Comparison of Copaxone® and Synthon's therapeutically equivalent glatiramer acetate.

Glatiramer acetate is indicated for the treatment of patients with relapsing forms of multiple sclerosis (RMS). In 2016, an alternative to the originator product was approved in the EU through the hybrid procedure regulatory pathway. This paper reviews the scientifically rigorous and multifaceted program undertaken to demonstrate the equivalence of this glatiramer acetate follow-on product (GTR) and the reference product Copaxone®, which resulted in the EU approval of GTR 20 mg/mL and 40 mg/mL. Establishing therapeutic equivalence for non-biological complex drugs is not trivial and requires a complex and multidisciplinary effort. Ultimately, there is not a single test or study that establishes therapeutic equivalence of two heterogeneous products. Instead, it requires a good understanding of the synthesis process together with a full set of data that includes comparative physicochemical testing, nonclinical in vitro and in vivo studies, and a comparative clinical study to allow for a valid conclusion that two products are therapeutically equivalent. The detailed understanding of glatiramer's synthesis process and its impact on the characteristics of glatiramer, combined with the results of a scientifically rigorous and multifaceted physicochemical and biological characterization program, and the clinical data from the 794-patient Phase III GATE study, demonstrate that GTR and Copaxone are therapeutically equivalent. The data further demonstrate that Synthon's manufacturing process consistently yields drug substance of the same quality as Copaxone and that switching from Copaxone to GTR is safe and well-tolerated.

Degradable PEG-folate coated poly(DMAEA-co-BA)phosphazene-based polyplexes exhibit receptor-specific gene expression.

A new cationic biodegradable polyphosphazene was developed, bearing both pendant primary and tertiary amine side groups, poly(2-dimethylaminoethylamine-co-diaminobutane)phosphazene (poly(DMAEA-co-BA)phosphazene). PEG and PEG-folate were coupled to polyplexes based on this poly(DMAEA-co-BA)phosphazene, leading to small (size 100 and 120nm, respectively) and almost neutral particles. In vitro tissue culture experiments showed a low cytotoxicity of both uncoated and coated polyplexes. However, the PEG coated polyplexes showed a 2-fold lower transfection activity in OVCAR 3 cells as compared to the uncoated polyplexes. On the other hand, the PEG-folate coated polyplexes had a 3-fold higher transfection than the PEGylated polyplexes. When free folate was added to the transfection medium, only the transfection activity of the targeted polyplexes was reduced, indicating internalization of the targeted PEG polyplexes via the folate receptor. Confocal laser scanning microscopy confirmed a lower binding and uptake of the PEGylated polyplexes by OVCAR-3 cells when compared to uncoated and folate-PEGylated polyplexes. While uncoated polyplexes induced aggregation of erythrocytes at polymer concentrations of 0.09microg/mL, the PEGylated systems could be incubated at ten times higher concentration before aggregation occurred indicating excellent shielding of the surface charge of the polyplexes by grafting of PEG. In conclusion, the targeted delivery of poly(DMAEA-co-BA)phosphazene bases polyplexes and their improved compatibility with erythrocytes makes them interesting for in vivo applications.

Bioavailability of selenium from fish, yeast and selenate: a comparative study in humans using stable isotopes.

