Zinc binding: a difference between human and bovine milk.

Gel chromatography indicated that most of the zinc in cow's milk was associated with high-molecular-weight fractions, whereas zinc in human milk was associated with low-molecular-weight fractions. A species difference in zinc-binding ligands may explain why symptoms of the genetic disorder of zinc metabolism, acrodermatitis enteropathica, can be alleviated by feeding human but not cow's milk.

A comparison of the effects of dietary selenium on selenoprotein expression in rat brain and liver.

In studies with rodents, when dietary supplies of the essential nutrient Se are restricted, in most tissues there are parallel substantial losses of the element and the important antioxidant selenoenzyme glutathione peroxidase (GPx) for which it is a cofactor. In brain, however, there appears to be both a sequestration of Se and a conservation of GPx activity when dietary Se is limited. To further explore the relation between these phenomena, we have undertaken a comparison of the effects of diets low, normal and high in Se on GPx activity, and labeling of selenoproteins following short-term (72 h) in vivo exposure to 75Se, in subcellular fractions from rat brain and liver, the latter serving as a representative tissue which does not retain Se and is depleted of most GPx activity following dietary restriction. Brains and livers from animals on the three diets showed different patterns of response with respect to both GPx activity and retention of the 75Se dose. The low-Se diet (0.006 ppm) substantially reduced GPx activity in liver but not brain, while high levels (1 ppm) did not increase GPx in either tissue relative to a normal (0.1 ppm) intake. The 75Se was retained in brain homogenates and subcellular fractions to the greatest extent by rats on the restricted diet, while in liver, retention was greater in rats fed the normal supplement than in animals on either the low- or high-Se diets. Levels of non-protein-bound 75Se were higher in brain than liver and increased with dietary Se in both tissues. When proteins in brain and liver homogenates and subcellular fractions where separated by one-dimensional SDS-PAGE and exposed to X-ray film, the resulting autoradiograms revealed the existence of seven distinct selenoprotein bands in brain and eight in liver. Different patterns of selenoprotein expression were observed in subcellular fractions isolated from both tissues. Dependence of levels of individual selenoproteins on diet paralleled the effects on 75Se retention. Dietary influences on expression of protein bands tentatively identified as GPx were more pronounced in liver than brain. All of these observations provide further evidence of the unique nature of Se metabolism in brain.

Cataract formation is prevented by administration of verapamil to diabetic rats.

The purpose of the present study was to examine the effects on cataractogenesis of daily sc administration of the Ca2+ antagonist drug verapamil to diabetic rats. Streptozotocin-induced diabetic rats were given verapamil half-way through the 8-week experimental period or during the full 8 weeks of diabetes. Verapamil administration had no effect on the high blood glucose values, low circulating insulin levels, or elevated triglyceride and cholesterol concentrations in the diabetic rats. Untreated diabetic rats had a 90% incidence of cataracts. Four weeks of verapamil administration reduced this incidence to 41%, and a full 8 weeks of drug treatment further lowered the incidence to 20%. Diltiazem, another Ca2+ antagonist, lowered the incidence of cataracts in the diabetic rats to a similar extent. Verapamil administration to the diabetic animals also partially protected against the presence of retinal microangiopathy in the diabetic animals. Lenticular hydration and lipid accumulation were only indirectly related to cataractogenesis in the diabetic rats and its protection by verapamil treatment. Lenticular electrolyte imbalance, particularly Ca2+, in the diabetic animals was closely correlated with cataract formation, and verapamil significantly reduced the alterations in these ion concentrations. The present results demonstrate the efficacy of verapamil as a protective agent against cataractogenesis and some retinal damage in diabetic animals. Most importantly, this occurs in the absence of any change in the glycemic status of the diabetic animals. The findings strongly support a role for lenticular Ca2+ imbalance in cataract development in diabetes and provide initial evidence to suggest its clinical use in the diabetic population at risk for blindness.

Analysis of flavins in ocular tissues of the rabbit.

