave aggregation only with adrenaline (5.4 microgram) and a prolonged template bleeding time (> min). Malondialdehyde formation was reduced after N-ethylmaleimide stimulation (2--6 nmol/10(9) platelets; control values 8--12 nmol) and serum thromboxane B2 values were reduced (33--101 ng/ml; control values 200--700 ng/ml). When the platelets were incubated with [3H]arachidonic acid the final metabolite of the lipoxygenase pathway (HETE) was produced in normal amounts but the production of thromboxane B2 and HHT was decreased whereas prostaglandin F2a, and E2 and probably D2 were increased. Evidence for enhanced production of prostaglandin D2 was also provided by the rise in the patient's platelet cyclic AMP levels following stimulation with arachidonic acid. The patient's washed platelets stimulated the production of 6-keto PGF 1a by aspirin-pretreated cultured bovine endothelial cells. The plasma levels of 6-keto PGF1a (439--703 pg/ml; normal 181 +/- 46 pg/ml) were raised. The decreased production of thromboxane B2, HHT and malondialdehyde and increased formation of prostaglandin F2a, E2, D2 and of 6-keto PGF1a are compatible with a partial platelet thromboxane synthetase deficiency and reorientation of cyclic endoperoxide metabolism. The markedly prolonged bleeding time would result not only from reduced formation of thromboxane A2 but also from increased production of the aggregation inhibiting prostaglandins PGI2 and PGD2.

Transformations of prostaglandin H2 in the cat brain.

Transformations of prostaglandin H2 were studied in the whole homogenate and subcellular fractions (microsomes, high-speed supernatant) of the cat brain. Prostaglandin H2 was converted enzymatically to prostaglandin E2, 6-ketoprostaglandin F 1 alpha, and thromboxane B2. While prostaglandin E2 and thromboxane B2 predominated, respectively, in the whole homogenate and the microsomes, 6-ketoprostaglandin F1 alpha was formed in both tissue preparations. Pretreatment of the microsomes with reduced glutathione stimulated the formation of prostaglandin E2 at the expense of 6-ketoprostaglandin F1 alpha and thromboxane B2. Imidazole and 15-hydroperoxyarachidonic acid interfered with the microsome-induced transformations of prostaglandin H2 to thromboxane B2 and 6-ketoprostaglandin F1 alpha, respectively. No prostaglandin D2 was detected even after treatment of the tissue with reduced glutathione. Prostaglandin H2, on the other hand, was converted to prostaglandin F2 alpha, but the yield of this compound in native tissue was equal to, or lower than, that in boiled tissue. We conclude that the feline brain is endowed with several enzymes (prostaglandin E2 isomerase, thromboxane A2 and prostaglandin I2 synthetases) competing for the added endoperoxide. All enzymes were located in the microsomes. No evidence was obtained indicating enzymic reduction of prostaglandin H2 to prostaglandin F2 alpha.

Regulation of pulmonary arachidonic acid metabolism by anti-inflammatory steroids.

Enzymes in cells from many tissues including the lung metabolize arachidonic acid to a variety of highly active local hormones such as prostaglandins and 'slow-reacting substances'. Many of these play a part in the inflammatory response which follows injury or trauma or, in the case of slow-reacting substance, asthmatic bronchoconstriction. Both non-steroidal and steroidal anti-inflammatory drugs inhibit the formation of some or all of these products. The non-steroidal drugs block prostaglandin formation but not the formation of products such as slow-reacting substances. The steroids block the formation of all products. Their mechanism of action, investigated in the guinea-pig perfused lung, involved inhibition of arachidonic acid liberation, probably by suppression of phospholipase activity. To achieve this steroids must first bind to receptors in lung tissue and initiate de novo RNA and protein synthesis. This culminates in the synthesis or secretion by some cells in the lung of a polypeptide of mol.wt. 10 000-15 000 with potent anti-phospholipase properties. The generation of this factor could partly explain why steroids are so effective in the treatment of many types of inflammatory disease, and in particular why they are so efficacious against asthma.

Is a slow reacting substance-like compound involved in rat platelet agglutination.

Endoperoxide analogs at doses 100-fold higher than those required to aggregate human platelet-rich plasma (PRP), were ineffective on rat PRP. Indomethacin (20 microM) and imidazole did not affect arachidonate-induced aggregation of rat PRP. On the other hand, prostaglandin (PG) E1 inhibited aggregation at doses similar to those effective on human PRP, while high doses of PGI2 failed to inhibit arachidonate aggregation in the rat. Eicosatetraynoic acid (10 microgram) blocked the second phase of aggregation; the Ca2+-ionophore A 23187 potentiated the arachidonate effect. Thus, it appears that endoperoxides or thromboxane A2 may not be involved in rat platelet aggregation and that the formation of aggregants from arachidonate shares many properties with the biosynthesis of slow reacting substance, a metabolite of arachidonic acid containing a sulphate group. To test this, rat PRP was incubated with labeled sulphate and aggregated with arachidonate. After column and thin layer chromatography a labeled lipid was identified having a mobility higher than phospholipids but lower than PGF2 alpha. Treatment with arylsulphatase decreased radioactivity by at least 70%.