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Background: An in vitro digestion and Caco-2 cell model may predict iron bioavailability to humans; however, direct comparisons are lacking., Objective: The objective was to test the differences in iron bioavailability between 2 maize varieties and 2 bean varieties (white beans and colored beans) by comparing human, Caco-2, and algorithm results., Design: Two randomized, 2 x 2 factorial experiments compared women's iron absorption from 2 maize varieties (ACR and TZB; n = 26) and 2 bean varieties (great northern and pinto; n = 13), each fed with and without ascorbic acid (AA) from orange juice. Nonheme iron bioavailability was determined from 2-wk retention of extrinsic radioiron tracers and was compared with Caco-2 cell and algorithm results from identical meals., Results: Without AA supplementation, women absorbed only about 2% of the iron from the maize or bean meals. The results were unaffected by the variety of either maize or beans. Adding AA (15-20 molar ratios of AA:iron) roughly tripled the iron absorption (P < 0.0001) from all test meals. Although the Caco-2 model predicted a slightly improved bioavailability of iron from ACR maize than from TZB maize (P < 0.05), it accurately predicted relative iron absorption from the maize meals. However, the Caco-2 model inaccurately predicted both a considerable difference between bean varieties (P < 0.0001) and a strong interaction between bean varieties and enhancement by AA (P < 0.0001). The algorithm method was more qualitatively than quantitatively useful and requires further development to accurately predict the influence of polyphenols on iron absorption., Conclusions: Caco-2 predictions confirmed human iron absorption results for maize meals but not for bean meals, and algorithm predictions were only qualitatively predictive.

Four different colored beans (white, red, pinto, and black beans) were investigated for factors affecting iron bioavailability using an in vitro digestion/human Caco-2 cell model. Iron bioavailability from whole beans, dehulled beans, and their hulls was determined. The results show that white beans contained higher levels of bioavailable iron compared to red, pinto, and black beans. These differences in bioavailable iron were not due to bean-iron and bean-phytate concentrations. Flavonoids in the colored bean hulls were found to be contributing to the low bioavailability of iron in the non-white colored beans. White bean hulls contained no detectable flavonoids but did contain an unknown factor that may promote iron bioavailability. The flavonoids, kaempferol and astragalin (kaempferol-3-O-glucoside), were identified in red and pinto bean hulls via HPLC and MS. Some unidentified anthocyanins were also detected in the black bean hulls but not in the other colored bean hulls. Kaempferol, but not astragalin, was shown to inhibit iron bioavailability. Treating in vitro bean digests with 40, 100, 200, 300, 400, 500, and 1000 microM kaempferol significantly inhibited iron bioavailability (e.g., 15.5% at 40 microM and 62.8% at 1000 microM) in a concentration-dependent fashion. Thus, seed coat kaempferol was identified as a potent inhibitory factor affecting iron bioavailability in the red and pinto beans studied. Results comparing the inhibitory effects of kaempferol, quercitrin, and astragalin on iron bioavailability suggest that the 3',4'-dihydroxy group on the B-ring in flavonoids contributes to the lower iron bioavailability.

Elemental iron powders are widely used to fortify flour and other cereal products. Our objective was to test the hypothesis that baking enhances the bioavailability of elemental iron powders by oxidizing Fe(0) to Fe(2+) or Fe(3+). An in vitro digestion/Caco-2 cell culture model and a piglet model were used to measure bioavailability. Bread flour, either unfortified or fortified with hydrogen-reduced (HR) iron powder or FeSO(4) (300 mg Fe/kg flour), was baked into bread. For the in vitro studies, bread samples were treated with pepsin at pH 2, 3, 4, 5, 6, or 7 and subsequently incubated with pancreatic enzymes at pH 7 in a chamber positioned above monolayers of cultured Caco-2 cells. Ferritin formation in the cells was used as an index of iron bioavailability. Ferritin formation in cells fed HR Fe bread was similar to cells fed FeSO(4) bread when the peptic digestion was conducted at a pH 2 but lower when the peptic phase was conducted at pH 3, 4, 5, 6, or 7 (P < 0.05). Pig diets containing 35% dried bread were prepared and fed to cross-bred (Hampshire x Landrace x Yorkshire) anemic pigs in two studies. The rate of increase in hemoglobin Fe over the feeding period was used to calculate relative biological value (RBV), an index of iron bioavailability. In the first pig study, RBV of HR Fe added to flour prior to baking was 47.9% when compared to FeSO(4) fortified flour (P < 0.05). In the second pig study, a third treatment consisting of unfortified bread with HR iron added during diet mixing (after bread baking) was included. RBVs of the HR Fe diet (Fe added after baking) and HR Fe diet (Fe added before baking) were 40.1% and 53.5%, respectively, compared to the FeSO(4) diet. Differences in RBV between the HR Fe (before and after baking) and FeSO(4) (before baking) treatment groups were significant, but the difference between the before and after HR treatment groups was not significant. We conclude that bread baking does not enhance the bioavailability of elemental iron powders.

