The inhibition of bovine ceruloplasmin oxidase activity by thiomolybdates in vivo and in vitro: a reversible interaction

The administration of pharmacological doses (greater than 100 mg Mo) of trithiomolybdate or tretrathiomolydbate to ruminants causes a transient apparent decrease in the ceruloplasmin oxidase activity of plasma and a more persistent increase in copper bound to plasma albumin. Sephadex gel-filtration and/or dilution of "inhibited" samples taken from an infused animal or of plasma treated with thiomolybdate in vitro restores activity back to pretreatment levels. The increase in albumin bound copper does not appear to be related to ceruloplasmin breakdown. It is concluded that, contrary to a recent report, the inhibition of ceruloplasmin oxidase activity is reversible and thus unlikely to be of pathological relevance, since circulatory thiomolybdate concentrations in molybdenotic animals are likely to be very low. It is recommended that thiomolybdate preparations used for in vitro and in vivo studies should be carefully purified by Sephadex chromatography.     

Behavior of [185W]thiotungstates injected into sheep and the influence of copper: their fate and the effect of the compounds upon plasma copper

[185W]trithio- and tetrathiotungstates (0.5 mg W) were injected intravenously into sheep. The compounds circulated in plasma bound reversibly to plasma proteins, particularly to albumin. After the first few minutes, levels declined exponentially with a T 1/2 of 12-14 hr. The initial movement of [185W]trithiotungstate from the plasma compartment was delayed transiently by the immediate injection of copper (2-6 mg); the longer-term metabolism was unaffected. The final fate of the compounds appeared to be hydrolysis and excretion in urine as [185W]tungstate. 185W from [185W]trithiotungstate appeared more rapidly than from [185W]tetrathiotungstate, but in both the rate was unaffected by copper injections. Since the appearance in urine did not correspond to the disappearance from plasma, it was suggested that the hydrolysis occurred in extravascular tissues and that the liver might be the site. A control experiment showed that [185W]tungstate in plasma was very rapidly cleared (and appeared in urine). At higher W levels (25-50 mg W per sheep per day), systematic copper metabolism was perturbed since plasma copper levels rose. The experiments demonstrated that in sheep the behavior and the effects of thiotungstates and thiomolybdates are sufficiently similar for 185W to be used as a more convenient alternative to 99Mo for longer-term studies on the interaction of the compounds with copper metabolism in animals.     

The uptake and intracellular distribution of [35S]trithiomolybdate in bovine liver in vivo

[35S] trithiomolybdate was administered intravenously to a group of four steer at two dose rates, 1 and 26 mg Mo per animal. Radioactivity appeared rapidly in the liver and was distributed in all the subcellular fractions examined. Examination by Sephadex G-100 gel-filtration of the cytosol fraction showed that distinct 35S-binding protein peaks were present. The protein-bound radioactivity was displaceable and was identified as [35S]thiomolybdates. No radioactivity eluted with metallothionein, but 35S was associated with the high molecular weight copper fraction, eluted in the void volume of the column, which increased transiently after the administration of the higher dose. It was suggested that the presence of protein-bound thiomolybdates in the liver gave rise to new ligands, which altered the equilibrium of copper between the different metal-binding proteins. This might be similar to the alteration in the copper-binding of albumin produced by the presence of thiomolybdates.     

99Mo metabolism in sheep after the intravenous injection of [99Mo] thiomolybdates

[99Mo]di-, tri-, and tetrathiomolybdate (5.4 to 62.5 mg of Mo) were given by intravenous injection to sheep maintained on a sulfur-supplemented (3 g of S/kg) diet. All the compounds were metabolized very rapidly over the first 15 min postinjection, but relatively slowly thereafter, with a t1/2 of about 30 hr for dithiomolybdate and 40 h for tri- and tetrathiomolybdate. The [99Mo] metabolites present in plasma were identified by Sephadex G-25 chromatography. The main fate of the compounds injected appeared to be stepwise transformation to molybdate and subsequent rapid urinary elimination. Over 90-100 hr more than 90% of the radioactivity was excreted in urine, compared to less than 5% in the feces. The trichloroacetic acid (TCA) solubility of plasma copper and the diamine oxidase activity of ceruloplasmin was depressed, particularly over the first 15 min postinjection and a more persistant TCA-insoluble Cu fraction was apparent.     