Riboflavin is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), coenzymes required for the activity of flavoenzymes involved in the transfer of electrons in oxidation-reduction reactions. Flavins are light sensitive and rapidly degrade when exposed to light in the near ultraviolet and visible wavelengths. Some of the byproducts of flavin photodegradation are toxic. A quantitative survey of flavins in rabbit ocular tissues is reported. Adult male Dutch-Belt Rabbits were fed purified diets containing 3, 30, or 300 mg riboflavin/kg for 1 month. A method of aqueous extraction and high-performance liquid chromatography with fluorescence detection was used to measure riboflavin, FMN, and FAD in cornea, lens cortex, lens nucleus, retina, and blood. The retina contained the highest flavin concentration. In all tissues, the primary flavin was FAD followed by FMN and riboflavin. The highest concentration of riboflavin occurred in the cornea followed by the retina, lens cortex, and lens nucleus. A trend toward increasing concentrations of riboflavin occurred in the retina and blood in response to excess dietary riboflavin, but the concentration changes were not statistically significant. The highest concentration of FAD and FMN occurred in the retina followed by the cornea and the lens cortex and nucleus. The relative contribution of riboflavin, FMN, and FAD to the total flavin pool was markedly different in the various tissues of the eye. The proportion of tissue flavins present as riboflavin decreased from anterior to posterior. It was highest in the cornea followed by lens and retina. The pattern of distribution for FMN was: cornea greater than retina greater than lens cortex and nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Parenteral nutrition is associated with intestinal morphologic and functional changes in humans.

BACKGROUND: Numerous animal studies have demonstrated intestinal villus atrophy occurs when luminal nutrition is withheld and total parenteral nutrition (TPN) is provided. Intestinal morphologic and functional changes have not been well studied in humans during TPN. METHODS: Eight normal volunteers were hospitalized in the Clinical Research Center for 3 weeks. The subjects received TPN as an exclusive means of nutritional support for 14 days followed by 5 days of enteral refeeding with either a standard or a glutamine and arginine-supplemented formula. Endoscopic jejunal biopsies were taken before and after TPN and after enteral refeeding. Intestinal morphology was examined by light and transmission electron microscopy. Mucosa DNA, RNA, and protein concentrations were measured. Lactose breath hydrogen and intestinal permeability testing (urinary lactulose and mannitol excretion after an oral dose) were performed before and after TPN and after enteral refeeding. RESULTS: Total mucosal thickness decreased after TPN (645 +/- 19 to 512 +/_ 19 microns, p = .003) and increased significantly towards baseline after enteral refeeding (575 +/- 19 microns, p = .04). The change was related solely to villus height; crypt depth was unaffected. Villus cell count decreased from 179 +/- 15 to 163 +/- 12 after TPN (p = .03) and increased after enteral refeeding to 176 +/- 21 (p = .06). Crypt cell count was unaffected by TPN or refeeding. A nonsignificant decrease in the mitotic index after TPN was seen. Intracellular edema developed during TPN and resolved with enteral refeeding. The urinary lactulose-mannitol ratio increased with TPN [0.06 +/- 0.03 to 0.11 +/- 0.05 after TPN and 0.14 +/_ 0.09 after short-term enteral refeeding (p = .05)], indicating increased intestinal permeability. The urinary lactulose-mannitol ratio was significantly greater after refeeding with standard formula than the free amino acid peptide formula with glutamine and arginine (0.20 +/- 0.05, vs 0.08 +/- 0.01, p = .05). No significant differences were noted in mucosal RNA, DNA, protein, DNA-protein or RNA-DNA rations or breath hydrogen after lactose ingestion after either TPN or enteral refeeding. No significant difference in plasma glutamine was found during TPN (462.7 +/ 38.7 vs 491.8 +/- 46.1 mumol/L) or after enteral refeeding (457.3 +/- 51.4 mumol/L). CONCLUSIONS: Intestinal morphologic and functional changes occur in human for whom TPN is the sole nutritional source, although the findings in humans are substantially less significant than observed in animal models. The loss of mucosal structure may be sufficient to cause increased intestinal permeability, the clinical significance of which remains to be defined. Enteral nutrition is important in restoring and probably preventing morphologic intestinal changes associated with TPN, and a peptide and free amino acid-based formula supplemented with glutamine and arginine may have some added role. Our findings also suggest sepsis is associated with gut adaptation rather than degradation.

Cardiac contracile protein ATPase activity in a diet induced model of noninsulin dependent diabetes mellitus.

The effects on cardiac function of feeding a diet high in sucrose to male Wistar rats over an extended period of time (15 months) was examined. This diet produced a diabetic condition which resembled noninsulin dependent diabetes mellitus. Resting hyperglycemia, high circulating insulin and triglyceride levels were observed in these animals. Further, the sucrose fed animals were overweight in comparison to chow fed control animals. Contractile protein Ca2+-ATPase activity was measured as a biochemical estimate of cardiac contractile function. Myosin and actomyosin Ca2+-ATPase activities of isolated myofibrillar fractions from hearts of experimental animals were depressed in comparison to chow fed control rats. Myosin K+-EDTA activity was also altered. The results demonstrate for the first time a defect in contractile protein Ca2+-ATPase activity in rat hearts using a model of noninsulin dependent diabetes mellitus. As the animals were euthyroid, thyroid hormone alterations in these animals were unlikely to influence the results. The results also demonstrate that insulin could not be a direct factor associated with cardiac pathology in diabetes. Instead, cardiac dysfunction may be associated with other, as yet undefined, metabolic abnormalities which accompany the diabetic state.

