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D M Templeton

Publications and source records attributed to D M Templeton.

At least 37 records · Page 2Linked to original sources
PubMed1996

Sample collection guidelines for trace elements in blood and urine. IUPAC Commission of Toxicology.

This paper presents an organized system for element-specific sample collection and handling of human blood (whole blood, serum or plasma, packed cells or erythrocytes) and urine also indicating a proper definition of the subject and sample. Harmonized procedures for collection, preparation, analysis and quality control are suggested. The aim is to assist scientists worldwide to produce comparable data which will be useful on a regional, national and international scale. The guidelines are directed to the elements aluminium, arsenic, cadmium, chromium, cobalt, copper, lead, lithium, manganese, mercury, nickel, selenium and zinc. These include the most important elements measured for their occupational or clinical significance, and serve as examples of principles that will guide development of methods for other elements in the future.

Blood Specimen Collection↗
PubMed1996

Inhibition of mitogenesis and c-fos induction in mesangial cells by heparin and heparan sulfates.

When rat renal mesangial cells (RMC) or vascular smooth muscle cells are released from quiescence by serum stimulation they express c-fos mRNA transiently at 30 to 60 minutes and progress in synchrony to S phase. Heparin causes significant suppression of [3H]-thymidine incorporation into DNA in S phase and a decrease and delay of entry of cells into S/G2. Added at the time of serum stimulation, heparin (1 microgram/ml or less) causes a decrease in the subsequent expression of c-fos mRNA in RMC, and a similar effect is observed with heparan sulfate chains isolated from RMC-cultures themselves. Although these cells internalize and degrade heparin, the timing of the maximal effect indicates an extracellular action of heparin. In keeping with this idea, 125I-heparin binds specifically to a single class of high affinity sites on the cell surface. The effect of heparin on c-fos induction may be independent of interaction with cytokines or cytokine receptors; its magnitude is not diminished when heparin-binding substances are removed from serum by heparin-Sepharose. Furthermore, direct activation of protein kinase C (PKC) with a phorbol ester in the absence of serum likewise induces c-fos and 1 microgram/ml heparin inhibits this response by 65%. Phorbol ester caused an increase in the proportion of histone H1-active PKC associated with the cell membrane fraction, from approximately 25% to 70% of total activity. Heparin affected neither the total activity of the kinase nor the proportion associated with the membrane. When PKC was inhibited with staurosporine, only very low levels of c-fos were induced by serum. We conclude that low concentrations of heparin and heparan sulfate suppress the mitogenic response of mesangial cells to serum and inhibit c-fos mRNA induction through an effect of cell surface-bound glycosaminoglycan on a signalling pathway downstream of PKC.

Animals↗
PubMed1996

Collagen type I enhances endothelin-mediated contraction and induces nonproliferating phenotype in mesangial cells.

Accumulation of glomerular extracellular matrix is a characteristic accompaniment of mesangial cell proliferation in progressive renal disease. We examined how growth on several matrices affected the proliferative phenotype of cultured rat mesangial cells. Compared with growth on plastic, Matrigel, or mesangial matrix, collagen type I caused a decreased cell number at 72 h, decreased total DNA per culture, and a decrease in the incorporation of [3H]thymidine during S phase in cells released from quiescence. These antiproliferative and antimitogenic effects of collagen type I required growth on a collagen gel; soluble collagen or collagen fragments were without effect. Because a number of agents elicit both proliferative and contractile responses in mesangial cells, we examined the effect of growth on collagen on contractility. Compared with plastic, cells grown on collagen type I were more contractile, showed a higher Ca2+ signal in response to endothelin, and responded to endothelin with a more rapid myosin light-chain kinase-dependent phosphorylation of myosin light chain. We conclude that growth on a collagen type I gel uncouples contractility from a proliferative response in mesangial cells, suppressing proliferation while enhancing contraction and Ca2+ signaling in response to endothelin.