The effect of dietary molybdenum supplementation on tissue copper concentrations,mohair fibre and carcass characteristics of growing Angora goats

The purpose of this experiment was to better characterize the effects of the interaction between copper (Cu), molybdenum (Mo) and sulphur (S) in the diet on growth, metabolism and fibre characteristics in Angora goats. 15 Angora goats aged 9 months and weighing 21.5 kg on average were used in a ten-week study and allocated to three dietary treatments: Treatment C (10 MJ metabolisable energy, 178 g crude protein, 5.5 mg Cu, 0.57 mg Mo, and 3.4 g S): Treatment M1 (with 7.5 mg Mo) or Treatment M2 (with 15 mg Mo) per animal per day. Dose-dependent increases in the concentrations of Mo (P < 0.01) and Cu (P < 0.05) in plasma were recorded in response to increased dietary intake of Mo. Supplementation of the control diet with increased concentrations of Mo did not produce effects (P > 0.05) on growth rate, feed conversion efficiency, carcass weight or mohair fibre yield and diameter.Haematological status and concentration of Cu in liver and Cu and S in fibre at the end of the study were also not affected (P > 0.05).Concentrations of trichloroacetic acid (TCA) soluble “available” copper in plasma were not significantly different although significant (P < 0.05 and P < 0.01) reductions in the ratio of “available” to total Cu concentrations were observed. This effect was stabilised and maintained after 30 days. It is suggested that the additional Cu in plasma was largely TCA insoluble and possibly in the form of thiomolybdate complexes which may be poorly excreted and not available for uptake to the metabolic sites. It is evident that adequate “available” Cu was present in plasma and that exposure to elevated Mo intake was not severe or long enough to produce clinical symptoms or to affect growth, haematological status or fibre production.     

Spectral and kinetic studies on the binding of trithiomolybdate to bovine and canine serum albumin in vitro: the interaction with copper

Spectral studies showed that copper and trithiomolybdate participated in a three-way interaction with bovine and canine serum albumin. The interaction with the proteins was affected by increased pH and ionic strength. Kinetic studies of binding equilibria indicated that [³⁵S] trithiomolybdate bound to both albumins at a single site. The affinity of the site, but not the capacity of the protein, was increased by copper. It was concluded that the site was distinct from the N-terminal copper (and nickel) binding site, which is present on BSA but absent from CSA. Whether or not the N-terminal site has a role in copper transport is discussed. Reversible thiomolybdate-copper-protein interactions of this type may play a fundamental role in the pathogenesis of Mo-induced syndromes, since as the normal binding patterns are perturbed the interprotein equilibria are altered and the copper distribution patterns are modified.     

Excretion of copper complexed with thiomolybdate into the bile and blood in LEC rats

Copper (Cu) accumulating in a form bound to metallothionein (MT) in the liver of Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease, was removed with ammonium tetrathiomolybdate (TTM), and the fate of the Cu complexed with TTM and mobilized from the liver was determined. TTM was injected intravenously as a single dose of 2, 10 or 50 mg TTM/kg body weight into LEC and Wistar (normal Cu metabolism) rats, and then the concentrations of Cu and molybdenum (Mo) in the bile and plasma were monitored with time after the injection. In Wistar rats, most of the Mo was excreted into the urine, only a small quantity being excreted into the bile, while Cu excreted into the urine decreased. However, in LEC rats, Cu and Mo were excreted into the bile and blood, and the bile is recognized for the first time as the major route of excretion. The Cu excreted into both the bile and plasma was accompanied by an equimolar amount of Mo. The relative ratio of the amounts of Cu excreted into the bile and plasma was 40/60 for the low and high dose groups, and 70/30 for the medium dose group. The systemic dispositions of the Cu mobilized from the liver and the Mo complexed with the Cu were also determined for the kidneys, spleen and brain together with their urinal excretion. Although Mo in the three organs and Cu in the kidneys and spleen were increased or showed a tendency to increase, Cu in the brain was not increased at all doses of TTM.     

