Mark L. Failla

Hepatocyte-specific localization and copper-dependent trafficking of the Wilson’s disease protein in the liver

Wilsons disease is an inherited disorder of copper metabolism characterized by hepatic cirrhosis and neuronal degeneration. In this current study, a polyclonal antiserum specific for the Wilsons disease ATPase was used to examine the hepatic expression of this protein. Immunoblot analysis of lysates from human and rat liver detected a single 165-kDa protein, which by immunofluorescence was present only in hepatocytes and localized predominantly to the trans-Golgi network and exclusively in this compartment under low hepatic copper concentrations. Although hepatic copper concentration had no effect on the steady-state levels of the Wilsons disease protein, copper administration in vivo resulted in redistribution of this protein to a cytoplasmic vesicular compartment localized toward the hepatocyte canalicular membrane. The relative abundance of the Wilsons disease protein in the liver was found to be greatest in the fetus before the onset of biliary copper excretion. Taken together, these studies reveal a novel posttranslational mechanism of copper homeostasis in vivo consistent with the proposed function of the Wilsons disease protein in holoceruloplasmin biosynthesis and biliary copper excretion and of relevance to the broad clinical heterogeneity observed in this disease.

Estimation of carotenoid bioavailability from fresh stir-fried vegetables using an in vitro digestion/ Caco-2 cell culture model

We previously developed an in vitro model to estimate the relative bioavailability of carotenoids from a meal prepared using commercial baby foods. The general applicability of this model was tested using a stir-fried meal consisting of fresh spinach, fresh carrots, tomato paste, and vegetable oil. After in vitro digestion of the cooked meal, the aqueous fraction was separated from residual oil droplet and solids by centrifugation to quantify micellarized carotenoids. The percentages of lutein, lycopene, alpha-carotene, and beta-carotene transferred from the meal to the micellar fraction were 29.0 +/- 0.6, 3.2 +/- 0.1, 14.7 +/- 0.3, and 16.0 +/- 0.4, respectively. Carotenoid transfer from the meal to the aqueous fraction was inhibited when bile extract was omitted from the intestinal phase of digestion. The bioavailability of the micellarized carotenoids was validated using differentiated cultures of Caco-2 human intestinal cells. All four carotenoids were accumulated in a linear manner throughout a 6-hr incubation period. Metabolic integrity was not compromised by exposure of cultures to the diluted aqueous fraction from the digested meal. The addition of 500 µmol/L alpha-tocopherol to test medium significantly improved the stability of the micellar carotenoids within the tissue culture environment. These results support the utility of the in vitro digestion procedure for estimating the bioavailability of carotenoids from foods and meals.

Accumulation and retention of micellar β-carotene and lutein by Caco-2 human intestinal cells

Despite the interest in the diverse roles of dietary carotenoids in human health, little is known about the transfer of these plant pigments from foods to micelles during digestion and their subsequent transfer across the intestinal epithelium. We conducted this study to characterize the intestinal uptake of micellarized carotenoids using monolayers of differentiated Caco-2 human intestinal cells. Crystalline beta-carotene (BC) and lutein (LUT), solubilized in mixed micelles for delivery to cells, were stable in a tissue culture environment for 20 hours. Cellular accumulation of micellar BC and LUT was proportional to the media content of carotenoids at </=2 micromol/L and the length of exposure. Cellular accumulation of BC routinely exceeded LUT and was due in part to the enhanced efflux or possible metabolism of LUT. Cellular BC content increased in a curvilinear manner when cultures were incubated in micellar medium containing 2 to 27 micromol/L BC prepared from water miscible beadlets; cellular BC content was maximum when medium BC was >/=18 micromol/L. There was no indication that high levels of BC in medium or within cells adversely affected micellar LUT accumulation. These data support the use of the Caco-2 human cell line as a model for studying the intestinal uptake, absorption, and possible interactions of dietary carotenoids.

Marginal copper-restricted diets produce altered cardiac ultrastructure in the rat.