Alkaloids↗
PubMed1995

Modulation by iron loading and chelation of the uptake of non-transferrin-bound iron by human liver cells.

Hepatic non-transferrin-bound Fe (NTBI) flux and its regulation were characterized by measuring the uptake of Fe from [59Fe]/nitrilotriacetate (NTA) complexes in control and Fe-loaded cultures of human hepatocellular carcinoma cells (HepG2). Exposure to ferric ammonium citrate (FAC) for 1 to 7 days resulted in a time- and dose-dependent increase in the rate of NTBI uptake. In contrast to previous studies showing a dependence of the rate of Fe uptake on extracellular Fe, this was positively correlated with total cellular Fe content. The Fe3+ chelating agents deferoxamine (DFO), 1,2-dimethyl-3-hydroxypyrid-4-one (CP 020) and 1,2-diethyl-3-hydroxypyrid-4-one (CP 094) prevented or diminished the increase in NTBI transport when present during Fe loading and reversed the stimulation in pre-loaded cells in relation to their abilities to decrease intracellular iron. Although saturation of the Fe uptake process was not achieved in control cells, kinetic modelling to include linear diffusion-controlled processes yielded estimated parameters of Km = 4.3 microM and Vmax = 2.6 fmol/micrograms protein/min for the underlying process. There was a significant increase in the apparent Vmax (31.2 fmol/micrograms protein per min) for NTBI uptake in Fe-loaded cells, suggesting that Fe loading increases the number of a rate-limiting carrier site for Fe. Km also increased to 15.2 microM, comparable to values reported when whole liver is perfused with FeSO4. We conclude that HepG2 cells possess a transferrin-independent mechanism of Fe accumulation that responds reversibly to a regulatory intracellular Fe pool.

Biological Transport↗
PubMed1995

Iron-chelation therapy with oral deferiprone in patients with thalassemia major.

BACKGROUND: To determine whether the orally active iron chelator deferiprone (1,2-dimethyl-3-hydroxy-pyridin-4-one) is efficacious in the treatment of iron overload in patients with thalassemia major, we conducted a prospective trial of deferiprone in 21 patients unable or unwilling to use standard chelation therapy with parenteral deferoxamine. METHODS: Hepatic iron stores were determined yearly by chemical analysis of liver-biopsy specimens or magnetic-susceptibility measurements. Detailed clinical and laboratory studies were used to monitor safety and compliance. RESULTS: The patients received deferiprone therapy for a mean (+/-SE) of 3.1 +/- 0.3 years. Ten patients in whom previous chelation therapy with deferoxamine had been ineffective had initial hepatic iron concentrations of at least 80 mumol per gram of liver, wet weight -- values associated with complications of iron overload. Hepatic iron concentrations decreased in all 10 patients, from 125.3 +/- 11.5 to 60.3 +/- 9.6 mumol per gram (P < 0.005), with values that were less than 80 mumol per gram in 8 of the 10 patients (P < 0.005). In all 11 patients in whom deferoxamine therapy had previously been effective, deferiprone maintained hepatic iron concentrations below 80 mumol of iron per gram. CONCLUSIONS: Oral deferiprone induces sustained decreases in body iron to concentrations compatible with the avoidance of complications from iron overload. The risk of agranulocytosis associated with deferiprone may restrict its administration to patients who are unable or unwilling to use deferoxamine.

Administration, Oral↗
PubMed1994

Reversed-phase high-performance liquid chromatography of non-transferrin-bound iron and some hydroxypyridone and hydroxypyrone chelators.

