Immunomodulation following zinc supplementation during chelation of lead in male rats

Influence of zinc supplementation (30 and 45 mg kg^–1, orally once for 5 days) during chelation of lead (0.3 mmol kg^–1, chelating agent, i.p., once for 5 days) on some selected variables of the immune system was investigated in male rats. Treatment with CaNa₂EDTA either alone or in combination with zinc (30 mg kg^–1) produced a significant recovery in lead induced alteration in primary antibody forming cells to T-dependent antigen and the delayed-type hypersensitivity response to bovine albumin. However, biologically significant recovery was observed only with zinc at a dose of 45 mg kg¹. It is assumed that zinc depletion during lead exposure and chelation treatment lead to harmful effects on cellular proliferation by inhibiting DNA synthesis and various enzymes during mitosis. The zinc supplementation fulfills this requirement during proliferation and clonal expansion of immunocompetent cells augmenting the immune system.     

Chelation in metal intoxication XXVI

The effect of pretreatment and simultaneous treatment with thiamine on therapeutic efficacy of calcium disodium edetate (CaNa₂EDTA) in lead intoxication was investigated in rats. The animals exposed to Pb as Pb (CH₃COO)₂·3 H₂O through drinking water (0.1%) for 8 wk were treated with either saline, thiamine-HCl (sc), CaNa₂EDTA (ip), or thiamine-HCl plus CaNa₂EDTA, for 3 d or thiamine-HCl for 3 d followed by thiamine, then HCl plus CaNa₂EDTA for a further 3 d. The Pb exposure caused significant accumulation of Pb in liver, kideny, and brain, inhibition in the activity of blood δ-amino-levulinic acid dehydratase (δ-ALAD), and increase in levels of urinary δ-aminolevulinic acid, homovanillic acid (HVA), vanillyl mandelic acid (VMA), brain HVA and VMA. The brain δ-ALAD and lipomide dehydrogenase remained unaffected by Pb. Thiamine significantly enhanced the urinary excretion of Pb by CaNa₂EDTA, but only marginally influenced the efficacy of CaNa₂EDTA to either mobilize tissue Pb or reverse the biochemical alterations.     

The effects of triethyl lead on the development of hippocampal neurons in culture

Triethyl lead is the major metabolite of tetraethyl lead, which is used in industrial processes and as an antiknock additive to gasoline. We tested the hypothesis that low levels of triethyl lead (0.1 nmol/L to 5mol/L) interfere with the normal development of cultured E18 rat hippocampal neurons, possibly through increases in intracellular free calcium ion concentration, [Ca²⁺]_in. The study assessed survival and differentiation using morphometric analysis of individual neurons. We also looked at short-term (up to 3.75-h) changes in intracellular calcium using the calcium-sensitive dye fura-2. Survival of neurons was significantly reduced at 5 mol/L, and overall production of neurites was reduced at 2 mol/L. The length of axons and the number of axons and dendrites were reduced at 1 mol/L. Neurite branching was inhibited at 10 nmol/L for dendrites and 100 nmol/L for axons. Increases in intracellular calcium were observed during a 3.75-h exposure of newly plated neurons to 5 mol/L triethyl lead. These increases were prevented by BAPTA-AM; which clamps [Ca²⁺]_in at about 100 nmol/L. Culturing neurons with BAPTA-AM and 5 mol/L triethyl lead did not reverse the effects of triethyl lead, suggesting that elevation of [Ca²⁺]_in is not responsible for decreases in survival and neurite production. Triethyl lead has been shown to disrupt cytoskeletal elements, particularly neurofilaments, at very low levels, suggesting a possible mechanism for its inhibition of neurite branching at nanomolar concentrations.Abbreviations BAPTA-AM 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester - [Ca²⁺]_in intracellular free calcium ion concentration - DMSO dimethyl sulfoxide - E18 embryonic day 18 - FBS fetal bovine serum - fura-2AM fura-2 acetoxymethyl ester - HBSS Hanks' Balanced Salt Solution - MEM Eagle's Minimum Essential Medium     

A method for studying the cellular location of lead in lichens

Abstract: A number of possible displacing agents have been tested for their ability to release extracellular bound lead from Cladonia portentosa. Metal cations (Ni and Cu), thiol-rich reagents (glutathione, ammonium pyrrolidinedithiocarbamate) and some chelating agents (EGTA, EDTA at pH 9·5 or pH 2·7) were ineffective or caused membrane damage. Disodium EDTA at pH 4·5 was found to be an effective displacing agent, although the Na caused some release of intracellular K without membrane damage. This displacement procedure showed that no Pb supplied in the laboratory entered the protoplast in 2 h. The effect of Ca, Cu, Pb, and Sr on membrane integrity was investigated.     