Sodium iron(III) ethylenediaminetetraacetate (NaFeEDTA) has considerable promise as an iron fortificant because of its high bioavailability in foods containing iron absorption inhibitors. In this study, uptakes of iron from NaFeEDTA, FeSO4, and FeCl3 by Caco-2 cells were compared in the absence or presence of ascorbic acid (AA), an iron absorption enhancer; at selected pH levels; and in the absence or presence of an iron absorption inhibitor, bathophenanthroline disulfonic acid (BPDS). Ferritin formation in the cells was used as the indicator of iron uptake. Uptake from all three Fe sources was similar in the absence of AA. Adding AA at a 5:1 molar excess as compared to Fe increased uptake by 5.4-, 5.1-, and 2.8-fold for FeSO4, FeCl3, and NaFeEDTA, respectively. The smaller effect of AA on uptake from NaFeEDTA may be related to the higher solubility of NaFeEDTA and/or the strong binding affinity of EDTA for Fe3+, which may prevent AA and duodenal cytochrome b from effectively reducing EDTA-bound Fe. Uptake was inversely related to the pH of the media over a range of 5.8-7.2. Because uptake by DMT-1 is proton-coupled, the inverse relationship between pH and Fe uptake in all three iron sources suggests that they all follow the DMT-1 pathway into the cell. Adding BPDS to the media inhibited uptake from all three iron compounds equally. Because BPDS binds Fe2+ but not Fe3+ and because only Fe2+ is transported by DMT-1, the finding that BPDS inhibited uptake from NaFeEDTA suggests that at least some iron dissociates from EDTA and is reduced just as simple inorganic iron at the brush border membrane of the enterocyte. Taken together, these results suggest that uptake of iron from NaFeEDTA by intestinal enterocytes is regulated similarly to uptake from iron salts.

Sodium iron ethylenediaminetetraacetate (NaFeEDTA) has superior iron bioavailability especially in foods containing iron absorption inhibitors. However, mechanisms involved in the absorption and subsequent partitioning of iron complexed with EDTA are poorly understood. Our objectives were to compare retention and tissue distribution of iron administered to rats either as FeSO4 or NaFeEDTA, either orally (OR) or subcutaneously (SC). Weanling rats were fed semipurified diets supplemented with either FeSO4 or NaFeEDTA for 7 days. They were then given a meal containing 59Fe-labeled FeSO4 or NaFeEDTA, or they were injected SC with these two forms of radiolabeled Fe. 59Fe retention was measured by whole body counting. Urine was collected and counted at 24 h intervals throughout the counting period. Tissue samples were analyzed for nonheme iron and 59Fe activity. Absorption of iron from FeSO4 or NaFeEDTA was similar (57.7 and 53.4%, respectively). Seventy-seven percent of the injected Na59FeEDTA was excreted in the urine within 24 h, whereas only 0.5, 0.8, and 1.4% of the injected 59FeSO4, oral 59FeSO4, and oral Na59FeEDTA, respectively, was excreted in the urine. The nonheme iron content was lower in the liver and spleen, by 56.8 and 28.4%, respectively, among rats consuming the NaFeEDTA diet as compared to rats fed FeSO4. We conclude that iron is dissociated from EDTA prior to or during intestinal absorption and that some fraction of the dissociated EDTA is absorbed separately from the iron.