Search for Fc and C3b receptors on black-eyed RCS rat RPE cells.

In the retina, the membranous outer segments shed from the photoreceptors are phagocytized by the adjacent retinal pigment epithelial cells. These cells are some of the most active phagocytic cells in the body and like photoreceptors must survive the lifetime of the organism. The initiation of engulfment by the pigment epithelial cells occurs by an unidentified mechanism. The ingestion of particles by many, but not all phagocytic cells, is mediated by Fc and C3 receptors located on the external plasma membrane. The present experiment reports our unsuccessful attempt to demonstrate either the Fc or C3b receptor on the plasma membrane of RCS rat pigment epithelial cells.

Nutrient-genetic drive hypothesis of biological expression.

The cellular change from an ordered to anaplastic state occurs as a part of a normal biological process which works to increase the chance of survival of the genome. This change of state is driven primarily by changes in the micronutrient environment. The anaplastic state of cells provides a framework for dramatic changes in biological expression, and offers a means for the introduction of new species. Neoplasms are a product of this natural mechanism for survival of the genome with unfortunate consequences for the host cell population.

The response of trout and zebrafish embryos to low and high boron concentrations is U-shaped.

Fish in the embryo-larval stage of development have been shown to be sensitive to boron (B) at both ends of the dose-response curve (1,2). The present study evaluated the health effects of low and high B concentrations on rainbow trout (Oncorhynchus mykiss), a cold water species, and zebrafish (Danio rerio), a warm water species. Rainbow trout embryos were incubated from day 1 until 2 wk posthatch in Type 1 ASTM ultrapure-grade water (12.5 degrees C) supplemented with only B (0-500 microM) as boric acid, or together with CaCO3 (0-2 mM) to increase water hardness. Embryonic growth was stimulated by B in a dose-dependent manner at all Ca concentrations (p < 0.001). Chronic exposures below 9 micromol B/L impaired embryonic growth and above 10 mmol B/L caused death (p < 0.001). Thus, the safe range of exposure for the rainbow trout was between the adverse effect concentrations of 9 micromol B/L and 10 mmol B/L. Zebrafish were maintained for 6 mo in ultrapure water containing <0.2 micromol B/L to determine the effect of low-level exposure. High-level exposure was assessed by exposing zygotes, derived from parents maintained at 46 micromol B/L, to graded concentrations of boric acid up to a concentration of 75 mmol B/L from fertilization until they were free feeding (96 h). Fertilization occurred, but zygotes failed to survive when water contained <0.2 micromol B/L (p < 0.001). Death occurred at and above 9.2 mmol B/L. Thus, the safe range of B exposure for zebrafish was between the adverse effect concentrations of 0.2 micromol B/L and 9.2 mmol B/L. The dose-response for both species was thus U-shaped.

Cellular changes in boric acid-treated DU-145 prostate cancer cells.

Epidemiological, animal, and cell culture studies have identified boron as a chemopreventative agent in prostate cancer. The present objective was to identify boron-induced changes in the DU-145 human prostate cancer cell line. We show that prolonged exposure to pharmacologically-relevant levels of boric acid, the naturally occurring form of boron circulating in human plasma, induces the following morphological changes in cells: increases in granularity and intracellular vesicle content, enhanced cell spreading and decreased cell volume. Documented increases in beta-galactosidase activity suggest that boric acid induces conversion to a senescent-like cellular phenotype. Boric acid also causes a dose-dependent reduction in cyclins A-E, as well as MAPK proteins, suggesting their contribution to proliferative inhibition. Furthermore, treated cells display reduced adhesion, migration and invasion potential, along with F-actin changes indicative of reduced metastatic potential. Finally, the observation of media acidosis in treated cells correlated with an accumulation of lysosome-associated membrane protein type 2 (LAMP-2)-negative acidic compartments. The challenge of future studies will be to identify the underlying mechanism responsible for the observed cellular responses to this natural blood constituent.

Differential effects of riboflavin and RRR-alpha-tocopheryl acetate on the survival of newborn RCS rats with inheritable retinal degeneration.