Abstract To determine if chronic ingestion of a diet containing a marginally low level of Cu could cause deleterious alterations in cardiac ultrastructure, male offspring were nursed by dams fed a diet containing either 6.7 or 2.8 mg Cu/kg from midgestation through lactation before weaning to the same diet. Conventional measures of Cu status, including growth, relative heart weight, tissue concentrations of Cu, ceruloplasmin activity, and tissue activity of Cu, Zn-superoxide dismutase (SOD) were similar in both dietary treatment groups at 5.5 months of age. However, significant increases in the number and volume of lipid droplets and an increased incidence of pathological abnormalities in mitochondria and basal laminae were observed in sections of hearts from rats chronically fed the diet containing 2.8 mg/kg Cu. Reduction of the dietary level of Cu from 2.8 to 1.3 mg/kg from 4 to 5.5 months of age caused significant reductions in the concentration of Cu in serum and liver, but Cu content, Cu, Zn-SOD activity, pathological scores, and morphometric parameters in hearts were not modified by the greater restriction of dietary Cu in adult rats. This study suggests that abnormalities in cardiac ultrastructure occurred in rats chronically fed diets marginally low in Cu, despite minimal changes in conventional biochemical indicators of Cu status.

Ascorbate Offsets the Inhibitory Effect of Inositol Phosphates on Iron Uptake and Transport by Caco-2 Cells

Abstract Differentiated monolayer cultures of Caco-2 human intestinal cells were used as a model to examine interactions between various dietary factors related to the intestinal uptake and absorption of nonheme Fe. Caco-2 cells accumulated 91–98 pmol Fe/mg protein from uptake buffer containing 12 nmol of Fe(III)-nitrilotriacetate during a 1-hr incubation at 37°C. Addition of a 10-fold molar excess of inositol hexa-phosphate (IP6) and its lesser phosphorylated derivatives (IP3, IP4, and IP5) decreased cellular uptake and transport of Fe from the lumenal compartment. Addition of ascorbic acid (AA) to the solution containing IPs stimulated Fe uptake and transport in a manner dependent upon the ratio of AA to IP and inversely proportional to the degree of phosphorylation of inositol (i.e., IP3 > IP4 > IP5 > IP6). A mixture of essential amino acids had minimal impact on Fe uptake in either the absence or presence of IPs. Cellular acquisition of Fe from solutions containing IPs was further enhanced by simultaneous addition of essential amino acids and AA. The stimulatory influence of ascorbic acid on Fe uptake from solutions containing IP6 was associated with an increase in the level of ferrous ion. These data further support the usefulness of Caco-2 cells as a model for investigating the effects of various dietary factors on mineral bioavailability.

Considerations for determining ‘optimal nutrition’ for copper, zinc, manganese and molybdenum

Defining optimal dietary intakes of Cu and Zn throughout the life cycle continues to present a considerable challenge for nutrition scientists. Although the daily intake of these micronutrients is below that currently recommended for many groups, traditional biochemical indicators of nutritional status for these trace metals largely remain within the normal range. Thus, it is unclear whether the recommended daily intakes may be unnecessarily high, or if the commonly-used markers simply lack the necessary sensitivity and specificity that are required for accurately assessing Cu and Zn status. The increasing number of reports that daily supplements with these trace metals enhance the activities of selective metalloenzymes and specific cellular and organ processes further points out the need to differentiate between meeting the requirement and providing optimal nutriture. Results from recent studies suggesting that alternative molecular and functional markers possess sufficient sensitivity to better assess Cu and Zn status are discussed. Likewise, recent studies evaluating the impact of very low and excessive levels of dietary Mn and Mo on selective biochemical and metabolic indicators are reviewed.

Differentiation of Human U937 Promonocytic Cells Is Impaired by Moderate Copper Deficiency

Copper (Cu) deficiency suppresses macrophage activities in animals and humans. Our previous studies indicated that the Induction of Cu deficiency in differentiated U937 monocytic cells impairs respiratory burst and bactericidal activities and lipopolysaccharide-mediated secretion of inflammatory mediators. The current investigation examined the roles of Cu in the monocytic differentiation process. Human U937 promonocytic cells were exposed to a high affinity Cu chelator (5 µM 2,3,2-tetraamine [tet]) for 24 hr before inducing differentiation by treatment with 1,25-dihydroxyvitamin D3 plus Interferon-γ (DI). This procedure decreased cell Cu by 55% without compromising cellular Zn, Fe, or general metabolic activities. Lower Cu status significantly attenuated the expression of maturation markers Mac-1 (CD11b), ICAM-1 (CD54), and LPS-R (CD14). This change was associated with a marked suppression in respiratory burst activity and killing of Salmonella. To examine if the adverse effect of inadequate Cu on the DI-induced differentiation represented a more general defect, U937 cells were treated with phorbol 12-myrlstate 13-acetate (PMA). Lower Cu status also suppressed PMA-mediated differentiation of U937 cells. Supplemental Cu, but not Zn or Fe, blocked the tet-induced declines in cell Cu, expression of maturation markers, and respiratory burst and bactericidal activities. These results demonstrate that Cu is essential for the monocytic differentiation process that contributes to the competency of the hosts defense system.