We have generated transgenic maize plants expressing Aspergillus phytase either alone or in combination with the iron-binding protein ferritin. Our aim was to produce grains with increased amounts of bioavailable iron in the endosperm. Maize seeds expressing recombinant phytase showed enzymatic activities of up to 3 IU per gram of seed. In flour paste prepared from these seeds, up to 95% of the endogenous phytic acid was degraded, with a concomitant increase in the amount of available phosphate. In seeds expressing ferritin in addition to phytase, the total iron content was significantly increased. To evaluate the impact of the recombinant proteins on iron absorption in the human gut, we used an in vitro digestion/Caco-2 cell model. We found that phytase in the maize seeds was associated with increased cellular iron uptake, and that the rate of iron uptake correlated with the level of phytase expression regardless of the total iron content of the seeds. We also investigated iron bioavailability under more complex meal conditions by adding ascorbic acid, which promotes iron uptake, to all samples. This resulted in a further increase in iron absorption, but the effects of phytase and ascorbic acid were not additive. We conclude that the expression of recombinant ferritin and phytase could help to increase iron availability and enhance the absorption of iron, particularly in cereal-based diets that lack other nutritional components.

Although it has been shown that iron absorption from NaFeEDTA, a promising iron fortificant, is effectively down-regulated in iron-loaded rats, effects of prolonged exposure to high dietary levels of NaFeEDTA are not well understood. The objectives of this study were to determine whether rats can adapt to a high dietary level of NaFeEDTA by down-regulating iron absorption, and to determine effects on tissue iron distribution, with or without an iron absorption inhibitor. Male Sprague-Dawley rats were exposed to diets supplemented with FeSO4 or NaFeEDTA at 1200 mg of Fe/kg of diet, with or without tea, for 27 days. Iron absorption measured by whole-body counting before and after exposure showed that rats adapted to the high dietary level of FeSO4 or NaFeEDTA by down-regulating iron absorption to a similar extent. However, nonheme iron concentrations in liver and spleen were about 35-50% lower, whereas the concentration in kidney was about 300% higher in rats fed NaFeEDTA, compared to rats fed FeSO4. Tea had no major impact on iron absorption or iron status, regardless of iron source. Our results showed that although iron absorption was down-regulated similarly, body iron distribution was markedly different between rats exposed to FeSO4 and those exposed to NaFeEDTA. Further studies are warranted to determine the effects of prolonged exposure to dietary NaFeEDTA on kidney iron accumulation and kidney function.

The calcium, zinc, and iron bioavailabilities of human milk with commercial and noncommercial human milk fortifiers (HMFs) were evaluated under a variety of conditions: peptic digestion at pH 2 and pH 4, supplementation of ascorbic acid, and addition of three calcium salts. The noncommercial HMFs consisted of casein phosphopeptides (CPPs), alpha-lactalbumin, colostrum, and hydrolyzed whey protein concentrate (WPC). They were mixed with human milk (HM) and calcium, zinc, and iron were added. Ascorbic acid (AA) was added in certain studies. The commercial HMFs were Nestlé FM-85, Similac HMF (SHMF), and Enfamil HMF (EHMF). All HMFs were compared to S-26/SMA HMF. Results showed that the peptic pH (2 vs. 4) had no effect on mineral bioavailability. Addition of different calcium salts had no effect on calcium cell uptake and cell ferritin levels (an indicator of iron uptake), however, the addition of calcium glycerophosphate/gluconate increased zinc uptake by Caco-2 cells. Addition of AA significantly increased ferritin levels, with no effect on calcium or zinc uptake. Among the commercial HMFs, FM-85 was significantly lower in zinc uptake than S-26/SMA, and HM+EHMF was significantly higher than HM+S-26/SMA. Cell ferritin levels were significantly higher for HM+S-26/SMA than for all other commercial fortifiers. None of the commercial HMFs were different from HM+S-26/SMA in calcium uptake.