OBJECTIVE: To measure the bioavailability of selenium from cooked and raw fish in humans by estimating and comparing apparent absorption and retention of selenium in biosynthetically labelled fish with labelled selenate and biosynthetically labelled selenium in brewers yeast. DESIGN: The intervention study was a parallel, randomised, reference substance controlled design carried out at two different centres in Europe. SETTING: The human study was carried out at the Institute of Food Research, Norwich, UK and at TNO Nutrition and Food Research, Zeist, The Netherlands. SUBJECTS: In all, 35 male volunteers aged 18-50 y were recruited; 17 subjects were studied in Norwich (UK) and 18 in Zeist (Netherlands). All of the recruited subjects completed the study. INTERVENTIONS: Biosynthetically labelled trout fish (processed by two different methods), biosynthetically labelled brewers yeast and isotopically labelled selenate were used to estimate selenium apparent absorption and retention by quantitative analysis of stable isotope labels recovered in faeces and urine. Subjects consumed the labelled foods in four meals over two consecutive days and absorption was measured by the luminal disappearance method over 10 days. Urinary clearance of isotopic labels was measured over 7 days to enable retention to be calculated. RESULTS: Apparent absorption of selenium from fish was similar to selenate and there was no difference between the two processing methods used. However, retention of fish selenium was significantly higher than selenate (P<0.001). Apparent absorption and retention of yeast selenium was significantly different (P<0.001) from both fish selenium and selenate. CONCLUSION: Fish selenium is a highly bioavailable source of dietary selenium. Cooking did not affect selenium apparent absorption or retention from fish. Selenium from yeast is less bioavailable.

Water-soluble biodegradable cationic polyphosphazenes for gene delivery.

Polyphosphazenes bearing cationic moieties were synthesized from poly(dichloro)phosphazene, which in turn was obtained by thermal polymerization of hexachlorocyclotriphosphazene in 1,2,4-trichlorobenzene. Next, either 2-dimethylaminoethanol (DMAE) or 2-dimethylaminoethylamine (DMAEA) side groups were introduced by a substitution reaction. The polymers were purified by dialysis against water and tetrahydrofuran, lyophilized and evaluated as polymeric transfectants. The polyphosphazenes were able to bind plasmid DNA yielding positively charged particles (polyplexes) with a size around 80 nm at a polymer/DNA ratio of 3:1 (w/w). The polyphosphazene-based polyplexes were able to transfect COS-7 cells in vitro with an efficiency comparable to a well-known polymeric transfectant [poly(2-dimethylaminoethyl methacrylate), pDMAEMA]. The toxicity of both polyphosphazenes was lower than pDMAEMA. The transfection efficiency for the poly(DMAE)phosphazene-based polyplexes was about threefold higher in the absence of serum than in the presence of 5.0% fetal bovine serum. This is probably caused by unfavorable interactions of the polyplexes with serum proteins. In contrast, the poly(DMAEA)phosphazene-based polyplexes showed a threefold lower transfection activity in the absence of serum. For this system, serum proteins likely masked the toxicity of the polyplexes, as shown by the XTT cell viability assay and confocal laser scanning microscopy studies. Preliminary degradation studies indicate that the polymers were indeed degradable. The half-life at pH 7.5 and 37 degrees C was around 7 days for poly(DMAE)phosphazenes and 24 days for poly(DMAEA)phosphazenes. This study shows that polyphosphazenes are a suitable and promising new class of biodegradable polymeric carriers for gene delivery.

Upgrading of maatjes herring byproducts: production of crude fish oil.

Fish oil has been extracted from byproducts of the maatjes (salted) herring production using a pilot plant consisting of a mincer, heat exchanger, and three-phase decanter. The crude herring oil obtained had an initial peroxide value (PV), anisidine value (AV) and free fatty acids (FFA) level of only 3 mequiv of peroxide/kg of lipid, 8.9, and 2.9%, respectively. 5,8,11,14,17-Eicosapentaenoic acid (EPA) and 4,7,10,13,16,19-docosahexaenoic acid (DHA) were present in considerable amounts (99 and 91 g/kg, respectively). During storage of the oil, no photooxidation could be detected. Storage at room temperature led to significant autoxidation over time, apparent from primary and tertiary oxidation products, measured by a decrease of hydroperoxides and an increase of fluorescent compounds (FC). Storage at 50 degrees C resulted in significant increases in secondary (AV) and tertiary oxidation (FC) products. At all storage conditions, the FFA contents remained low (<3%) and the alpha-tocopherol content remained constant. These results open the possibility for fish oil production of good quality using salted herring byproducts.

Characterisation of fatty acids in biological oil samples using comprehensive multidimensional gas chromatography.