99mTc(CO)3-15-[N-(Acetyloxy)-2-picolylamino]pentadecanoic acid: a potential radiotracer for evaluation of fatty acid metabolism

99mTc(CO)3-15-[N-(Acetyloxy)-2-picolylamino]pentadecanoic acid (1a) was prepared by incorporating [99mTc(CO)3]+ into 15-[N-(hydroxycarbonylmethyl)-2-picolylamino]pentadecanoic acid (2a). The overall radiochemical yield of 1a after HPLC purification was 60-63%. Radiotracer 1a was found to be chemically stable when incubated in human plasma for 4 h at 37 degrees C. Tissue distribution studies showed that high radioactivity accumulated in the heart with rapid clearance. The maximum heart-to-blood uptake ratio was 1.87 at 5 min after a tail-vein injection. Radioactive metabolites were analyzed in urine samples of mice and corresponded to a 9.3:1 ratio of 99mTc(CO)3-5-[N-(acetyloxy)-2-picolylamino]pentanoic acid (1b) to 99mTc(CO)3-3-[N-(acetyloxy)-2-picolylamino]propionic acid (1c), indicating that 1a is mainly metabolized to 1b via beta-oxidation in the body. These results suggest that 1a is a promising radiotracer for evaluation of fatty acid metabolism in myocardium.     

Sulphate recycling and metabolism in sheep and cattle.

Merino wethers and Brahman x Shorthorn steers, offered lucerne or spear grass hay, were used to study the movements of sulphate through pools in plasma and ruminal liquor. The irreversible loss of sulphate from ruminal liquor was 60 and 76% of sulphur ingested for both species fed lucerne and spear grass respectively. The irreversible loss of sulphate from the plasma averaged 67 and 56% of sulphur ingested for animals fed lucerne and spear grass respectively. Daily recycling of sulphate to the rumen of sheep was 98 mg sulphur on the lucerne diet and 3.9 mg sulphur on the spear grass diet. Sulphate recycling in cattle fed lucerne was 533 mg sulphur; in cattle fed spear grass the value was 234 mg sulphur. Over 6 days following an intravenous injection of [35S]sulphate into sheep and cattle fed lucerne, 5-10% of the dose was excreted in the faeces and c. 10% was retained. Corresponding values for animals fed spear grass were 23-31% in faeces and 40-51% of the dose retained. After intraruminal injections of [35S]sulphate, animals fed lucerne excreted 15-18% of the dose in the faeces and retained 25-30% of the dose over 6 days. Values for animals fed spear grass were 22-26% in faeces and 62-70% retained. It was concluded that sulphate recycling to the rumen is a limiting factor in microbial synthesis for sheep fed low-quality roughage, and that secretion of endogenous sulphur into the postruminal tract of ruminants is of importance in the metabolism of sulphate.     

Contribution of the apo[a] phenotype to plasma Lp[a] concentrations shows considerable ethnic variation.