Iron chelates, namely, ferrous bisglycinate and ferric EDTA, are promising alternatives to iron salts for food fortification. The objectives of this study were to compare iron uptake from radiolabeled ferrous sulfate, ferrous ascorbate, ferrous bisglycinate, ferric chloride, ferric citrate, and ferric EDTA by Caco-2 cells with different iron status and in the presence of divalent metal cations. Iron-loaded Caco-2 cells, with reduced DMT-1 and elevated HFE mRNA levels, down-regulated uptake from ferrous ascorbate and bisglycinate but not from ferric compounds. Nevertheless, iron uptake from all compounds was markedly inhibited in the presence of 100-fold molar excess of Co2+ and Mn2+ cations, with ferrous compounds showing a greater percent reduction. Our results suggest that ferrous iron is the predominant form of iron taken up by intestinal epithelial cells and the DMT-1 pathway is the major pathway for uptake. Iron uptake from chelates appears to follow the same pathway as uptake from salts.

Adding human milk fortifiers (HMF) to human milk (HM) is one way of overcoming the nutrient deficits found in the latter. In this study, the bioavailabilities of calcium, zinc, and iron in S-26/SMA HMF added to HM were compared with those in HM fortified with various bovine milk proteins: alpha-lactalbumin, colostrum, caseinate, casein phosphopeptides, and whey protein concentrate. The bioavailability of each mineral was assessed using an in vitro digestion/Caco-2 cell culture model. Calcium and zinc uptake by the cells was traced with radioisotopes; iron uptake was assessed via cell ferritin levels. Samples were prepared on an equal protein content basis and with added calcium, but no zinc or iron was added. Results revealed that calcium uptake from HM + S-26/SMA was not different from any of the HM fortified with the bovine milk proteins, except for unfortified HM and HM + colostrum in which calcium uptake was significantly lower (-89 and -38%, respectively). Uptake of zinc and iron were significantly higher for HM + S-26/SMA than for the other HM + fortifiers.

We developed a rapid in vitro digestion/Caco-2 cell culture model for assessing relative bioavailabilities of iron in foods and meals. The objective of the present study was to determine how closely our Caco-2 model reflects the human response. Meals described in published reports of studies on effects of varying levels of ascorbic acid (AA) and tannic acid (TA) on iron absorption by human subjects were carefully replicated. Iron absorption ratios (iron absorption from meals containing AA or TA divided by iron absorption from identical meals without these enhancers or inhibitors) were determined using the Caco-2 model. Ferritin formation by the Caco-2 cells was used as an indicator of iron absorption. Response patterns of effects of AA and TA on absorption ratios (AR) calculated from Caco-2 and human data were very similar: AA increased ARs in a dose-response manner and TAs decreased AR. The natural logs of the ARs determined in Caco-2 and human studies were correlated: R = 0.935 (P = 0.012) and 0.927 (P = 0.007) for AA and TA, respectively. When results from meals with AA and TA were pooled, a linear relation between the natural logs of ARs from Caco-2 and human studies was observed (R = 0.968, P < 0.001). We conclude that our Caco-2 model accurately predicts the human response to AA and TA in the meals we tested. If future work reproduces the precision and accuracy shown in this paper for predicting iron bioavailability to humans, then the implications for saving time and resources in iron bioavailability measurements are considerable.

NaFeEDTA is a promising fortificant for use in plant foods, because it is less susceptible to iron absorption inhibitors and has fewer undesirable impacts on sensory quality than ferrous sulfate. However, the hypothesis that iron absorption from NaFeEDTA is effectively downregulated in iron-overload conditions has not been thoroughly tested. Therefore, the objective of this study was to compare downregulation of iron absorption from ferrous sulfate and NaFeEDTA in intact iron-loaded rats. Male Sprague-Dawley rats were fed diets containing either ferrous sulfate (35 mg Fe per 1 kg diet) or elemental iron (30,000 mg Fe per 1 kg diet) for 29 d to achieve basal or iron-loaded status. While body weights and hemoglobin concentrations were the same for basal and iron-loaded rats, nonheme-iron concentrations in liver, spleen, and kidney were all significantly higher in iron-loaded rats, indicating elevated iron status. Percentage of iron absorption from (59)Fe-labeled ferrous sulfate and NaFeEDTA, determined from whole-body retention of (59)Fe activity, was 64.7 and 49.4% in basal rats but decreased to 12.8 and 10.2% in iron-loaded rats, respectively. The reductions in percentage of iron absorption from both iron sources in rats as a result of iron loading were comparable (about -80% for both iron sources). Our results suggest that iron absorption from NaFeEDTA and ferrous sulfate is downregulated to a similar extent in iron-loaded rats. Hence, NaFeEDTA is no more likely than ferrous sulfate to exacerbate iron overload in subjects with adequate body iron stores.

