Liquid chromatographic determination of urea in water-soluble urea-formaldehyde fertilizer products and in aqueous urea solutions: collaborative study.

Water soluble urea-formaldehyde (UF) fertilizers, manufactured by complex reaction of urea and formaldehyde, typically contain varying amounts of unreacted urea. A liquid chromatography method for the analysis of urea in these products, and in aqueous urea solutions, was collaboratively studied. An amine chromatography column was used to separate the unreacted urea from numerous UF reaction products present in these liquid fertilizers. Unreacted urea was determined by using external urea standards with UV detection at 195 nm. The standards and test samples were prepared in the mobile phase of 85% (v/v) acetonitrile in water. Ten laboratories analyzed 5 different UF-based commercial products containing unreacted urea in the range of 6 to 17% by weight, and 5 different concentrations of urea in water equivalent to commercial products of that nature. The aqueous urea solutions contained 2-20% urea (w/w). The range of s(R) values for the 5 UF-based commercial fertilizers was 0.49-1.02 and the %RSD(R) was 1.94-6.14. The s(R) range for the 5 urea solutions was 0.10 to 0.79 and the %RSD(R) range was 2.54 to 4.88. The average recovery of urea from the aqueous urea solutions was 96-103%. Therefore, this method is capable of monitoring urea nitrogen manufacturers' label claims and total nitrogen claims in those cases where urea is the sole source of plant food nitrogen. Based on the collaborative study data, the authors recommend this method be approved for AOAC Official First Action status.

In vivo incorporation of [14C]leucine into brain protein of mice treated with methylmercury and thiol complexes of methylmercury.

The effect of methylmercury and thiol complexes of methylmercury on inhibition of protein synthesis was evaluated. Mice were injected (i.p.) with the following treatments: methylmercuric chloride, methylmercury-glutathione, methylmercury-cysteinylglycine and control (vehicle) for 10 days. Ten animals from each group were injected with [14C]leucine 90 min prior to death. The brains were removed and the extracted protein was subjected to liquid scintillation analysis. Mice receiving the methylmercury and methylmercury-glutathione treatments exhibited significantly greater weight loss than the control while the methylmercury-cysteinylglycine treatment was not significantly different than the control. Incorporation of [14C]leucine into brain protein was significantly depressed in the methylmercury (81% of control) and the methylmercury-glutathione (79% of control) treatments. Protein synthesis in mice receiving the methylmercury-cysteinylglycine complex although not significantly different than the methylmercury treatments was only 92% of the control mice.

The toxicity, distribution and elimination of methylmercury in mice following intracerebral injection.

Intracerebral injection of methylmercury (CH3Hg) into the mouse brain resulted in significant weight loss and the appearance of characteristic neurological disturbances associated with CH3Hg intoxication. Neurological effects appeared dependent upon a minimum injected dose of 16 micrograms of CH3Hg corresponding to a CH3Hg concentration in the brain of 32 micrograms/g. Methylmercury was rapidly eliminated from the brain resulting in 40% and 5% remaining in the brain at 10 min and 7 days, respectively. The half-lives of CH3Hg in the tissues/organs were relatively short, ranging from 1.6 days for the cerebellum to 9.9 days for the liver and intestine. At the 10 min interval following injection, 22% of the injected 203Hg was found in the red blood cells which declined to 3% at the end of 7 days. The kidney concentration of 203Hg rapidly increased to 8% of the injected dose at 4 hr and remained at 5% of the body CH3Hg burden after 8 hr. The rapid elimination of 203Hg from the brain following intracerebral injection indicates that the blood brain barrier does not play a significant role in the retention of CH3Hg.

Influence of dietary zinc and cadmium on iron bioavailability in mice and rats: oyster versus salt sources.

Two feeding studies with young mice and one in situ intestinal perfusion study with adult rats were conducted to evaluate the influences of intrinsic (oyster) and extrinsic sources of cadmium and zinc on iron metabolism. When oyster was included in the diets, less cadmium accumulated in small intestines whether the cadmium was supplied as cadmium chloride or cadmium intrinsic to oyster. Increasing the zinc concentration of diets containing 2 ppm cadmium reduced cadmium retention in the small intestine regardless of whether the zinc supplied was intrinsic to oyster or as zinc carbonate. Dietary cadmium (20 ppm) reduced iron retention in the small intestine. Increasing dietary intrinsic zinc from 290 to 450 ppm reduced iron retention in small intestine whereas zinc carbonate did not. Inclusion of oyster in low cadmium diets reduced iron retention in the liver. Short-term in situ studies indicated salt sources of cadmium and zinc reduce uptake of iron from the intestine. Iron concentrations in the blood peaked between 20 and 55 min after exposure whether the iron was supplied alone or in combination with cadmium or zinc. The results suggest foods containing high concentrations of cadmium and zinc may reduce the availability of iron.

Toxicity, distribution, and elimination of thiol complexes of methylmercury after intracerebral injection.

Intracerebral injection of CH3Hg and CH3Hg complexed with glutathione (GSH), cysteine (cys), cysteinylglycine (cys-gly), and homocysteine (homocys) resulted in differences in toxicity. Criteria based on neurological indices, mortality, and weight loss indicated that the cys-gly complex of CH3Hg was significantly less toxic than CH3Hg or the other complexes. The other complexes of CH3Hg (GSH, homocys, and cys) were also found to be less toxic than CH3Hg. The selenium status of the animal did not seem to significantly influence the toxicity of CH3Hg and the complexes. While CH3Hg complexed to cys-gly was significantly less toxic than CH3Hg alone, there were no differences observed in the CH3Hg half-life values or in the distribution of these compounds in the kidneys, brain, liver, and blood. It was observed, however, that the CH3Hg--cys-gly complex had higher fecal excretion on d 3 and 4 following intracerebral injection.