The effect of EDTA and EDDS on lead uptake and localization in hydroponically grown Pisum sativum L.

Pisum sativum plants were treated for 3 days with an aqueous solution of 100 μM Pb(NO₃)₂ or with a mixture of lead nitrate and ethylenediaminetetraacetic acid (EDTA) or [S,S]-ethylenediaminedisuccinic acid (EDDS) at equimolar concentrations. Lead decline from the incubation media and its accumulation and localization at the morphological and ultrastructural levels as well as plant growth parameters (root growth, root and shoot dry weight) were estimated after 1 and 3 days of treatment. The tested chelators, especially EDTA, significantly diminished Pb uptake by plants as compared to the lead nitrate-treated material. Simultaneously, EDTA significantly enhanced Pb translocation from roots to shoots. In the presence of both chelates, plant growth parameters remained considerably higher than in the case of uncomplexed Pb. Considerable differences between the tested chelators were visible in Pb localization both at the morphological and ultrastructural level. In Pb+EDTA-treated roots, lead was mainly located in the apical parts, while in Pb+EDDS-exposed material Pb was evenly distributed along the whole root length. Transmission electron microscopy and EDS analysis revealed that in meristematic cells of the roots incubated in Pb+EDTA, large electron-dense lead deposits were located in vacuoles and small granules were rarely noticed in cell walls or cytoplasm, while after Pb+EDDS treatment metal deposits were restricted to the border between plasmalemma and cell wall. Such results imply different ways of transport of those complexed Pb forms.     

Different effects of inorganic and triethyl lead on growth and ultrastructure of lily pollen tubes

Summary Pollen tubes ofLilium longiflorum growingin vitro were treated for 1 h with inorganic lead (Pb) and with triethyl lead (TriEL) and studied by light and electron microscopy. Pb was considerably more toxic in relation to inhibition of pollen tube growth (EC₅₀=6 M Pb) than was TriEL (EC₅₀=60 M TriEL). On the other hand, at almost the entire concentration range tested (25-500 M) TriEL caused aberrant tubes and tube swellings. Pb did not cause tube swellings, even at highly growth-impairing concentrations. Pb (60 M) predominantly affected the ultrastructure of the growing cell walls without impairing the distribution of the cell organelles in the tube tips. In contrast, 50 and 100 M TriEL did not visibly influence cell wall ultrastructure but it severely damaged dictyosomes; 100 M TriEL also disturbed the original order of cell organelles in the tube tips. Cortical microtubules were selectively and completely destructed by TriEL at concentrations (50 M) where no effect on polar organization of the tube tips occurred but they remained unimpaired by 60 M Pb, indicating selective and effective interaction of TriEL with these cell organelles.Abbreviations EC⁵⁰ effective lead concentration causing 50% inhibition of pollen tube growth - MTs microtubules - Pb inorganic lead - TriAL trialkyl lead - TriEL triethyl lead     

Isolated epithelial cells of the toad bladder. Their preparation, oxygen consumption, and electrolyte content

Epithelial cells of the toad bladder were disaggregated with EDTA, trypsin, hyaluronidase, or collagenase and were then scraped free of the underlying connective tissue. In most experiments EDTA was complexed with a divalent cation before the tissue was scraped. Q _OO2, sucrose and inulin spaces, and electrolytes of the isolated cells were measured. Cells disaggregated by collagenase or hyaluronidase consumed O₂ at a rate of 4 µl hr^-1 dry wt^-1. Q _OO2 was increased 50% by ADH (100 U/liter) or by cyclic 3'',5''-AMP (10 mM/liter). Na⁺-free Ringer''s depressed the Q _OO2 by 40%. The Q _OO2 of cells prepared by trypsin treatment or by two EDTA methods was depressed by Na⁺-free Ringer''s but was stimulated relatively little by ADH. Two other EDTA protocols produced cells that did not respond to Na⁺ lack or ADH. The intracellular Na⁺ and K⁺ concentrations of collagenase-disaggregated cells were 32 and 117 mEq/kg cell H₂O, respectively. Cation concentrations of hyaluronidase cells were similar, but cells that did not respond to ADH had higher intracellular Na⁺ concentrations. Cells unresponsive to ADH and Na⁺ lack had high sucrose spaces and low transcellular membrane gradients of Na⁺, K⁺, and Cl^-. The results suggest that trypsin and EDTA disaggregation damage the active Na⁺ transport system of the isolated cell. Certain EDTA techniques may also produce a general increase in permeability. Collagenase and hyaluronidase cells appear to function normally.     