The objective of this study was to isolate and characterize the meat factor(s) that enhances nonheme iron bioavailability using various analytical and in vitro cell culture techniques. Nonheme iron bioavailability was measured via radiolabeled iron uptake or ferritin formation in Caco-2 cells. Fish haddock fillet was cooked and lyophilized to be used as the muscle tissue of choice because of its low intrinsic iron content. It was demonstrated that the low pH of the stomach (pH 2.0) was the primary factor responsible for initiating the enhancing effect of fish on nonheme iron uptake. Subsequently, cooked fish samples were titrated with HCl to pH 2.0 and incubated for 1 h without digestive enzymes to release the factor(s) from the fish. The supernatant of this acidic digest was then used as a starting material for the meat factor isolation procedures. Fractions generated through Sephadex G-25 size exclusion increased Caco-2 cell iron uptake approximately 9-fold. Subsequent chromatography of these fractions via C18 reverse-phase HPLC were conducted, and enhancing activity was observed only in the "injection peak." This observation coupled with protein measurement and amino acid composition analysis revealed that the active fractions contained negligible amounts of proteins or amino acids. Active fractions were highly enriched with carbohydrates. Subsequent chromatography via high performance anion exchange chromatography with pulsed amperometric detection yielded 3 active peaks that increased Caco-2 cell iron uptake 3.4- to 4.9-fold. Our results indicate that specific carbohydrates contribute to the enhancing effect of meat on iron uptake by the enterocyte. These carbohydrates may be oligosaccharides originating from glycosaminoglycans in the extracellular matrix of muscle tissue.

Epidemiological studies have shown that consumption of carotenoid-rich fruits and vegetables is associated with a reduced risk of developing chronic diseases. beta-Carotene, alpha-carotene, and beta-cryptoxanthin are precursors of vitamin A, a nutrient essential for human health. However, little is known about the bioavailability of carotenoids from whole foods. This study characterized the intestinal uptake performance of carotenoids using monolayers of differentiated Caco-2 human intestinal cells and mimicked human digestion to assess carotenoid absorption from carrots and corn. Results showed that Caco-2 cellular uptake of beta-carotene and zeaxanthin was higher than that of lutein. Uptake performances of pure carotenoids and carotenoids from whole foods by Caco-2 cells were both curvilinear, reaching saturated levels after 4 h of incubation. The time kinetics and dose response of carotenoid uptake presented a similar pattern in Caco-2 cells after plating for 2 and 14 days. Furthermore, the applicability of this new model was verified with whole grain corn, showing that cooked corn grain significantly enhanced carotenoid bioavailability. These results support the feasibility of the in vitro digestion cell model for assessing carotenoid absorption from whole foods as a suitable and cost-effective physiological alternative to current methodologies.

Spinach (Spinacia oleracea) cv Whitney was tested for iron bioavailabilty using an in vitro human intestinal cell culture ferritin bioassay technique previously developed. Spinach was cultured in a growth chamber for 33 days, harvested, and freeze-dried. Total iron in the samples was an average of 71 micrograms/g dry weight. Spinach was digested in vitro (pepsin and 0.1 M HCl followed by pancreatin and 0.1 M NaHCO3) with and without the addition of supplemental ascorbic acid. Caco-2 cell cultures were used to determine iron bioavailability from the spinach mixtures. Production of the iron-binding protein ferritin in the Caco-2 cells showed the supplemental ascorbic acid doubled bioavailability of iron from spinach. The data show fresh spinach is a poor source of iron, and emphasize the importance of evaluation of whole meals rather than single food items. The data support the usefulness of the in vitro/Caco-2 cell ferritin bioassay model for prescreening of space flight diets for bioavailable iron.