The pursuit of orally available Fe(III) chelating agents has resulted in several clinical trials of 1,2-dimethyl-3-hydroxypyrid-4-one (CP20). Chromatography of this and related Fe chelators on silica-based columns has proven difficult due to unwanted interactions with the stationary phase, including with contaminating Fe bound to silanol groups. By addition of Fe3+ (50 microM ferric ammonium citrate) to an acidified aqueous mobile phase, we have successfully separated a series of hydroxypyridones-including CP20-and the related pyrones maltol and ethylmaltol by HPLC on microBondapak C18. Complexation occurs with these agents even at low pH, and they elute in an order consistent with the partition coefficients of their Fe(III) complexes. By the reverse strategy of adding ethylmaltol to the mobile phase, chelatable Fe was chromatographed and the peak response at 500 nm was linear down to a detection limit below 0.5 microM. This method was applied to pooled serum and to serum spiked with Fe after filtration at 10 kDa cut-off. The direct determination of non-transferrin-bound Fe at micromolar concentrations in serum is possible with this approach.

Chromatography, High Pressure Liquid↗
PubMed1994

Uptake of non-transferrin-bound iron by both reductive and nonreductive processes is modulated by intracellular iron.

Non-transferrin-bound iron (NTBI) uptake occurs in a variety of cells by a saturable, specific and temperature-sensitive process. Our previous studies indicated that NTBI uptake by cardiac myocytes and Hep G2 cells was reversibly up-regulated by iron deposition. In the present work we have characterized this up-regulation and examined its mechanism by comparing the uptake of oxidized (Fe3+) and ascorbate-reduced (Fe2+) forms of iron. Iron loading markedly enhanced the uptake of iron both in the presence and absence of ascorbate, but the increment was greater when ascorbate was absent. This up-regulation is partially inhibited by actinomycin D and cycloheximide, indicating a requirement for protein synthesis. Uptake by the iron-loaded cells was less sensitive to thiol-alkylating agents and competing metal ions, but was more sensitive to proteolysis. Iron loading causes an increase in both Km and Vmax for uptake of both Fe2+ and Fe3+, although the values differ, suggesting distinct rate-limiting steps for uptake of Fe2+ and Fe3+. Consistent with this idea, uptake of the two ions showed differential sensitivity to thiol reagents, competing metal ions and monensin. The Fe(2+)-specific chelators bathophenanthroline disulfonate and ferrozine markedly inhibited iron uptake whether ascorbate was present or not, indicating that Fe3+ uptake is dependent on reduction to the ferrous state. This requirement for reduction was independent of the iron status of the cells, demonstrating that the process of up-regulation is not due to the appearance of a new mechanism for translocation of Fe3+ without reduction. Taken together, the evidence favors a model of NTBI transport where an obligate and rate-determining reduction of Fe3+ occurs prior to or during uptake, followed by translocation through an Fe2+ carrier. The distinct translocation mechanisms of uptake in the presence and absence of ascorbate suggest that exogenous Fe2+ does not access the carrier available to the nascent ferrous ion derived from the reductase and is consistent with close coupling between the reduction and the translocation processes. In iron-loaded cells with increased rates of NTBI transport, a similar mechanism prevails.

Alkylating Agents↗
PubMed1994

Tentative reference values for nickel concentrations in human serum, plasma, blood, and urine: evaluation according to the TRACY protocol.

Published reports of Ni concentrations in human serum or plasma, whole blood, and urine have been reviewed in order to establish a database of reference values. In keeping with the TRACY program as previously applied to Hg, reports were evaluated in the categories of description of sample population, specimen collection and processing, analytical methods, and data presentation. Based on these considerations, eight studies of Ni in serum were deemed suitable for establishing reference levels in the general population. In five of these studies, the mean values for serum Ni concentration were < 0.3 microgram/l and the upper limits were < or = 1.1 micrograms/l. Six studies of Ni in urine were found suitable, and in four of these the mean values of Ni were < or = 2.0 micrograms/l and the upper limits were < or = 6.0 micrograms/l. Fewer studies on Ni in whole blood have been reported, and the Ni content of blood remains uncertain.

Humans↗
PubMed1994

Isotope-specific analysis of Ni by ICP-MS: applications of stable isotope tracers to biokinetic studies.