Comprehensive multidimensional gas chromatography can adequately resolve very complex mixtures of analytes such as the fatty acid mixtures which are contained in, e.g., fish and vegetable oils. Well-ordered patterns are obtained in the two-dimensional separation plane which can be used to tentatively identify peaks when no standard is available. The technique which can also be used for quantification, i.e., quantitative ratio analysis, should be especially useful for fingerprinting purposes. Unravelling the composition of complex mixtures such as fish oils appears to be highly rewarding.

Identification of fish species after cooking by SDS-PAGE and urea IEF: a collaborative study.

A collaborative study, to validate the use of SDS-PAGE and urea IEF, for the identification of fish species after cooking has been performed by nine laboratories. By following optimized standard operation procedures, 10 commercially important species (Atlantic salmon, sea trout, rainbow trout, turbot, Alaska pollock, pollack, pink salmon, Arctic char, chum salmon, and New Zealand hake) had to be identified by comparison with 22 reference samples. Some differences in the recoveries of proteins from cooked fish flesh were noted between the urea and the SDS extraction procedures used. Generally, the urea extraction procedure appears to be less efficient than the SDS extraction for protein solubilization. Except for some species belonging to the Salmonidae family (Salmo, Oncorhynchus), both of the analytical techniques tested (urea IEF, SDS-PAGE) enabled identification of the species of the samples to be established. With urea IEF, two laboratories could not differentiate Salmo salar from Salmo trutta. The same difficulties were noted for differentiation between Oncorhynchus gorbuscha and Oncorhynchus keta samples. With SDS-PAGE, three laboratories had some difficulties in identifying the S. trutta samples. However, in the contrast with the previous technique, SDS-PAGE allows the characterization of most of the Oncorhynchus species tested. Only Oncorhynchus mykiss was not clearly recognized by one laboratory. Therefore, SDS-PAGE (Excel gel homogeneous 15%) appears to be better for the identification, after cooking, of fish such as the tuna and salmon species which are characterized by neutral and basic protein bands, and urea IEF (CleanGel) is better for the gadoid species, which are characterized by acid protein bands (parvalbumins). Nevertheless, in contentious cases it is preferable to use both analytical methods.

A standardized method of identification of raw and heat-processed fish by urea isoelectric focusing: a collaborative study.

A urea-isoelectric focusing (urea-IEF) method of identifying fish species in processed fishery products was investigated as an interlaboratory collaborative study. The technique was optimized with respect to (i) protein extraction conditions, composition of the extraction solution (urea and SDS solutions), determination of protein concentrations of the fish extracts (five tested methods); (ii) nature of gel (with carrier ampholytes and Immobilines), conditions of rehydration of commercial dry gels, urea concentration; (iii) staining conditions, Coomassie blue and silver staining. The results of various experiments were compared to select the most appropriate methodology, with respect to the discrimination power of differentiating species with the minimal influence of heat processing, reproducibility, speed, and ease of application. The method recommended meets the requirements of food control and customs laboratories.

Diffusibility of selenate, selenite, seleno-methionine, and seleno-cystine during simulated gastrointestinal digestion.

The present study was undertaken to evaluate the in vitro availability of chemically varying forms of selenium (Se), supplemented in cow's milk. Two inorganic (selenite and selenate) and two organic (seleno-methionine [Se-Met] and seleno-cystine [Se-Cys]) Se sources were evaluated. The in vitro availability was estimated by the diffusibility of Se during simulated gastrointestinal digestion. First, the diffusibility was compared after adding a constant amount of Se as either selenate, selenite, seleno-methionine, or Se-Cys in milk samples. Se-Met and selenate were found to be significantly more diffusible than seleno-cystine and selenite under the simulated gastrointestinal conditions. The tendency for superior in vitro availability of selenate and Se-Met compared to selenite and Se-Cys was confirmed for a supplementation range of 5-40 ng/g of Se. This study suggests that the high diffusibility of selenate and Se-Met in a simulated gastrointestinal environment may contribute to their high absorption in vivo.

Bioavailability of calcium of fresh cheeses, enteral food and mineral water. A study with stable calcium isotopes in young adult women.