The iron bioavailability of human milk (HM) is substantially greater than that of cow's milk (CM), but the factor responsible for this high bioavailability is unknown. This study evaluated the effects of various HM and CM fractions on iron bioavailability. Milk was separated into fat, casein and whey fractions by ultracentrifugation. Whey was further fractionated by ultrafiltration with a 10-kDa membrane to produce a 10-kDa retentate (10kR) and a 10-kDa filtrate (10kF). Samples were prepared by mixing various combinations of the fractions, bringing the samples to prefractionation weight with minimum essential medium (MEM), and adding iron (10 micro mol/L) as ferrous sulfate. Samples were divided into two aliquots: one was subjected to in vitro digestion, the other was not. Bioavailability was assessed by applying the samples to Caco-2 cell monolayers and incubating for 24 h. Ferritin formation in the cells was used as an index of iron uptake. Removing the fat from undigested HM samples doubled the ferritin formation, but removing the whey or casein had no effect. Results with digested HM samples were similar, except that removing the whey decreased ferritin formation by 48%. Removing the fat from digested CM samples had no effect, but removing the casein doubled the ferritin formation. Removing the 10kF from HM reduced ferritin formation by 60%, but removing the 10kR had no effect. These data suggest that a low-molecular-weight factor (<10 kDa) in human milk enhances iron absorption.

Twenty elite early-maturing (75-90 days) tropical maize varieties grown in three diverse agroecologies in West Africa were evaluated to identify varieties with high kernel-Fe and -Zn and bioavailable Fe levels. Bioavailable iron was assessed using an in vitro digestion/Caco-2 cell model. Significant (P < 0.001) varietal differences were observed in mean kernel-Fe and -Zn levels. The ranges were 15.5-19.1 mg kg(-)(1) for Fe and 16.5-20.5 mg kg(-)(1) for Zn. Genetic component accounted for 34% of the total variation in kernel-Zn and for 11% of the variation in kernel-Fe levels. Mean bioavailable Fe in varieties ranged between 4% below and 49% above the reference control variety. A significant negative relationship was detected between kernel-P concentration and bioavailable Fe (R = -0.36; P < 0.004; n = 60). Two varieties, ACR90POOL16-DT and ACR86TZESR-W, were identified as the most promising for further evaluation to determine their efficacy as improved sources of iron in target populations.

Adequate bioavailable Fe intake is essential for optimal growth and intellectual development of infants and children. Fruit juices are nutritious and popular drinks for infants and children and are known to contain Fe uptake inhibitors (e.g., polyphenolic compounds) and a dominant promoter, ascorbic acid. Ascorbic acid is naturally present in fruit juices and is added during processing to almost all juices found in supermarkets. With these facts taken into account, an in vitro digestion/Caco-2 cell culture model was developed to compare the effects of apple, pear, white grape, red grape, prune, grapefruir, and orange juices on iron bioavailability. In two series of experiments, juices from a local supermarket were combined with FeCl(3) or commercial infant cereal fortified with elemental iron and subject to simulated gastric and intestinal digestion. Caco-2 cell ferritin formation in response to exposure to the digests served as the measure of Fe uptake. The pear, apple, grapefruit, orange, and white grape juice significantly increased Fe bioavailability from FeCl(3). For the infant cereal studies, the apple, orange, pear, and white grape juices increased the Fe bioavailability of the infant cereal. In contrast, the red grape juice and prune juice had profound inhibitory effects on iron bioavailability. These inhibitory effects were likely due to high levels of polyphenolic compounds that bind and thereby prevent absorption of soluble Fe. These inhibitory compounds appeared to counteract the promotional effects of ascorbic acid as they were in considerable molar excess relative to ascorbic acid and Fe in the digest. From a nutritional standpoint, the results suggest that individuals in need of optimal Fe absorption should avoid red grape and prune juice or at least vary the types of juices consumed. Alternatively, individuals seeking to limit Fe uptake (e.g., hemochromatitics and astronauts) may be able to utilize red grape or prune juice as effective inhibitors of Fe uptake. Consumers should be aware that the compounds that inhibit Fe availability are also linked to anticancer benefits; thus, a dietary balance of the above juices may be optimal.

