Biomedical subjects

D R Van Campen

Publications and source records attributed to D R Van Campen.

17 recordsLinked to original sources

PubMed1998

Decreased citrate improves iron availability from infant formula: application of an in vitro digestion/Caco-2 cell culture model.

We have applied an in vitro digestion/Caco-2 cell culture model to the assessment of iron availability from human milk and a generic cow's milk-based infant formula. Experiments were designed to determine the availability of iron from human milk relative to infant formula and whether known promoters of iron absorption would increase Caco-2 cell iron uptake and availability from the infant formula. In addition, we sought to determine if decreasing the citrate concentration in the infant formula would increase the iron uptake. Although approximately twice as much iron was in solution from digests of the infant formula relative to that of human milk, smaller or equal amounts of iron were taken up from the infant formula relative to the human milk digest. These results are qualitatively similar to in vivo studies. Addition of known iron uptake promoters to infant formula did not enhance Caco-2 cell iron uptake from the infant formula digest, indicating that the iron in the infant formula existed predominantly in a tightly bound unavailable form(s). Enzymatic pretreatment of the infant formula with citrate lyase and oxalacetate decarboxylase decreased the citrate concentration by 67% and resulted in a 64% increase of iron in solution, which corresponded to a 46% increase in the cell iron uptake. Iron uptake from the "low citrate" formula plus cysteine was 102% greater relative to the nontreated formula. The results indicate that too much citrate can reduce iron uptake, particularly if it is present at concentrations greater than promoters such as ascorbic acid and cysteine.

Animals↗

PubMed1997

Iron uptake is enhanced in Caco-2 cell monolayers by cysteine and reduced cysteinyl glycine.

Human and animal studies have shown that amino acids and peptides influence iron absorption from the intestinal lumen. This study was conducted using Caco-2 cell monolayers as the experimental model to determine whether similar effects on iron absorption occur. Conditions were chosen to mimic the pH of the intestinal lumen and the most likely order whereby ferric and ferrous forms of iron would combine with various amino acids and dipeptides resulting from protein digestion. We demonstrated the enhancing effect of cysteine and reduced cysteinyl glycine on iron uptake by Caco-2 cells. The addition of glutathione to the transport media had no effect on uptake from ferrous or ferric iron complexes, nor did it affect iron solubility. Cysteine and reduced cysteinyl glycine increased iron solubility when added to a solution containing insoluble iron. This effect is different from that of ascorbate, which must be combined with soluble ferric iron at pH 2 to reduce and solubilize iron. Taken together, these observations are evidence that cysteine and reduced N-terminal cysteine peptides are capable of enhancing iron uptake from soluble and insoluble ferric iron. These results qualitatively reflect those observed in human studies. Our results indicate that glutathione requires digestion to Cys or Cys-Gly in order to promote iron uptake. The similarity between this study and human studies further reinforces that the Caco-2 cell model is a useful tool in studies of iron absorption and bioavailability.

Absorption↗

PubMed1996

Caco-2 cell iron uptake from meat and casein digests parallels in vivo studies: use of a novel in vitro method for rapid estimation of iron bioavailability.

We developed a model for assessing iron bioavailability from foods which combines simulated peptic and intestinal digestion with measurement of iron uptake by Caco-2 cell monolayers. Our objective was to further validate this model by determining if meat enhances Caco-2 cell iron uptake relative to casein. Caco-2 cell monolayers were covered with Hank's balanced salt solution (HBSS) buffered with HEPES, pH 7.4. An upper chamber was created over the cells by fitting the bottom of a Costar Transwell insert with a 12,000-14,000 molecular weight cut-off dialysis membrane. This membrane allowed low molecular weight iron complexes to diffuse into the media bathing the cells and prevented damage to the cells from the digestive enzymes. Prior to digestion, each sample (homogenate of beef, chicken, fish or casein) was mixed with 59FeCl3 to achieve an iron concentration of 10 mumol/L. Following pepsin digestion (pH2), pH was adjusted to 7.4, pancreatic enzymes and bile extract were added to each digest, and an aliquot was then introduced into the upper chamber of the culture dish. During this intestinal digestion period, 59Fe uptake occurred from iron that dialyzed into the lower chamber. The 59Fe uptake from beef, chicken and fish digests was 300-400% of the 59Fe uptake from a casein digest. Our results parallel human absorption studies indicating that meat enhances iron absorption. The results suggest that digestion products of the meat proteins were at least partially responsible for the enhancement of iron uptake. Overall, this study supports the usefulness of our model as a means of assessing iron bioavailability.