The interaction between calcium and the activation of Na+, K+-ATPase by noradrenaline

Summary The interaction of noradrenaline, various cation chelators and calcium on Na⁺, K⁺-ATPase from rat cerebral cortex plasma membranes was studied. It was shown that chelation of inhibitory cations by EGTA, EDTA and dipyridyl activated Na⁺, K⁺-ATPase to the same extent as noradrenaline but at higher concentrations; increasing concentrations of EGTA depressed the activation by noradrenaline; calcium in the form of a calcium-EGTA buffer depressed Na⁺, K⁺-ATPase at physiological concentrations; the inhibition of Na⁺, K⁺-ATPase by calcium is dependent on the magnesium concentration in the assay and the inhibition by calcium was partially reversed by noradrenaline.     

Organic and inogranic lead inhibit neurite growth in vertebrate and invertebrate neurons in culture

Summary Neurons from brains of chick embryos and pond snails (Lymnaea stagnalis) were cultured for 3 to 4 d in the presence of no toxins, inorganic lead (PbCl₂), or organic lead (trielthyl lead chloride). In chick neurons, inorganic lead reduced the percentage of cells that grew neurites (IC₅₀=270μM total lead, approximately 70 nM free Pb²⁺) but did not reduce the number of neurites per cell or the mean neurite length. Triethyl lead reduced the percentage of cells that grew neuites (IC₅₀=0.24 μM) and the mean neurite length (extrapolated IC₅₀=3.6 μM) but did not reduce the number of neurites per cell. InLymnaea neurons, inorganic lead reduced the percentage of cells that grew neurites (IC₅₀=13 μM total lead; approximately 10 nM free Pb²⁺). Triethyl lead reduced the percentage of cells that grew neurites (IC₅₀=0.4 μM) and exerted significant toxicity at 0.2 μM. The two forms of lead affected neurite growth in qualitatively different ways, which suggests that their mechansms of action are different. These experiments were supported by grants from the Environmental Protection Agency, Washington, DC, and the National Institutes of Environmental Health Science, Research Triangle Park, NC.     

重金属铅与两种淡水藻的相互作用

为了研究重金属铅与淡水藻类之间的相互作用,采用不同Pb2+浓度处理铜绿微囊藻(Microcysis aeruginosa Kutz.)和斜生栅藻[Scenedesmus obliquus(Turp.)Kutz.],分别对两种藻的生物量、藻液电导率、O-·2含量、过氧化物酶(POD)、过氧化氢酶(CAT)活性以及藻对Pb2+的吸收作用等进行了测定,并通过扫描电镜观察了不同Pb2+浓度处理下两种藻细胞的表面结构。结果显示:(1)Pb2+浓度低于3 mg/L促进铜绿微囊藻生长,高于9 mg/L抑制其生长;但在3—12 mg/L范围内,Pb2+均明显抑制了斜生栅藻的生长,说明斜生栅藻对Pb2+毒性的敏感程度要高于铜绿微囊藻。(2)受到铅离子的胁迫,两种藻细胞膜通透性均有一定改变,扫描电子显微镜的照片观察,两种藻细胞表面的絮状物随着Pb2+的升高而增多,尤其是斜生栅藻细胞结构改变明显,多数细胞变形破裂;同时,O-·2含量升高,POD、CAT活性早期均可随Pb2+的增加而上升,表明氧自由基的产生增多以及由其引起的细胞生理生化改变可能是铅离子作用于藻细胞的主要机制。(3)两种淡水藻对Pb2+均有吸收作用,单位量藻细胞内,斜生栅藻对Pb2+的吸收能力好于铜绿微囊藻。所有结果提示:斜生栅藻不仅可以作为对重金属敏感的指示生物来监测水体Pb2+污染程度;同时由于斜生栅藻比铜绿微囊藻具有更好的Pb2+吸收能力,因此还可以利用斜生栅藻作为处理水体Pb2+的生物材料。     