Inductively coupled plasma mass spectrometry (ICP-MS) offers excellent detection limits and isotopic analysis of Ni in aqueous standards, but is prone to interferences--mainly from Ca-containing polyatomics--when biological matrices are analyzed for Ni. We have used multivariate calibration with principal components analysis (PCA) to correct for mass overlaps in serum digests. The resulting detection limit for Ni is below 1 microgram/l and the within-run imprecision is 6% at 1.46 micrograms Ni/l. In urine, the higher Ca content renders routine application of PCA problematic. We evaluated several methods of pre-concentration, and have developed a method of Ca oxalate precipitation that allows direct analysis of Ni in the diluted supernatant. The stable isotope 62Ni and the radiosotope 63Ni were co-administered i.v. to rats and the serum and urinary clearances were determined by liquid scintillation counting and ICP-MS. Ni measurements by both methods were in excellent agreement, and serum clearance fit a double exponential decay consistent with the two-compartment model of Onkelinx et al. [24]. A human volunteer ingested 61Ni (20 micrograms Ni/kg body wt.) in water after an overnight fast. Identical serum levels, peaking near 35 micrograms/l at 2 h, were measured by electrothermal atomic absorption spectrometry and ICP-MS with PCA. Urinary excretion of 61Ni measured by ICP-MS demonstrated absorption of 30% of the administered dose. We conclude that Ni isotopes can be measured in body fluids by ICP-MS at levels that allow stable isotope tracer studies in humans.

Adult↗
PubMed1994

Iron-balance and dose-response studies of the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in iron-loaded patients with sickle cell disease.

Several life-threatening complications of the common disorder sickle cell disease require management with red blood cell transfusions and, hence, long-term iron-chelating therapy. The efficacy of the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) has not previously been determined in patients with sickle cell disease. We compared the efficacy of L1 to that of standard-dose subcutaneous deferoxamine in four regularly transfused patients with homozygous sickle cell disease, who had evidence of severe iron overload and a history of poor compliance with deferoxamine. Determination of 24-hour urinary iron excretion conducted over 5 days immediately after transfusion showed that the mean daily urinary iron excretion induced by L1 at 75 mg/kg/d (0.48 +/- 0.23 mg/kg) was equivalent to that induced by deferoxamine at 50 mg/kg/d (0.39 +/- 0.06 mg/kg). In two of three patients studied, a significant (P < .025) increase in mean daily urinary iron excretion was achieved when the dose of L1 was increased to 100 mg/kg/d. Total iron balance studies, which quantitated both urinary and stool iron excretion on L1 and deferoxamine, determined that mean total daily iron excretion induced by deferoxamine (0.88 +/- 0.05 mg/kg) was significantly greater (P < .05) than that induced by L1 (0.53 +/- 0.17 mg/kg), attributable to the significantly greater stool iron excretion during deferoxamine treatment (0.50 +/- 0.16 mg/kg/d) compared with that measured during L1 treatment (0.12 +/- 0.08 mg/kg/d, P < .01). Stool iron excretion accounted for a significantly greater percentage of total iron excretion during deferoxamine treatment (59% +/- 20%) than during L1 treatment (23% +/- 14%, P < .01). These iron balance studies are the first to compare total iron excretion induced by L1 with that achieved by deferoxamine. They demonstrate that the mean total daily iron excretion during L1 treatment (0.53 +/- 0.17 mg/kg) is sufficient to maintain net negative iron balance in most regularly transfused patients with sickle cell disease. Because long-term compliance with L1 has been shown previously to be superior to that with deferoxamine in patients with homozygous beta-thalassemia, the use of L1 should increase the long-term effectiveness of iron chelation in patients with sickle cell disease.

Administration, Oral↗
PubMed1994

Copper complexation by 3-hydroxypyridin-4-one iron chelators: structural and iron competition studies.