Apolipoprotein[a] polymorphism has been investigated by sodium dodecyl sulfate polyacrylamide (5.37%) gel electrophoresis and immunoblotting using a standardized sample load in four ethnic groups: German, Ghanaian, Chinese, and San (Kalahari Bushmen). A total of 10 different apparent molecular weight (Mr) polymorphs, designated 1 to 10 with increasing Mr, were detected in greater than 99% of all individuals tested (German, 99%; Ghanaian, 99%; Chinese, 100%; San 100%). A null allele is therefore at most an infrequent variant in all populations. Polymorphs 6-10 were common to all four populations, while polymorphs 1-5 appeared to be relatively rare variants not universally detected in each group in the present study. The Chinese had the highest proportion of double-band phenotypes and the observed frequencies were not significantly different from those expected according to simple Mendelian inheritance, whereas the observed apo[a] phenotype distributions of the other three groups did not concur with those expected for Hardy Weinberg equilibrium. The German and Ghanaian groups displayed similar distributions of apo[a] phenotypes while the Chinese and San had significantly higher frequencies of polymorphs 9 and 10. Mean plasma Lp[a] concentrations in Ghanaians (36.2 +/- 31.5 mg/dl) were almost 2-fold greater than in Germans (18.7 +/- 23.1 mg/dl) and ca 1.65-fold greater than in either Chinese (22.9 +/- 18.3 mg/dl) or San (21.1 +/- 19.3 mg/dl). A strong inverse correlation was observed between apo[a] Mr and plasma Lp[a] concentration in Germans but this was much less pronounced in Ghanaians. While the mean plasma Lp[a] levels associated with polymorphs 1-6 were similar in both Germans (43.4 +/- 30.0 mg/dl) and Ghanaians (49.2 +/- 37.6 mg/dl), those Ghanaians with any combination of the polymorphs 9 and 10 had an almost 3-fold greater mean plasma Lp[a] level (20.6 +/- 11.3 mg/dl) than their German counterparts (7.8 +/- 5.7 mg/dl). It is therefore apparent that: 1) differences in apo[a] allele frequencies are not primarily responsible for differences in Lp[a] levels between populations; and 2) the greatest ethnic variation is observed in plasma Lp[a] concentrations associated with the high molecular weight apo[a] polymorphs.     

The effect of trithiomolybdate and some selenium compounds upon 109Cd labeled rat metallothionein in vitro: the possibilities for cadmium chelation therapy.

The main binding protein for 109Cd was metallothionein after in vitro incubation of various tissue cytosol preparations obtained from rats supplemented with zinc. The exception was heart cytosol where the label was associated with higher molecular weight proteins. The metallothionein-bound 109Cd was sensitive to trithiomolybdate and moved too higher molecular weight proteins, presumably because of the creation of new stronger ligands by the association of thiomolybdate with these proteins. The 109Cd binding was affected by selenate, selenite, and selenide while molybdate, sulphate, and thiosulphate were ineffective. It is proposed that thiomolybdates should be investigated for use in the therapy of in vivo cadmium toxicity because they can remove the accumulated metal from metallothionein.     

Determination of cyclizine and norcyclizine in plasma and urine using gas—liquid chromatography with nitrogen selective detection

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-a]-[1,4]-benzodiazepine-3-carboxamide [I] and its 4-hydroxy metabolite, 8-chloro-6-(2-chlorophenyl)-4-hydroxy-4H-imidazo-[1,5-a][1,4]-benzodiazepine-3-carboxamide [II] in whole blood, plasma or urine. The assay for both compounds involves extraction into diethyl ether—methylene chloride (70:30) from blood, plasma, or urine buffered to pH 9.0. The overall recoveries of [I] and [II] are 92.0 ± 5.4% (S.D.) and 90.3 ± 4.9% (S.D.), respectively. The sensitivity limit of detection is 50 ng/ml of blood, plasma, or urine using a UV detector at 254 nm. The HPLC assay was used to monitor the blood concentration—time fall-off profiles, and urinary excretion profiles in the dog following single 1 mg/kg intravenous and 5 mg/kg oral doses, and following multiple oral doses of 100 mg/kg/day of compound [I].     