True fractional Ca absorption from six foods was measured in twelve normal healthy women, aged 20-29 years. The tested foods were commercially available fresh cheese, fresh cheese prepared by new technology and rich in Ca, similar cheese with added Fe, enteral food, mineral water alone and combined with a spaghetti meal. The aim of the study was to investigate: (1) Ca absorption from a new Ca-rich fresh cheese and to compare it with that from the traditional commercial type of fresh cheese; (2) the effect of Fe enrichment of the new cheese on Ca absorption; (3) Ca absorption from the mineral water and the enteral product and to compare it with that from the dairy products; (4) the effect of a meal combined with the mineral water on Ca absorption. All test foods were consumed by all subjects according to a design with two Latin squares. Each treatment of 2 d was followed by a wash-out period of 2 weeks. Ca absorption was measured using a double stable-isotope (44Ca and 48Ca) extrinsic labelling technique. Mean fractional Ca absorption from the new fresh cheese was not significantly different from that from the traditional type (37.7 (SD 10.2)% v. 42.2 (SD 11.6)%). The addition of Fe to the new cheese did not significantly influence Ca absorption. Ca-absorption values from the mineral water (37.0 (SD 9.8)%) and from the enteral product (42.6 (SD 11.4)%) were not significantly different from those from the dairy products (37.7-42.2%, SD 10.2-11.6%). The co-ingestion of a spaghetti meal with the mineral water significantly enhanced Ca absorption from 37 (SD 9.8)% to 46.1 (SD 11.7)%. It is concluded that a new process leading to a fresh cheese with a higher Ca concentration does not alter Ca bioavailability compared with the standard technology and for a constant Ca supply. Thus this new fresh cheese would probably provide more Ca than the standard one. The fractional Ca-absorption values for mineral water and the enteral product indicate that these products can make an interesting contribution to Ca supply for populations with a low Ca intake and patients with specific diseases respectively.

Estimation of selenium bioavailability from human, cow's, goat and sheep milk by an in vitro method.

The trace element selenium (Se) has been recognized to be essential for human health. The dependence of infants on milk as their principal food source, generally low in Se content, makes them more vulnerable to inadequate Se intake. The present study compared the Se availability as estimated by a simulated gastrointestinal digestion procedure, of human milk and some common ruminant milks, namely cow, goat and sheep milk. The Se availability of human milk (11.1%) was significantly higher compared to that of cow (6.8%), goat (6.2%) and sheep milk ( < 2%). Further study suggested that the Se availability may be related to the gastric digestibility of protein. The high Se availability of human milk might be attributed to the high gastric digestibility of human milk protein. It was found that removal of the milk fat fraction increases the Se availability.

Modification of an in-vitro method for estimating the bioavailability of zinc and calcium from foods.

A continuous flow in-vitro method for estimating the bioavailability of minerals and trace elements was modified. This modified method includes a simulated gastric digestion with pepsin, gradual pH change during the first 30 min of dialysis in an Amicon stirred cell, and a further 2 h of continuous dialysis accompanied by intestinal digestion with pancreatin-bile extract. The percentage of continuously dialysed minerals or trace elements (dialysability) is used to express the bioavailability. Comparison of different in-vitro methods by using the dialysability of zinc and calcium from milk- and soy-based formula samples revealed that with the modified method the results are closer to the in-vivo situation and could be used as a relative index for predicting the bioavailability of some minerals and trace elements.

Comparison of renal toxicity after long-term oral administration of cadmium chloride and cadmium-metallothionein in rats.