The dystrophic RCS rat is one of the most important animal models available for investigating retinal degeneration. In addition to the characteristic progressive loss of neural retina the strain is hampered by a high rate of mortality during the first week of life. Death rate during this period is greatly influenced by diet. A 69% reduction in mortality was achieved by supplementing a purified diet with double the amount of AIN-76 vitamin mix. The objective of this study was to identify vitamin(s) in the AIN-76 mix responsible for the enhanced survival. The experiment determined the effect on survival of independently doubling the concentration of each vitamin present in the AIN-76 vitamin mix. This was done by single addition of individual vitamins to a complete purified diet. Survival was determined in litters whose mothers and grandmothers had been provided the supplemented diets as their sole source of food. Supplementation with riboflavin increased mortality by 19%, whereas RRR-alpha-tocopheryl acetate supplementation reduced the mortality by 73%. The effect of RRR-alpha-tocopheryl acetate was equivalent to that achieved by supplementation with complete vitamin mix. First-week survival of pups (born alive) rose from 72.3% +/- 11.0 to 92.5% +/- 3.8 when the level of vitamin E was increased from 50 to 100 IU/kg diet.

Isolation of a protein from human milk that enhances zinc absorption in humans.

Humans absorb zinc more readily from human milk than from the milk of other species. The basis for this species specificity has not been elucidated. This report describes the isolation of a human milk protein which enhances zinc absorption. The consumption of bovine cow's milk supplemented with the human protein resulted in an elevation in human plasma zinc of 101% compared to a 50% rise following the ingestion of cow's milk alone. The identification of this biologically active protein in human milk establishes in part the basis for the species difference in zinc bioavailability.

Elemental concentrations in ocular tissues of various species.

An investigation was undertaken to expand the data base for elemental concentrations within eye tissues of different species. This report provides data on the distribution of calcium, copper, iron and zinc in human, dog, bovine, bird, amphibian and fish ocular tissues. The variation between different eyes of the same individual and different individuals was calculated for each metal. Elemental concentrations between the left and right eyes of an individual were usually closer in value than between two eyes of different individuals.

Growth and survival of RCS dystrophic rats fed modifications of the AIN-76 diet.

The RCS dystrophic rat is a hooded, pigmented-eyed strain widely used as a model for retinal degeneration. In addition to progressive photoreceptor loss, this strain suffers from unexplainable high mortality during the suckling period when reared on commercial cereal-based diets. Supplementation of these diets with 25% sunflower seeds greatly reduces this problem. This report presents the results of a study undertaken to test the effectiveness of the purified AIN-76 diet in controlling the high mortality. Growth of F1 rats and percent survival of the F2 offspring were determined for RCS dystrophic rats provided one of 5 diets: (Control) modified AIN-76 diet (15% additional sucrose in place of starch, nd vitamin and mineral mixes made up in cellulose rather than powdered sucrose); control with double the content of vitamin mix; control with double the content of fat; control with double the content of mineral mix; and control with double the content of protein. The percent survival of the F2 offspring was increased from 73% to 92% by vitamin supplementation. Doubling the mineral or protein content proved lethal to 54% and 79% of offspring on these respective diets. Increasing the vitamin content also resulted in an improvement in the initial growth of females. Increased fat, mineral or protein content all decreased the growth rate of males. Likewise, growth of females was decreased when the mineral and protein content of the diet was raised.

Elevated body zinc in rats with inherited retinal dystrophy.

Carcass zinc concentrations were determined in Royal College of Surgeon (RCS) rats carrying the gene for retinal dystrophy. Analysis of zinc was undertaken on both mutant RCS (rdy/rdy) and cogenic control RCS (rdy/+) newborn male pups using atomic absorption spectrophotometry. The carcass of mutant rats contained a higher concentration of zinc but not copper. A relationship between the altered zinc concentrations and subsequent retinal degeneration remains to be determined.

Boron stimulates embryonic trout growth.