Copper and molybdenum absorption by rats given ammonium tetrathiomolybdate

Previous studies have shown that the tetrathiomolybdate ion [MoS_4^2?] is a potent antagonist of Cu metabolism. Effects of orally administered MoS_4^2? on the absorption and tissue distribution of ⁶⁴Cu in rats have now been investigated. Four or 12 mg Mo/kg diet, when given as MoS_4^2?, strongly inhibited ⁶⁴Cu absorption and modified the fate of absorbed Cu, decreasing hepatic and renal uptake but increasing plasma retention of ⁶⁴Cu. These effects were not induced by equivalent dietary concentrations of Mo as MoS_4^2? or when S^2? was given as CaS. Clinical and biochemical effects induced by orally administered MoS_4^2? were abolished by increasing dietary concentrations of Cu. Such treatment also inhibited the absorption and tissue retention of ⁹⁹Mo derived from ⁹⁹MoS_4^2?. Intraperitoneal administration of Cu ameliorated clinical effects attributable to MoS_4^2? but neither inhibited ⁹⁹Mo absorption nor the appearance of systemic defects in Cu metabolism. Since the absorption of MoS_4^2? (or its derivatives) from the gastrointestinal tract is inhibited by Cu, it is evident that the site of its action as an antagonist influencing either the absorption or the subsequent metabolic fate of Cu depends upon the ratio Cu/MoS_4^2? in the diet.     

Stable isotope pilot study of exchangeable copper kinetics in human blood plasma

To develop further our understanding of initial dietary copper metabolism, a method has been developed to separate plasma copper that is bound to albumin, from that bound to ceruloplasmin. This method has been tested using plasma samples from a pilot study involving six human volunteers who consumed 3mg oral doses of the stable isotope (65)Cu and gave blood samples at timed intervals up to 7 days. The results suggest that this method can be used to monitor dynamic fluctuations in newly absorbed copper over a short time frame.     

The Effects of Acute Waterborne Exposure to Sublethal Concentrations of Molybdenum on the Stress Response in Rainbow Trout,Oncorhynchus mykiss

To determine if molybdenum (Mo) is a chemical stressor, fingerling and juvenile rainbow trout (Oncorhynchus mykiss) were exposed to waterborne sodium molybdate (0, 2, 20, or 1,000 mg l^-1 of Mo) and components of the physiological (plasma cortisol, blood glucose, and hematocrit) and cellular (heat shock protein [hsp] 72, hsp73, and hsp90 in the liver, gills, heart, and erythrocytes and metallothionein [MT] in the liver and gills) stress responses were measured prior to initiation of exposure and at 8, 24, and 96 h. During the acute exposure, plasma cortisol, blood glucose, and hematocrit levels remained unchanged in all treatments. Heat shock protein 72 was not induced as a result of exposure and there were no detectable changes in total hsp70 (72 and 73), hsp90, and MT levels in any of the tissues relative to controls. Both fingerling and juvenile fish responded with similar lack of apparent sensitivity to Mo exposure. These experiments demonstrate that exposure to waterborne Mo of up to 1,000 mg l^-1 did not activate a physiological or cellular stress response in fish. Information from this study suggests that Mo water quality guidelines for the protection of aquatic life are highly protective of freshwater fish, namely rainbow trout.     

In Vitro Incorporation of Molybdate into Demolybdoproteins in Escherichia coli

When Escherichia coli was grown in the presence of tungstate, inactive forms of two molybdoenzymes, nitrate reductase and formate dehydrogenase, accumulated and were converted to their active forms upon incubation of cell suspensions with molybdate and chloramphenicol. The conversion to the active enzymes did not occur in cell extracts. When incubated with [(99)Mo]molybdate and chloramphenicol, the tungstate-grown cells incorporated (99)Mo into protein components which were released from membranes by procedures used to release nitrate reductase and formate dehydrogenase and which migrated with these activities on polyacrylamide gels. Although neither activity was formed during incubation of the crude extract with molybdate, (99)Mo was incorporated into protein components which were released from the membrane fraction under the same conditions and were similar to the active enzymes in their electrophoretic properties. The in vitro incorporation of (99)Mo occurred specifically into these components and was equal to or greater than the amount incorporated in vivo under the same conditions. Molybdenum in preformed, active nitrate reductase and formate dehydrogenase did not exchange with [(99)Mo]molybdate, demonstrating that the observed incorporation depended on the demolybdo forms of the enzymes. We conclude that molybdate may be incorporated into the demolybdo forms both in vivo and in vitro; some unknown additional factor or step, required for active enzyme formation, occurs in vivo but not in vitro under the conditions employed.     