A simple method for simultaneously determining cell-associated Ca and Zn in Caco-2 cells is described. Calcium and zinc uptake was measured via radioisotopes (45)Ca and (65)Zn. Preliminary studies revealed that (65)Zn, a positron (beta(+)) and gamma emitter, contributed to (45)Ca counts in a liquid scintillation counter (LSC). However, (45)Ca, being a true beta emitter, did not contribute to the counts in a gamma counter (gammaC). To differentiate the counts of (45)Ca from those of (65)Zn, first a (65)Zn-labeled cell suspension was read in a gammaC and an LSC, thus obtaining the relationship between the radioactive counts obtained from the gammaC and LSC. This information defined the linear relationship between gammaC (65)Zn counts per minute (CPM) and LSC (65)Zn CPM. Because the (45)Ca and (65)Zn counts obtained in the LSC are additive, giving total LSC CPM, the value of LSC (45)Ca CPM was obtained by subtracting LSC (65)Zn CPM from total LSC CPM for the dual-labeled cell sample, obtaining then LSC (45)Ca CPM. To determine the absolute activity or disintegrations per minute (DPM) of each isotope in the dual-labeled sample, the linear relationship between DPM and CPM was determined for each isotope. The method is simple and straightforward for the determination of (45)Ca counts from a sample also containing (65)Zn, using gamma and liquid scintillation counters.

An in vitro digestion/Caco-2 model was used to compare iron bioavailability from 15 selected Fe-dense and normal genotypes of unpolished rice from the International Rice Research Institute. Iron uptake was determined using Caco-2 cell ferritin formation in response to exposure to a digest of the cooked rice. Iron bioavailabilities from all rice genotypes were ranked as a percent relative to a control variety (Nishiki). Iron concentration in the rice samples ranged from 14 to 39 microg/g. No correlation was observed between Fe uptake and grain-Fe concentration. Furthermore, phytic acid levels were not correlated with Fe bioavailability. Genotypes with low Fe bioavailability (Tong Lan Mo Mi, Zuchein, Heibao, and Xua Bue Nuo) were noticeably more brown to purple in color. The results suggest that certain unknown compounds related to rice grain color may be a major factor limiting Fe bioavailability from unpolished rice.

The objective of this study was to document the effects of phytic acid, tannic acid, and zinc on iron uptake in an in vitro digestion/Caco-2 cell culture model. The effects of phytic acid and tannic acid on iron uptake were measured at increasing molar ratios of FeCl3 to phytic acid or tannic acid. Maximal inhibition of iron uptake by phytic acid occurred at a 1:10 ratio of Fe to phytic acid. Dialyzable Fe decreased with a low Fe to phytic acid ratio but increased with Fe:phytic acid ratios greater than 1:3 indicating that more iron was soluble at higher phytic acid levels but less available. As in human studies, heme iron was less inhibited by phytic acid than nonheme iron. Tannic acid was a more potent inhibitor of nonheme iron uptake, as maximal inhibition (97.5%) of iron uptake occurred at a ratio of 1:1 or less. The addition of ZnCl2 to the digest at ratios of 1:0.5 and 1:1 decreased iron uptake by 57 and 80%, respectively. Overall, the results agree qualitatively with studies in humans and demonstrate the relative effects of these compounds on iron uptake in this model system. This study provides key information for determining iron availability under more complex meal conditions.