Analysis of Variance↗

PubMed1996

Ferrous iron uptake but not transfer is down-regulated in Caco-2 cells grown in high iron serum-free medium.

Caco-2 cells in culture provide an attractive model for the study of human iron absorption. Because iron status has a marked effect on human iron absorption, we devised serum-free growth conditions that allow manipulation of Caco-2 cell iron stores while maintaining growth. Caco-2 cells were cultured in serum-free media containing 0-20 micromol/L added iron. Intracellular ferritin, measured by radioimmunoassay, increased 100-fold with the addition of 20 micromol/L iron to the serum-free growth medium. Iron uptake and transfer across Caco-2 cell monolayers were measured from balanced salt solutions of ferrous and ferric forms of iron. Uptake from ferrous, but not ferric, iron was inversely related to cell ferritin concentration and culture medium iron concentration. Kinetic analysis of uptake data from solutions of ferrous and ferric iron revealed saturable and nonsaturable components for ferrous iron, but only a nonsaturable component for ferric iron. Uptake by the nonsaturable pathway was not affected by cell ferritin concentration for either form of iron. Maximal uptake from a ferrous iron solution via the saturable pathway was nearly 100% greater in cells cultured under low compared with high iron conditions. Iron transfer across Caco-2 monolayers was not proportional to iron uptake, but was related to monolayer permeability. Iron uptake by Caco-2 cells was a reliable indicator of relative iron availability. We observed no difference in iron transfer that was related to the iron status of the cell monolayer. The lack of this effect suggests that this model may be inadequate for studies of iron transfer.

Caco-2 Cells↗

PubMed1995

Bathophenanthrolene disulfonic acid and sodium dithionite effectively remove surface-bound iron from Caco-2 cell monolayers.

Iron uptake by Caco-2 cell monolayers is commonly assessed by incubating the cells under radiolabeled iron solutions, removing the radiolabeled solution, rinsing to stop uptake and measuring the radioactivity retained by the cells. It is therefore essential to differentiate between iron that is nonspecifically bound to the cell surface from that which has been taken up by the cell. We report here on a method for removal of surface-bound iron from Caco-2 cell monolayers. We used a 140 mmol/L NaCl, 10 mmol/L PIPES, pH 6.7 solution containing 5.0 mmol/L sodium dithionite (Na2S2O4) and 5.0 mmol/L bathophenanthroline disulfonic acid to reduce, remove and chelate iron bound to the cell surface. We validated our method by demonstrating the removal of 97% of an insoluble iron complex from the apical surface of Caco-2 cell monolayers. Our data indicate that the removal solution does not damage the apical membrane and thereby does not have access to intracellular iron; thus only surface bound iron is removed. The remaining cell-associated iron represents that which has been transported into the cell. We present data on the uptake and nonspecific binding of iron from iron complexes of both ferrous and ferric forms, and show that iron removal treatment resulted in uptake measurements that agree more closely with accepted principles of iron uptake by intestinal epithelium. The iron removal method used in this study should provide investigators with a valuable tool for accurately determining iron uptake by epithelial cells in culture.

Biological Transport↗

PubMed1994

Enhanced Fe(3+)-reducing capacity does not seem to play a major role in increasing iron absorption in iron-deficient rats.

Some eucaryotic organisms, including many plants, yeast and mice, have a higher iron uptake during iron deficiency because the capacity to reduce Fe3+ from the environment to Fe2+ is greatly enhanced. To determine whether this occurs in rats, a common experimental model for iron absorption in humans, we compared the in vivo capacity to reduce intraluminal Fe3+ in iron-deficient and normal rats. We also measured potential Fe(3+)-reducing components within the intestinal lumen and on the mucosal surface. Iron-reducing capacity was higher in iron-deficient rats, by a significant (P = 0.026) but modest 20%, in parallel with higher mucosal weight (R2 = 0.501, P = 0.003). In vitro iron reduction by lumen contents was correlated with mucosal weight, even though mucosal tissue was not present in the assays. This capacity was not related to ascorbic acid, glutathione or other nonprotein sulfhydryls. Mucosal ferric reductase activity was higher in iron-deficient rats in parallel with higher tissue weight, but the specific activity did not differ and the higher total activity was not associated with the brush border fraction. The role of endogenous Fe3+ reduction in regulating iron absorption should be investigated in humans and in other experimental models.