Cholesterol metabolism in copper deficient rats

The influence of copper deficiency on the appearance of newly synthesized cholesterol in the plasma lipids of rats was examined following ³H mevalonate injection. At 181 days copper deficient rats exhibited a highly significant increase in plasma cholesterol concentration. Copper deficiency was associated with a greatly enhanced appearance of ³H in newly synthesized cholesterol and cholesteryl esters in the plasma lipids. A concomitant decrease in ³H incorporation into liver lipids was also observed. The results suggest that copper deficiency markedly influences the clearance of hepatic cholesterol to the plasma pool, and a highly significant correlation was observed between plasma copper concentrations and ³H incorporation into plasma cholesterol. The results are discussed in terms of a possible role for copper in lipoprotein metabolism, bile acid metabolism, and the uptake of cholesterol by extra-hepatic tissues.     

Indices of iron and copper status during experimentally induced,marginal zinc deficiency in humans

This study examined the effect of diet-induced, marginal zinc deficiency for 7 wks in 15 men (aged 25.3 +/- 3.3 yrs; mean +/- SD) on selected indices of iron and copper status. The regimen involved low-zinc diets based on egg albumin and soy protein with added phytate and calcium such that mean [phytate]/[Zn] and [phytate] X [Ca]/[Zn] molar ratios were 209 and 4116, respectively, for 1 wk, followed by 70 and 2000, respectively, for 6 wks. Subjects were then repleted with 30 mg Zn/d for 2 wks. Plasma copper, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activity in plasma and red blood cells (RBC), hemoglobin, hematocrit, and serum ferritin were determined weekly on fasting blood samples. Significant reductions (p less than 0.05) after 7 wks in RBC Cu,Zn-superoxide dismutase (49.5 +/- 7.2 vs 33.6 +/- 6.3 U/mg Hb) and serum ferritin (69.2 +/- 38.7 vs 53.8 +/- 33.7 micrograms/L) occurred; no comparable decline was noted for plasma Cu, hemoglobin, or hematocrit. Significant (p less than 0.05) but less consistent changes were also observed in plasma superoxide dismutase activity. None of the changes were associated with the decreases in plasma, urinary and hair zinc concentrations, and alkaline phosphatase activity in RBC membranes. Results indicate that the biochemical iron and copper status of the subjects was marginally impaired, probably from the dietary regimen that induced marginal zinc deficiency.     

Plasma appearance of labeled beta-carotene, lutein, and retinol in humans after consumption of isotopically labeled kale

The bioavailability of carotenoids from kale was investigated by labeling nutrients in kale with 13C, feeding the kale to seven adult volunteers, and analyzing serial plasma samples for labeled lutein, beta-carotene, and retinol. Ingested doses of labeled carotenoids were 34 micromol for beta-carotene and 33 micromol for lutein. Peak plasma concentrations, areas under the plasma concentration-time curves (AUCs), and percentages of dose recovered at peak plasma concentrations were calculated. Average peak plasma concentrations were 0.38, 0.068, and 0.079 microM for [13C]lutein, [13C]beta-carotene, and [13C]retinol, respectively. Average AUC values (over 28 days) were 42.8, 13.6, 13.2 microM h for [13C]lutein, [13C]beta-carotene, and [13C]retinol, respectively. Percentages of dose recovered at peak plasma concentrations were 3.6, 0.7, and 0.7% for [13C]lutein, [13C]beta-carotene, and [13C]retinol, respectively. A positive relationship was observed between baseline plasma retinol levels and [13C]retinol plasma response. It is possible that this relationship was mediated either through some aspect of beta-carotene absorption or via the common pathways of metabolism for postdose and endogenous retinoid.