Human lipoproteins as a vehicle for the delivery of beta-carotene and alpha-tocopherol to HepG2 cells.

Abstract Highly differentiated human cell lines represent a useful in vitro model for the study of carotenoid uptake, metabolism, and function. Carotenoids are usually introduced into tissue culture media either in organic solvents or as micelles, whereas carotenoids are localized in lipoproteins in vivo. Initially, the stability of β-carotene and α-tocopherol in micelles and human lipoproteins under standard tissue culture conditions was compared. Recovery of β-carotene and α-tocopherol was 27% ± 2% and 73% ± 2%, respectively, after overnight incubation of micellar β-carotene and α-tocopherol in serum-free medium without cells. This marked loss of β-carotene was attenuated by inclusion of α-tocopherol in micelles. In contrast, recovery of β-carotene and α-tocopherol was 88%–95% when medium containing the total lipoprotein fraction isolated from β-carotene supplemented individuals was incubated overnight without cells. Cellular accumulation of β-carotene and α-tocopherol from medium containing total lipoproteins (1 mg/ml) was proportional to their concentrations in the lipoprotein fraction (r = 0.94 for β-carotene and 0.74 for α-tocopherol). Cells exhibited similar capability of acquiring β-carotene and α-tocopherol from medium containing either low- or high-density lipoproteins. These data show that lipoproteins represent a stable vehicle for delivery of β-carotein and α-tocopherol to HepG2 human liver cells.

Expression of ceruloplasmin gene in human and rat lymphocytes

The acute phase plasma protein ceruloplasmin (Cp) appears to play some role in host defense. The possibility that production of Cp in extrahepatic sites may also be essential for the activation, effector functions and cytoprotection of immune cells in localized environments has received minimal attention. Here, we have surveyed various types of human and rat immune cells for the presence of Cp mRNA using RT-PCR with primers that span exons 17-19 as an initial step in addressing this possibility. Validated Cp RT-PCR bands were obtained from RNA samples isolated from resting and activated human lymphocytes, CD4 and CD8 T-cells and B-cells. Semiquantitative RT-PCR indicated that Cp mRNA in immune cells is present at about 0.2% the level of Cp mRNA in HepG-2 human liver cell line. Various human cell lines derived from the immune system, rat splenic MNC and purified rat T-lymphocytes also constitutively express Cp gene.

Transferrin-iron and proinflammatory cytokines influence iron status and apical iron transport efficiency of Caco-2 intestinal cell line

Abstract Endogenous factors that regulate the absorption of dietary iron remain unknown. Differentiated cultures of Caco-2 human intestinal cells grown on membrane inserts were used to study the characteristics of transferrin-iron uptake across the basolateral surface, the effects of transferrin-iron uptake on cellular ferritin content, the transport of apical iron across the monolayer, and the influence of proinflammatory cytokines on these processes. Caco-2 cells accumulated transferrin-iron from the basolateral chamber by a temperature-dependent, saturable process that was enhanced in less differentiated cultures and attenuated by pre-exposure to high-iron medium. Exposure of Caco-2 cells to 10 μmol/L diferric transferrin for 36 hr increased cellular ferritin protein 3.4-fold and decreased the transport of apical 59 Fe to the basolateral compartment by 45%. Pretreatment of cells with a combination of interleukin-1β, interleukin-6, and tumor necrosis factor-α increased transferrin-iron uptake by 70% and cellular ferritin content by 54%. Also, cytokine treatment decreased apical iron transport across the monolayer by 40% without altering paracellular transport of mannitol. These results suggest that transferrin-iron and proinflammatory cytokines are capable of modulating the iron status and iron transport activity of intestinal epithelial cells.