Clinical trials of 1,2-dimethyl-3-hydroxypyridine-4-one (1) as an orally available iron chelator are presently underway in several centers. Discrepant reports of toxicity in human and animal studies have stimulated debate on the role of iron status and the availability of iron for chelation relative to other essential elements like copper in determining the clinical effects of 1. Therefore, we investigated the ability of 1, its 1,2-diethyl analog 2, and their iron chelates to complex copper. Both compounds formed tetracoordinate 2:1 Cu(II) complexes which X-ray structure analysis showed to be planar and coordinated through the oxygen atoms of the hydroxy ketone functionality. Potentiometric analysis revealed that these complexes dominated at physiological pH, although between pH 6 and 7 approximately equal amounts of the mono and bis complexes of Cu with 1 were present at equilibrium. Comparing the stepwise formation constants deduced from the stability constants of these complexes (log beta 2 = 21.7 +/- 0.8 (1) and 20.2 +/- 2.0 (2)) with those of their Fe(III) complexes (Motekaitis,R.J.;Martell,A.E.Inorg.Chim.Acta 1991, 183,71-80) leads to a prediction of insignificant copper complexation when equimolar iron is present and dissociation products are thermodynamically unimportant. However, displacement of Fe3+ occurred from both complexes with stoichiometric amounts of Cu2+, implicating the participation of metal hydrolysis products in the equilibria. We conclude that Cu(II) complexes of the 3-hydroxypyridin-4-one chelators are stable under physiological conditions and that copper can effect displacement of iron by these agents under circumstances where hydrolysis of the metals is important.

Antithyroid Agents↗
PubMed1994

Growth modulation and proteoglycan turnover in cultured mesangial cells.

Proliferation of mesangial cells is a common feature of renal disease, and conditioned media from glomerular epithelial and endothelial cells have been found to contain heparin-like molecules that suppress proliferation of rat mesangial cells (RMC). We have partially characterized the glycosaminoglycans that are labeled with 35SO4(2-) by RMC in culture at early passage and examined their ability to inhibit mitogenic stimulation of the cells. Four chondroitin/dermatan sulfate proteoglycans (CS/DSPG) were identified, the largest and smallest of which (Kd of 0.04 and 0.26 on Superose 6) were retained in the cell layer while the other two (Kd = 0.17 and 0.22) were secreted into the medium. Heparan sulfate proteoglycans (HSPG) with Kd values of 0.09, 0.13, and 0.39 were minor components of the cell layer, while a single heparan sulfate (Kd = 0.17) was recovered from the medium. After 16 h of labeling in serum-free medium, about 60% of macromolecular 35S was cell-associated and 40% was in the medium. Cell-associated label consisted of 7% CS/DSPG, 9% HSPG, and 84% free glycosaminoglycan chains (mostly CS/DS), whereas the medium contained 52% CS/DSPG, 17% HSPG, and approximately equal amounts of free HS and CS/DS chains. Bovine lung heparin (1 microgram/ml) decreased by 45% the incorporation of [3H]-thymidine into DNA after release of serum-starved RMC from growth arrest. Heparin acted prior to the G1/S interface; arrest of the cells in early S phase with aphidicolin abrogated the heparin response. The endogenous HSPGs had a slight antimitogenic effect on the RMC, but heparan sulfate chains from both the medium and cell layer had a potent effect. On an equivalent mass basis, only the free glycosaminoglycan chains were more potent than heparin in this regard, decreasing thymidine incorporation by over 90% when present at 1 microgram/ml. These results demonstrate that heparan sulfate glycosaminoglycans derived from mesangial proteoglycans are potential negative autocrine growth regulators. Proteoglycan metabolism releases these soluble heparan sulfate chains, determining the level of this activity.

Animals↗
PubMed1993

Subunit structure of bovine ESF (extracellular-matrix stabilizing factor(s)). A chondroitin sulfate proteoglycan with homology to human I alpha i (inter-alpha-trypsin inhibitors).