Animals↗

PubMed1991

Mucus and iron absorption regulation in rats fed various levels of dietary iron.

We tested two hypotheses: (1) that iron binding by secreted mucus enhances iron absorption (Quarterman, Digestion 37: 1, 1987) and (2) that iron binding by secreted mucus prevents excess iron absorption. Rats were fed diets containing 6, 200 or 500 mg Fe/kg diet (Fe-0, Fe-200 and Fe-500 rats, respectively) for 3 wk. Iron absorption was measured in fasted rats using 59FeCl3 in a 10-min in situ duodenal ligated-segment procedure. After draining the segment contents, the mucus layer was separated from the under-lying mucosal surface using Quarterman's agar cast technique. In comparison with that in Fe-200 rats, iron absorption in Fe-0 rats was markedly increased, but the 59Fe and the total mucus in the mucus layer were decreased. The 59Fe absorption and total mucus and total iron in the mucus layer were similar in Fe-500 rats and Fe-200 rats, but the 59Fe in the mucus layer was marginally lower in Fe-500 rats. There was no evidence that mucus enhanced iron absorption; it appeared to trap or bind iron proportionally to the amount of secreted mucus, suggesting protection against excess absorption. Mucus secretion and possibly synthesis were decreased in the Fe-0 rats.

Animals↗

PubMed1991

Ferric iron absorption in rats: relationship to iron status, endogenous sulfhydryl and other redox components in the intestinal lumen.

Based on the hypothesis that Fe+3 must be reduced before Fe absorption, we investigated luminal factors that might participate in the physiological Fe+3 reduction. Rats were fed diets containing 7 mg Fe/kg diet [adequate iron (+Fe]) for 3 wk prior to a 10-min test of 59Fe absorption from an in vivo ligated duodenal segment. During absorption of 59Fe, the oxidation-reduction potential became more reducing and the pH rose in segment contents. There were small but significant differences between the -Fe and +Fe rats. In one experiment, the lumen environment was modified by bile duct ligation and/or intestinal perfusion prior to the absorption test. Ascorbic acid, nonprotein sulfhydryl compounds, Fe+2 and total ionizable Fe were measured in luminal contents. Nonprotein sulfhydryl concentration was positively correlated with, and the best predictor of, Fe absorption in -Fe rats.

Animals↗

PubMed1982

Effect of phytic acid on the absorption, distribution, and endogenous excretion of zinc in rats.

Zinc metabolism in male rats was studied by combining nutritional balance methods with an analysis of 65Zn kinetics. The rats, two groups of 84 each, were fed zinc-adequate diets (33 ppm Zn) with either 0 (basal) or 2% phytic acid added as sodium phytate. A fourth-order exponential function described the time-course of 65Zn in plasma, and compartmental models were developed accordingly. Plasma zinc exchanged more rapidly with zinc in liver and kidneys than it did with zinc in testes, skeletal muscle, or bone. Total body zinc content (2.6 mg/100 g live body weight) measured chemically was about 9 times higher than estimates of exchangeable zinc in the body. Whole-body retention of 65Zn was higher and endogenous fecal zinc excretion was lower in rats fed phytate than in those fed the basal diet; these responses to phytate may reflect a homeostatic adjustment to decreased absorption of zinc. Respective values for apparent absorption and true absorption of zinc were 13 and 32% of zinc intake in rats fed phytate, and 19 and 46% of zinc intake in rats fed the basal diet. When whole grains or mature seeds constitute a major portion of the diet, the phytate: zinc molar ratio may approach that (60:1) used in our study. Whether or not phytic acid occurring naturally in foods affects zinc metabolism to the same extent as sodium phytate can not be determined from our study.

Absorption↗

PubMed1980

Availability to rats of iron from spinach: Effects of oxalic acid.

The availability to rats of iron from two varieties of spinach was determined. Also, the absorption of Fe was compared between FeCl3 and Fe-oxalate and the effects of adding 0.75% oxalate to the diet were determined. Absorption of iron from both varieties of spinach was comparable to that from FeCl3 and the iron was equally available from Fe-oxalate and FeCl3. The addition of 0.75% oxalic acid to the diet did not depress iron absorption and, if anything, appeared to enhance iron utilization by rats.

Animals↗

PubMed1975