Experimental production of hypocalcemia by EDTA infusion in calves: a critical appraisal assessed from the profile of blood chemicals and enzymes

Physiological studies of the effects of Ca²⁺ withdrawal using Na₂EDTA have been conducted with the prior basic assumption that Na₂EDTA-specific direct or indirect effects on the functions under study were negligible. The present study aimed at providing unequivocal confirmation of such assumption by establishing the pattern of response of blood constituents to intravenous infusions of Na₂EDTA in calves. Na₂EDTA infusion in calves allowed effective chelation of blood Ca²⁺, leading to a progressive hypocalcemia. Magnesium levels remained constant and concentrations of other ions (Na⁺, K⁺, Pi, H⁺, HCO₃⁻), although significantly altered (P ≤ 0.0001), remained within the normal range. Comparison of enzymes, urea, and creatinine changes precluded renal, hepatic, or muscular parenchymatous damages as being the cause of dysfunctions in the context of Na₂EDTA-induced hypocalcemia. It was not possible, however, to standardize the Na₂EDTA infusion characteristics (flow, volume) to obtain previsible Ca²⁺ decay in different animals. Conversely, monitoring of systemic arterial pressure (SAP) offered a precious tool to estimate the degree of hypocalcemia reached. Infusion rate must, therefore, be manipulated using careful on-line monitoring of SAP to obtain an experimental range of Ca²⁺ as large as possible. It was concluded that physiological data collected during Na₂EDTA perfusions can be reliably discussed in terms of Ca²⁺ dependence rather than in terms of either Na₂EDTA toxicity, electrolytes maladjustment, acid-base imbalance, impaired blood oxygenation, or hepatic, renal, myocardic, or skeletal muscle damages.     

Comparison of the effectiveness of 2,3-dimercaptopropanol (BAL) andmeso-2,3-dimercaptosuccinic acid (DMSA) as protective agents against mercuric chloride-induced nephrotoxicity in rats

The effectiveness of 2,3-dimercaptopropanol (BAL) andmeso-2,3-dimercaptosuccinic acid (DMSA) on HgCl₂-induced nephrotoxicity was studied in the rat. Seven groups of adult male rats were given a single sc toxic dose of HgCl₂ (0.68 mg/kg) followed by 0.9% saline (positive control group), BAL (15, 30, and 60 mg/kg) or DMSA (50, 100, and 200 mg/kg) administered ip at 0, 24, 48, and 72 h thereafter. Although the renal function of HgCl₂-exposed rats was slightly improved after BAL administration, Hg concentrations in the kidney were only reduced at 60 mg/kg. In addition, the protective effect of BAL was not dose-related. In contrast to BAL, DMSA was effective in increasing the urinary excretion of Hg and in reducing the renal Hg content. These results show that DMSA would be more effective than BAL in preventing or in protecting against inorganic Hg-induced nephrotoxicity.     

Inhibition of Spore Outgrowth and Vegetative Growth of Bacillus stearothermophilus by Dipicolinate,

Spores of Bacillus stearothermophilus germinated in the presence of sodium dipicolinate (DPA) but did not elongate after the emergence stage of outgrowth. Vegetative growth also was inhibited by Na₂DPA.     

Differential effects of metal chelators on Na plus, K plus-ATPase activity

Concentrations of EDTA greater than 1 mm inhibited the enzymatic activity of a Na⁺, K⁺-ATPase preparation isolated from beef brain by competing with ATP for Mg²⁺. EGTA, which does not chelate Mg²⁺ significantly at pH 7.4, had no such effect. Low concentrations of either EDTA or EGTA stimulated enzymatic activity. This effect was maximal at 5–6 μm of chelator. Stimulation of the reaction was observed only if the chelator was added to the incubation medium before initiation of the reaction by the addition of enzyme. If low concentrations of the chelators were added after enzyme, i.e., after the turnover cycle had begun, no effect was seen.     