Two chondroitin sulfate-containing complexes have been isolated from fetal bovine serum and shown to contain the serine protease inhibitor bikunin. A complex of 126 kDa contains bikunin linked by a chondroitin sulfate chain to a protein with homology to the HC2 component of the human inter-alpha-trypsin inhibitor. This complex represents the extracellular matrix stabilizing factor recently described as a bikunin-containing fraction necessary for expansion of the cumulus matrix [(1992) J. Biol. Chem. 267, 12380-12386]. A second complex of 236 kDa contains, in addition to bikunin and HC2, a bovine homolog of HC3 of the human pre-alpha-trypsin inhibitor. Thus, bovine bikunin is a chondroitin sulfate proteoglycan that achieves multifunctionality by linkage to proteins homologous to human serine antiproteinase complexes.

Alpha-Globulins↗
PubMed1993

Protective elevations of glutathione and metallothionein in cadmium-exposed mesangial cells.

Exposure of cultured rat mesangial cells to CdCl2 caused a dose- and time-dependent increase in intracellular glutathione that was significant at 0.5 microM and maximal at 1 microM Cd2+. The effect depended on glutathione synthesis and was masked by inhibiting synthesis with buthionine sulfoximine. The cells responded to slightly higher concentrations of Cd with a marked decrease in DNA synthesis, and reversible depletion of glutathione enhanced this sensitivity. Pre-induction of the thiol-rich protein metallothionein with ZnCl2 afforded a degree of protection to the glutathione-depleted cells. We conclude that the Cd-dependent elevation of glutathione in these cells may be protective at concentrations of Cd that can arise during acute environmental and occupational exposures.

Animals↗
PubMed1993

Effects of iron loading on uptake, speciation, and chelation of iron in cultured myocardial cells.

Accumulation of Fe in the myocardium in circumstances of transferrin saturation is associated with heart failure in Fe-loaded patients. To characterize the underlying causes of this phenomenon, we measured the flux as well as the speciation of Fe in normal and Fe-loaded cultures of rat myocardiocytes. Fe loading with low-molecular-weight Fe (ferric ammonium citrate) promoted a dose- and time-dependent increase in the rate of uptake of non-transferrin-bound Fe (NTBI) that was positively correlated (R = 0.9, p < 0.005) with cellular iron content. At concentrations sufficient to produce this up-regulation, membrane integrity was unaffected but the rate of spontaneous beating of the cells was decreased by 60%. The enhanced rate of NTBI uptake in Fe-loaded cells reverted to control rates after treatment with therapeutic concentrations of Fe chelators deferoxamine, 1,2-dimethyl-3-hydroxypyrid-4-one and 1,2-diethyl-3-hydroxypyrid-4-one under conditions where approximately 80% of the cellular Fe was removed by chelation. Fe loading of cultured myocytes also induced shifts in Fe speciation. Thus the ratio of Fe bound in hemosiderin-like precipitates to ferritin-bound Fe increased twofold, from a range of 0.84 to 1.44 in control cells to 1.96 to 3.3 in iron-loaded cells. This increased ratio was similar to that measured in the heart and liver of a thalassemic patient who underwent a double transplant for the failure of both organs, even though the Fe content of the heart (mean, 5.8 mg Fe/gm dry weight) was much less than that of the liver (28.1 mg/gm dry weight). These results suggest that increased rates of uptake of NTBI may exacerbate iron loading of the heart and contribute to iron-mediated cardiotoxicity, whereas the clinical benefits of chelation therapy may be enhanced by the down-regulation of NTBI uptake.

Animals↗
PubMed1993

Oral iron chelation with 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in iron loaded thalassemia patients.

Despite the successes of deferoxamine (DFO) in the treatment and prevention of iron overload, an effective orally available iron chelating drug is needed, since erratic compliance with irritating, cumbersome parenteral infusions still results in fatal iron accumulation in many patients. Disorders of increased iron absorption should also benefit from the development of safe and effective iron chelating agents. Individuals with non-transfusion-dependent thalassemia (thalassemia "intermedia"), exhibit excessive dietary iron absorption that can lead to serious iron loading by the second or third decade of life. An orally effective iron-chelating drug would have major therapeutic advantages for all these patients.

Administration, Oral↗