There is a clear lack of information on the toxicological risk of dietary intake of cadmium-metallothionein (CdMt). The present study aimed at establishing dose-dependent cadmium (Cd) disposition and to investigate differences in renal toxicity after long-term dietary exposure to CdMt or cadmium chloride (CdCl2) in rats. Male Wistar rats were fed diets containing 0.3, 3, 30, or 90 mg Cd/kg either as CdMt or as CdCl2 for 10 months. In rats fed 30 and 90 mg/kg Cd as CdCl2 the Cd concentrations in intestine, liver, and kidneys were all higher than in rats fed the same doses in the form of CdMt. The kidney/liver Cd concentration ratio was higher with CdMt than with CdCl2. At the lower Cd concentrations (0.3 and 3 mg/kg), no differences in Cd accumulation between CdMt and CdCl2 groups were observed and the kidney/liver Cd ratio was also similar. When based on the amount of CdMt per milligram Cd in the tissue, rats fed CdMt and those fed CdCl2 had a similar relative CdMt concentration in liver and kidney. First signs of renal injury, indicated by an increase of urinary lactate dehydrogenase (LDH) activity, were seen 4 months after exposure to 90 mg/kg Cd as CdCl2. After 8 and 10 months the renal effect of 90 mg/kg Cd as CdCl2 became more pronounced and urinary enzyme activities of LDH, N-acetyl-beta-D-glucosaminidase and alkaline phosphatase were all elevated. The only clinical effect of CdMt at the dose level of 90 mg/kg was a slight increase in urinary gamma-glutamyl transpeptidase activity at 8 and 10 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Mass spectrometric methods for studying nutrient mineral and trace element absorption and metabolism in humans using stable isotopes. A review.

Mass spectrometric methods for determining stable isotopes of nutrient minerals and trace elements in human metabolic studies are described and discussed. The advantages and disadvantages of the techniques of electron ionization, fast atom bombardment, thermal ionization, and inductively coupled plasma and gas chromatography mass spectrometry are evaluated with reference to their accuracy, precision, sensitivity, and convenience, and the demands of human nutrition research. Examples of specific applications are described and the significance of current developments in mass spectrometry are discussed with reference to present and probable future research needs.

Dietary iron lowers the intestinal uptake of cadmium-metallothionein in rats.

It has been shown that addition of extra calcium/phosphorus (Ca/P), zinc (Zn) and iron (Fe2+) to the diet results in a significant protection against cadmium (Cd) accumulation and toxicity in rats fed inorganic Cd salt. However, it is not clear whether the presence of these mineral supplements in the diet also protects against the Cd uptake from cadmium-metallothionein. The present study examines the influence of Ca/P, Zn and Fe2+ on the Cd disposition in rats fed diets containing either 1.5 and 8 mg Cd/kg diet as cadmium-metallothionein (CdMt) or as cadmium chloride (CdCl2) for 4 weeks. The feeding of Cd resulted in a dose-dependent increase of Cd in intestine, liver and kidneys. The total Cd uptake in liver and kidneys after exposure to CdMt was lower than after exposure to CdCl2. At the low dietary Cd level and after addition of the mineral supplement, the kidney/liver concentration ratio increased. However, this ratio was always higher with CdMt than with CdCl2, suggesting a selective renal disposition of dietary CdMt. The uptake of Cd from CdCl2 as well as from CdMt was significantly decreased by the presence of a combined mineral supplement of Ca/P, Zn and Fe2+. The protection which could be achieved was 72 and 75% for CdMt and 85 and 92% for CdCl2 after doses of 1.5 mg/kg and 8 mg/kg respectively. In a following experiment it was shown that the protective effect of the mineral mixture against CdMt was mainly due to the presence of Fe2+. It seems clear that Cd speciation and the mineral status of the diet have a considerable impact on the extent of Cd uptake in rats.

Comparative toxicity and accumulation of cadmium chloride and cadmium-metallothionein in primary cells and cell lines of rat intestine, liver and kidney.