Boron is present in our soil, water and air. Cyanobacteria require it for nitrogen fixation, and vascular plants require it for the formation of cell walls and membranes. I report here how boron affects the growth of embryonic rainbow trout (Oncorhynchus mykiss). Fertilized ovum from the Mt. Whitney rainbow trout strain were incubated at (12.5 degreesC) in Type 1 ASTM ultrapure grade water supplemented with boric acid (99.5% purity) during the 1995 and 1997 spawning seasons. Boron concentrations of the incubation solutions were determined by direct measurement using the curcumin procedure or inductively coupled plasma-mass spectrometry. In the 1995 study boron ranged from 1 to 936 micromol/L. Ca, Na and Mg salts were included in the incubation solutions to approximate concentrations in natural water. In the 1997 study fertilized eggs were incubated in ultrapure water supplemented with boric acid alone over a range from 2.2 to 90.6 micromol/L. The 1995 study used 144 embryos per B concentration and the 1997 study used 96 embryos per B concentration. Growth and teratogenicity were evaluated at the eye, hatch and 2-wk posthatch developmental stages. Boron stimulated growth in a dose-dependent manner in both studies (P < 0.001), and exposure was associated with an increase in B body concentration (P < 0.05). No teratogenic or microbicidal effects were apparent. These results are consistent with those expected of an element essential for vertebrate development. J. Nutr. 2488-2493, 128: 1998

Identification of FAD, FMN, and riboflavin in the retina by microextraction and high-performance liquid chromatography.

The presence of flavins in the retina has been known for some time. However, the small size of the tissue has made it difficult to quantify the levels of the individual flavins, riboflavin (RB), FMN, and FAD without pooling large numbers of retinas. A procedure to extract and quantitate RB, FMN, and FAD in retinal tissue from as few as four rat retinas has been developed. The procedure resolves these three classes of flavins and provides a recovery near 100%. For the analysis, HPLC using a reverse-phase column with cyclohexyl functional groups was coupled to a fluorescence detector. The microextraction-HPLC procedure was reproducible for the quantitative analysis of flavins in the retina and equally applicable for analysis of flavins in liver and plasma.

Sucrose-induced lipid, glucose, and insulin elevations, microvascular injury, and selenium.

Injury to microvessels caused by the chronic consumption of sucrose can be prevented by selenium (Se). The objective of this study was to determine the temporal sequence of changes in serum triglycerides, total cholesterol, glucose, and insulin induced by sucrose and their relationship to Se status and microvascular injury. Two groups of 24 Wistar rats were fed ad libitum diets in which the entire carbohydrate was either corn starch or sucrose. Two other groups were fed identical diets supplemented with 0.1 micrograms Se/g. At 6, 8, and 10 mo, eight rats from each group were fasted for 12 h and had blood taken. Rats were then given a glucose tolerance test and killed, and their retinal microvessels were evaluated for injury. After 6 mo, sucrose-fed rats had elevated triglycerides and total cholesterol. Abnormal glucose clearance and hyperinsulemia developed after 8 mo. Evidence of microvascular injury became apparent after 10 mo. These changes did not occur in rats provided the starch-based diets, and microvascular injury did not develop in the sucrose-fed rats provided supplemental Se. Glutathione peroxidase activity was normal in all groups throughout the 10-mo experiment. These results chronicle the sucrose-induced systemic insult and show that the protective effect of Se does not occur by diminishing this insult to the microvessels.

Reduced DNA synthesis in zinc deficiency: regional differences in embryonic rats.

Zinc deficiency during prenatal life results in a high incidence of gross malformations especially of CNS. Reduced 3H-thymidine incorporation into rat embryos has previously been reported in zinc deficient rats. The effect of zinc deficiency on regional DNA synthesis in the 12 day rat embryo was therefore investigated. Zinc deficiency was achieved by feeding normal pregnant rats a diet containing 0.4 ppm zinc from day 0 to day 12. Pair-fed controls received a diet containing 100 ppm zinc. Dams were injected with 3H-thymidine on day 12 and embryos removed 1 hour later. In embryos from zinc deficient dams, there was a lower incorporation of 3H-thymidine into DNA in the head regions than in comparable regions from ad libitum and pair-fed controls. Total DNA and RNA contents in the head and body regions of embryos from zinc deficient dams were lower than respective regions of pair-fed controls, but the greatest deficit occurred in the head region. Replacement of zinc 28 hours prior to injection of the label increased the low incorporation of 3H-thymidine/DNA in the head region of zinc deficient embryos. Autoradiographs of the head region indicated that reduced uptake of 3H-thymidine and reversal by zinc replacement occurred mainly in the developing CNS. These results demonstrate that the reduction in DNA synthesis resulting from zinc deficiency can be reversed by zinc alone, and that zinc deficiency in the mammalian system does not result in a general reduction in DNA synthesis in all tissues, but the head region is more vulnerable to reductions in zinc than the body region. The observation that DNA synthesis in the head region is reduced by zinc deficiency more than is the rest of the body may explain the increased vulnerability of the CNS to prenatal zinc deficiency.