The divalent cation dependence of liver glycogen synthase phosphatase activity

Summary In a previous report it was shown that EDTA inhibition of liver glycogen synthase phosphatase activity in preparations from normal, fed rats could be increased upon glucagon or cAMP treatment. This occurred without a change in the half-maximum inhibitory concentration of EDTA. Glucose administration to animals resulted in decreased EDTA inhibition. The inhibitory action of EDTA has been further characterized by comparing its action with that of other chelators (CDTA and EGTA) and examining the effects of various divalent cations on chelator inhibition. Both CDTA and EDTA which differ structurally were inhibitory at 5 mm concentrations whereas EGTA which is structurally similar to EDTA was not inhibitory at concentrations up to 10 mm. The lack of inhibition by EGTA could be explained by its weak affinity for Mg⁺⁺ in the preparation. A comparison of CDTA and EDTA revealed that CDTA was a more potent inhibitor than EDTA (I_0.5, 0.15 mm vs 0.3 mm). Glucagon and glucose treatment of rats resulted in changes in CDTA inhibition which closely paralleled those of EDTA. A large group of divalent cations were tested but only Mg⁺⁺, Ca⁺⁺, and Mn⁺⁺ both prevented and reversed CDTA or EDTA inhibition. Fifty percent reversal using either chelator occurred at calculated free-metal ion concentrations of approximately 2 µm, 0.08 µm and 0.0004 µm, respectively. Thus, it is clear that EDTA inhibition is due to its chelation effect and is not due to a nonspecific anionic effect.     

Optimization of Ionic and Chelated Iron and Its Interaction with Disodium Ethylenediaminetetraacetic Acid for Enhancement of Plant Regeneration in Rice (Oryza sativa L)

Interactive effects of Fe₂(SO₄)3 and Na₂EDTA on callus induction and plant regeneration in indica rice (Oryza sativa cv Pusa Basmati-1) was investigated. Callus induction and subsequent plant regeneration from seed was obtained on MS medium supplemented with 11.31 µM 2,4-D and 2.68 µM NAA + 8.87 µM BAP respectively. Both the callus induction and the plant regeneration media were supplemented with different levels of Fe₂(SO₄)₃, Na₂EDTA and their combinations. Callus induction, its morphogenic potential, average number of regenerated plants as well as the appearance of the regenerants was influenced by the levels of Fe₂(SO₄)₃ and Na₂EDTA. Embryogenic callus could be induced in the presence of Fe₂(SO₄)₃ / Na₂EDTA. Iron was essential for plant regeneration. Non-chelated iron could induce callus formation as well as plant regeneration, yet the chelated form was more suitable. A higher level of Na₂EDTA in the regeneration medium was detrimental. Differential requirement of Fe₂(SO₄)₃ at induction and regeneration level was observed. Method of medium preparation affected the regeneration response. Nearly three fold increase in the number of regenerated plants was achieved with 0.05 mM Fe₂(SO₄)₃ + 0.1 mM Na₂EDTA at callus induction and 0.1 mM Fe₂(SO₄)₃+ 0.1 mM Na₂EDTA at plant regeneration and standard method of autoclaving.     

Hemmung der Cytokinese und Bildung von Riesenzellen beiPoterioochromonas malhamensis durch organische Bleiverbindungen und andere Agenzien

Summary Organic lead compounds inhibit cytokinesis of the chrysophycean flagellatePoterioochromonas malhamensis leading to giant, multinucleate cells. This action on cytokinesis is compared with the long-time effects of various compounds with better known subcellular activities.Calcium (10 mM), and cytochalasin B (up to 100 g/ml) do not visibly influence cytokinesis. Caffeine (1 mM) totally inhibits multiplication of the algae whereas calcium has only a slight and cytochalasin has no effect on this parameter.The other reference-compounds (colchicine, sodium cacodylate, deuterium oxide, local anesthetics, and sodium dodecylsulfate) all inhibit cell multiplication, simultaneously leading to giant multinucleate cells, obviously by inhibition of cytokinesis.The most potent inhibitor of cytokinesis is triethyl lead which was shown to be 250× more effective than colchicine in respect to the molar concentrations.The comparison of the effects of tetraethyl lead and triethyl lead with the reference agents leads to the conclusion that organic lead compounds might inhibit cytokinesis ofPoterioochromonas malhamensis by disintegrating peripheral microtubules and/or by interfering with structures and functions of membranes.