The protective role of metallothionein (Mt) in the toxicity of cadmium (Cd) is controversial, since Cd bound to Mt is more nephrotoxic than ionic Cd after parenteral exposure and less hepatotoxic than ionic Cd after oral exposure. This study compared the uptake and toxicity in vitro of CdCl(2) and two isoforms of rat cadmium-metallothionein (CdMt-1 and CdMt-2) using primary rat kidney cortex cells, primary rat hepatocytes, liver hepatoma cell line H-35, kidney epithelial cell line NRK52-E and intestinal epithelial cell line IEC-18. The molar ratio of Cd was 2.1 and 1.4 mol Cd/mol Mt for CdMt-1 and CdMt-2, respectively. Monolayer cultures were incubated for 22 hr with CdCl(2), CdMt-1 or CdMt-2 and Cd accumulation was examined at Cd levels of 0.25-10 muM-Cd. Cells exposed to CdCl(2) accumulated more Cd in 22 hr than cells exposed to an equimolar amount of CdMt. For CdCl(2) the Cd accumulation is directly related to the Cd concentration in the medium; however, for CdMt an increase in Cd concentration in the medium above 2 muM had no effect on the Cd accumulation in the cells. At Cd concentrations above 2 muM, therefore, the difference in Cd accumulation between CdCl(2) and CdMt was greater (5-6 times) than at concentrations below 2 muM (1-2 times). Cytotoxicity was examined in the Cd-concentration range from 0.25 to 100 muM by determining the lactate dehydrogenase (LDH) release in the medium and the neutral red uptake in the cells. Under these culture conditions CdCl(2) was at least 100 times more toxic than CdMt-1 or CdMt-2 in all cell types tested. Primary hepatocyte cultures were 10 times more sensitive (50% LDH release at 1-2 muM) to CdCl(2) intoxication than primary cultures of renal cortical cells or the intestinal cell line (50% LDH release at 10-20 muM). Hepatic and renal cell lines were less sensitive (50% LDH release at 20-35 muM) than the corresponding primary cultures. No difference in sensitivity towards CdMt-1 or CdMt-2 was found for the various cell types tested. To investigate the influence of the molar Cd ratio of CdMt on cytotoxicity, the Cd content of CdMt-1 (2.1 mol Cd/mol Mt) was artificially raised in vitro to 5 mol/mol Mt. Compared with native CdMt, CdMt with a high molar Cd ratio in primary renal cultures showed a 15% increase in LDH release at a Cd concentration of 1500 muM in the medium. In conclusion, exogenous CdMt is far less toxic than CdCl(2) to cell cultures in a serum-free medium. Whereas CdCl(2) in all cases showed dose-dependent Cd accumulation, Cd accumulation due to CdMt exposure in all cell types tested reached a plateau at medium Cd concentrations of 2 muM. The low cellular Cd uptake of CdMt and the corresponding low cytotoxicity supports previously reported results in vivo, showing that the difference in toxicity between CdMt and CdCl(2) is associated with a difference in Cd distribution.

Cadmium accumulation and metallothionein concentrations after 4-week dietary exposure to cadmium chloride or cadmium-metallothionein in rats.

The distribution of cadmium was examined in rats fed diets containing either cadmium-metallothionein (CdMt) or cadmium chloride (CdCl2) for 4 weeks. The test diets contained 3, 10, or 30 mg Cd/kg diet (3, 10, or 30 ppm) as CdMt or 30 mg Cd/kg diet (30 ppm) as CdCl2. A second study was performed to establish the Cd content in liver and kidneys after exposure to low doses of both CdMt and CdCl2 (1.5 and 8 ppm Cd). The feeding of CdMt resulted in a dose- and time-dependent increase of the Cd concentration in liver, kidneys, and intestinal mucosa. Rats fed 30 ppm CdMt consistently showed less Cd accumulation in liver and intestinal mucosa than did rats fed 30 ppm CdCl2. However, renal accumulation in rats fed 30 ppm was similar until Day 28 regardless of Cd form. At lower dietary Cd levels (1.5 and 8 ppm), relatively more Cd is deposited in the kidneys, although even at these doses the kidney/liver ratio of Cd is still higher with CdMt than with CdCl2. Tissue metallothionein (Mt) levels in the intestinal mucosa were relatively constant but always higher after CdCl2 exposure than after CdMt exposure. Mt levels in both liver and kidney increased after CdCl2 or CdMt exposure during the course of study. Although Mt levels in liver were higher after CdCl2 intake (30 ppm) than after CdMt intake (30 ppm), renal Mt concentrations were the same for both groups. In fact on Day 7, CdMt administration resulted in slightly higher Mt levels than CdCl2 administration, suggesting a direct accumulation of exogenous CdMt in the kidneys. In conclusion, after oral exposure to CdMt in the diet there is a relatively higher Cd accumulation in the kidneys. However, the indirect renal accumulation via redistribution of Cd from the liver might be lower than after CdCl2 exposure. Which of these two phenomena is decisive in the eventual level of renal toxicity of Cd after long-term oral intake could determine the toxicological risk of the chronic intake of biologically incorporated Cd.