Se-dependent GSH-peroxidase isolated from black sea bass (Centropristis striata).

Black sea bass were injected i.p. with Na2SeO3 (Se-75). A GSH-dependent peroxidase was extracted from the liver via differential centrifugation and gel filtration chromatography. Sea bass liver GSH-peroxidase eluted coincidently with Se-75 and was estimated to have a molecular weight of 72,000. The nature of the selenium-protein bond in the peroxidase was investigated by dialysis vs sodium selenite, glutathione (a sulfhydryl reagent) and alkali. It was concluded that the Se-75 was associated with the peroxidase through a non-sulfhydryl linkage likely to be a C-Se bond. This study confirms that Se is found in a GSH-peroxidase fraction isolated from black sea bass and suggests that Se may play an important role in marine fish.

Iron metabolism of mice fed low levels of physiologically bound cadmium in oyster or cadmium chloride.

Three feeding studies were conducted with young mice to compare the toxicity of cadmium (Cd) occurring in oyster tissue to that of cadmium chloride (CdCl2). In the first experiment, 5 or 20 ppm dietary Cd as CdCl2 depressed blood hematocrit and hemoglobin concentrations, which were further depressed by the addition of up to 3% oyster to the diet. In the second experiment, 1.5 or 3.0 ppm dietary Cd as CdCl2 did not influence hematopoiesis after 14 days exposure. The percent of the Cd dose retained in the kidney was inversely related to dosage level, directly related in the liver and femur and unaffected by dosage level in the small intestine. The liver-to-kidney ratio of retained Cd increased markedly with dose. In the last experiment, 0, 1.8 or 3.6 ppm Cd as CdCl2 or 1.8 ppm Cd from oyster was fed to mice for 28 days. Diets containing intrinsic oyster Cd at 1.8 ppm were more effective than diets containing CdCl2 at 3.6 ppm Cd for depressing total serum iron (Fe) and percent transferrin saturation and for enhancing ceruloplasmin activity. Oyster Cd was retained at a much lower rate in all tissues compared to CdCl2. Oyster Cd was preferentially retained in the kidney compared to the liver. Intrinsic oyster Cd may be more potent in altering normal Fe metabolism than CdCl2. Interactions with other dietary constituents in oysters which may also influence Fe metabolism, particularly zinc (Zn) and copper (Cu), are discussed.

Comparison of pathways of copper metabolism in aorta and liver. A functional test of metallothionein.

Soluble fractions from chick liver and aorta were examined for copper-binding proteins. In liver a zinc-binding thionein appeared to be the major binding protein for copper. Aortic tissue contained only traces of this thionein protein. Unlike liver, moderate amounts of soluble copper in aorta showed no association with macromolecules. Chicks fed on copper-deficient diets for 8 days had one-third the liver copper concentrations of controls. Aortic copper concentration was decreased only slightly, but the activity of lysyl oxidase, a copper-dependent enzyme in aorta, was decreased significantly. Treating the deficient chicks with CuSO4 (1 mg/kg) restored liver copper rapidly. The increase correlated with the binding of copper to a 10 000-mol.wt. component in the soluble fraction. Aortic copper concentrations responded much less to the CuSO4 treatment, but lysyl oxidase activity was again measurable in the tissue. Radioactive isotopes of copper bound almost exclusively to the 10 000-mol.wt. component in liver and to components of mol.wt. 30 000 or above in aorta. Hardly any of the administered radioactivity appeared with the 10 000-mol.wt. components in aorta, and none was found with unbound copper. The 30 000-mol.wt. components in aorta showed superoxide dismutase activity that was sensitive to NaCN. They also showed the highest specific activity of copper of any other aorta component. A clear distinction was seen between the metabolism of copper in liver and aortic tissues. Whereas a copper thionein, metallothionein, was a major component in the liver pathway, it is doubtful that this protein plays a major role in the intracellular metabolism of copper in aortic tissue.

Copper and the synthesis of elastin and collagen.

Copper's role in connective tissue is linked to the enzyme lysyl oxidase. From a biochemical perspective, copper is a cofactor for the enzyme and a determinant of its activity in connective tissues. Lysyl oxidase catalyses a post-translational oxidation of certain lysine and hydroxylysine residues. The peptidyl aldehydes so formed become active centres for the formation of cross-links in collagen and elastin. Less well understood is how copper controls the steady-state activity of lysyl oxidase; the enzyme fails in copper deficiency. Giving copper to a deprived animal increases lysyl oxidase activity in aortic tissue. Such activation in vivo appears to require caeruloplasmin. Suspending aortic tissue in a copper-enriched growth medium also activates lysyl oxidase provided that tissue structure is kept intact. Activation in vitro occurs with the binding of copper to a large-molecular-weight component, presumably the enzyme. Binding will not occur if protein synthesis is blocked. These studies clearly show that the synthesis of mature elastin and collagen can be controlled by the availability of copper. They further suggest that transport of copper to aortic tissue and its engagement to lysyl oxidase are linked to the synthesis or lysyl oxidase, an extracellular carrier, or both.