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Verwendete Abkürzungen im Text CB Cytochalasin B - KE Karminessigsäure - KV kontraktile Vakuole - LV Leukosinvakuole - MT Mikrotubuli - SDS Na-Dodecyl-sulfat - TEL Tetraäthylblei - TriEL Triäthylblei     

Stimulatory effect of calcium chelators on Na+-Ca2+ exchange in cardiac sarcolemmal vesicles

The calcium chelators EGTA, EDTA and cyclohexanediamine tetraacetic acid (CDTA) enhance initial rates of Na_i⁺-dependent Ca²⁺ uptake by cardiac sarcolemmal vesicles. The affinity of the exchanger for calcium is increased in the presence of the chelators to an extent dependent on chelator concentration and on the range of free calcium concentrations over which the phenomenon is measured. For free Ca²⁺ in the range of 4 μM or less, the apparent K_m is lowered to approximately 1 μM. The Ca-chelator complex appears to be the species which causes stimulation. The effect is not due to sequestration of contaminating heavy metal ions in the sarcolemmal membrane preparations or the solutions used in experiments. Caution is suggested in the use of EGTA or EDTA as calcium buffers when measuring calcium dependence of phenomena involving calcium binding and transport, because the added chelator may alter the properties of the system.     

Biomphalaria glabrata: lysozyme activities in the hemolymph, digestive gland, and headfoot of the intermediate host of Schistosoma mansoni.

Lysozyme (mucopeptide N-acetylmuramylhydrolase EC 3.2.1.17) activity has been found in the hemolymph, digestive gland, and headfoot extracts of Biomphalaria glabrata, the intermediate host of Schistosoma mansoni. Partial purification of the bacteriolytic enzyme was attained by gel chromatography on Sephacryl S-200 and active lytic fractions were concentrated by Amicon filtration. The properties of the lytic enzymes from the three tissue extracts were identical. Enzyme activity was determined by the rate of lysis of cell wall suspension of Micrococcus lysodeikticus. Lysis of the cell walls was accompanied by a release of reducing sugar groups and N-acetylhexosamines. The enzyme was stable to heating at 100 C for 2 min and had an optimum activity at pH 4.5 to 5.0 in 0.066 M glycylglycine buffer. Low concentrations (5 mM) of NaCl, KCl, and LiCl increased the activity of the enzyme, whereas high concentrations (25 mM) of the same ions caused about 50% inhibition of the enzyme activity. MgCl₂ and CaCl₂ also inhibited the enzyme activity. Addition of 1 mM EDTA or EGTA resulted in about a twofold increase in enzyme activity. Double reciprocal plots of enzyme velocities and substrate concentrations yielded an apparent Michaelis-Menten constant (K_m) of 0.05 ± 0.01 mg/ml of M. lysodeikticus.     

Metabolism of inorganic sulphate in the isolated perfused rat liver. Effect of sulphate concentration on the rate of sulphation by phenol sulphotransferase

1. The metabolism of inorganic [³⁵S]sulphate (Na₂³⁵SO₄) was studied in the isolated perfused rat liver at three initial concentrations of inorganic sulphate in the perfusion medium (0, 0.65 and 1.30mm), in relation to sulphation and glucuronidation of a phenolic drug, harmol (7-hydroxy-1-methyl-9H-pyrido[3,4-b]indole). 2. [³⁵S]Sulphate rapidly equilibrated with endogenous sulphate in the liver. It was excreted in bile and reached, at the lowest concentration in the perfusion medium, concentrations in bile that were much higher than those in the perfusion medium; at the higher sulphate concentrations, these concentrations were equal. The physiological concentration of inorganic sulphate in the liver, available for sulphation of drugs, is similar to the plasma concentration. 3. At zero initial inorganic sulphate in the perfusion medium, the rate of sulphation was very low and harmol was mainly glucuronidated. At 0.65mm-sulphate glucuronidation was much decreased and considerable sulphation took place, indicating efficient competition of conjugation by sulphation. At 1.30mm-sulphate the sulphation increased still further. 4. The results suggest that an important factor in sulphation is the relatively high K_m of synthesis of adenosine 3′-phosphate 5′-sulphatophosphate (the co-substrate of sulphation) for inorganic sulphate, which is of the order of the plasma concentration of inorganic sulphate. The steady-state adenosine 3′-phosphate 5′-sulphatophosphate concentration may determine the rate of sulphate conjugation of drugs in the rat in vivo.