Interaction of dietary Ca, P, Mg, Mn, Cu, Fe, Zn and Se with the accumulation and oral toxicity of cadmium in rats.

The toxicity of Cd was examined in rats fed diets containing 30 mg Cd/kg as CdCl2 for 8 wk. The Cd-containing diets were supplemented with various combinations of the minerals Ca, P, Mg, Mn, Cu, Fe, Zn and Se in order to investigate the protective effect of these mineral combinations on Cd accumulation and toxicity. The mineral combinations were chosen such that the effect of the individual components could be analysed. At the end of the 8-wk feeding period, the Cd concentrations in the liver and renal cortex were 13.9 and 19.5 mg/kg body weight, respectively. The feeding of 30 mg Cd/kg diet alone resulted in well known Cd effects, such as growth retardation, slight anaemia, increased plasma transaminase activities and alteration of Fe accumulation. Only supplements that contained extra Fe resulted in a significant protection against Cd accumulation and toxicity. The most pronounced effect was obtained using a supplement of Ca/P, Fe and Zn, which resulted in a 70-80% reduction in Cd accumulation in the liver and kidneys, as well as a reduction in Cd toxicity. The protective effect of the mineral combinations was mainly due to the presence of Fe2+, but in combinations with Ca/P and Zn the effect of Fe was most pronounced. Compared with Fe2+ the protective effect of Fe3+ was significantly lower. Addition of ascorbic acid to Fe in both forms improved the Fe uptake, but consequently did not decrease Cd accumulation. Thus, the mineral status of the diet may have a considerable impact on the accumulation and toxicity of Cd, fed as CdCl2 in laboratory animals. For the risk assessment of Cd intake, special consideration should be given to an adequate intake of Fe.

Comparison of the toxicity of inorganic and liver-incorporated cadmium: a 4-wk feeding study in rats.

The toxicity of cadmium was examined in rats fed diets containing either tissue-incorporated cadmium or cadmium salt for 4 wk. The test diets contained 30 mg cadmium/kg either as cadmium chloride, or as cadmium incorporated in pigs' livers; the control group was fed a diet containing liver from a pig not treated with cadmium. Over 90% of the cadmium present in the pigs' livers was bound to metallothionein. Analysis of the diet and determination of the food consumption revealed that both cadmium-fed groups were exposed to similar dietary cadmium levels. There was no adverse effect on general health or survival. Feeding cadmium resulted in growth retardation and slightly decreased water intake. Moreover, both cadmium-treated groups showed clear signs of anaemia and increased plasma aspartate and alanine aminotransferase activities. For the group fed cadmium chloride, all of these effects were more pronounced than for the group fed cadmium incorporated in liver. Microscopic examination of the liver and kidneys, however, did not reveal any lesion that could be attributed to the cadmium treatment. After exposure to cadmium the spleen showed decreased extramedullary haematopoiesis, an effect that was also more pronounced after feeding of the cadmium chloride than after feeding liver-incorporated cadmium. The differences in the extent of the toxic effects between the inorganic and the tissue-incorporated cadmium were accompanied by differences in the cadmium concentrations in liver and kidneys: the feeding of cadmium incorporated in pigs' livers resulted in about half the accumulation of cadmium in the rats' livers that took place after intake of a diet containing cadmium chloride. In contrast a much less marked difference in cadmium accumulation was observed in the kidneys. Since humans are usually exposed to tissue-incorporated cadmium these findings deserve further investigation, with special attention to the observed difference in tissue accumulation.