Clusters of phosphoserine residues in casein bind iron with high affinity. Casein inhibits iron absorption in humans but partial hydrolysis of casein prior to ingestion diminishes this inhibition. The objective of this study was to test two hypotheses: 1. Partial hydrolysis of the peptide bonds in casein exposes phosphoserine residues to attack by intestinal alkaline phosphatase (IAP). 2. Hydrolysis of the phospho-ester linkage in phosphoserine residues in casein by IAP releases bound iron or inhibits iron chelation, thereby allowing its absorption. Test of hypothesis 1: Suspensions of sodium caseinate (SC), enzymatically hydrolyzed casein (EHC), and casein phosphopeptides (CPP) were subjected to an in vitro pepsin/pancreatin digestion and subsequently incubated in the presence of calf IAP. The rate of release of inorganic phosphate was measured with the following results (expressed as &mgr;mol phosphate released/unit of IAP/min): 0.081, 0.104, 0.139 for SC, EHC, and CPP, respectively. These results are consistent with hypothesis 1. Test of hypothesis 2: (59)Fe-citrate or (59)Fe-citrate + CPP in minimum essential media were spiked with a Na(2)WO(4) solution or water (Na(2)WO(4) is a known inhibitor of IAP) and placed on Caco-2 cell monolayers. Uptake of (59)Fe by the cells was used as an index of iron bioavailability. Na(2)WO(4) did not affect (59)Fe uptake from samples containing only iron but did slightly inhibit (by 10%) uptake from samples containing iron + CPP. These results are consistent with hypothesis 2 and provide a possible explanation for the observation that partial hydrolysis of casein improves iron bioavailability.

The objectives of this study were to compare iron availability from commercial preparations of FeSO(4), ferrous gluconate, ferrous fumarate, and a polysaccharide-iron complex using an in vitro digestion/Caco-2 cell culture model. In addition, we sought to determine if calcium carbonate and calcium acetate (common phosphate binding agents) inhibited iron availability from an oral iron supplement when digested simultaneously. Caco-2 cell ferritin formation following exposure to simulated gastric and intestinal digests of the iron supplements was used as a measure of iron uptake and availability. Plates without cell monolayers were included in each replication of the experiment to measure the total amount of soluble iron that resulted from the in vitro digestion. Significantly more iron was taken up from the FeSO(4), ferrous gluconate, and ferrous fumarate than the polysaccharide-iron complex. Similar results comparing FeSO(4) and the polysaccharide-iron complex have been observed in humans. In addition, less iron was taken up from digests with calcium carbonate relative to calcium acetate even though similar amounts of soluble iron were observed in these experiments. The results indicate that when iron supplements and phosphate binders are consumed simultaneously, calcium acetate may be the preferred phosphate binder to maximize iron availability.

We have adapted an in vitro digestion/Caco-2 cell model to assess Fe availability from foods, by using ferritin formation by Caco-2 cells as an indicator of Fe uptake. Ferritin formation by Caco-2 cells occurs in response to Fe uptake at concentrations of available Fe greater than that of the culture media to which the cells have been adapted. This methodology circumvents the need for using radioactive Fe and thus eliminates the costs and controversies associated with food radiolabeling. To validate this method, we measured ferritin formation in Caco-2 cells exposed to digests containing Fe of relatively high and low availability. Our objective was to determine if ferritin formation would be proportional to Fe uptake and sufficiently sensitive to be an indicator of Fe availability from food digests. Our model uses established in vitro digestion techniques coupled with uptake of Fe by Caco-2 cell monolayers. Measurement of cell ferritin was done by a commercially available RIA. Higher ferritin formation was observed in cells exposed to digests containing FeSO4 plus ascorbic acid vs, digests containing FeSO4 plus citric acid. Additional comparisons of Fe availability from digests of beef, fish, corn and green beans yielded results that demonstrate higher Fe availability (i.e., greater ferritin formation) from beef and fish digests than from digests of corn and green beans. Overall, the results document the promotional effects of ascorbic acid and animal tissue on Fe uptake as measured indirectly by ferritin formation. The results of this study indicate that ferritin formation by Caco-2 cell monolayers is highly sensitive and accurately measures food Fe availability in this in vitro system.

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.

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.

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.

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.

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.

In vivo studies indicate that aflatoxin B1 (AFB1) may affect the renal regulation of inorganic phosphate (P(i)), possibly by altering the renal response to parathyroid hormone (PTH). Therefore, the present study utilized opossum kidney (OK) cells, a mammalian renal epithelial cell line, to determine whether AFB1 exposure alters sodium-phosphate (Na(+)-P(i)) co-transport and the hormonal modulation thereof. OK cells are an established renal cell line with many properties analogous to the proximal renal epithelium, including receptors for PTH, insulin, and high levels of Na(+)-P(i) co-transport. PTH and insulin have been shown to decrease and increase Na(+)-P(i) co-transport, respectively, in OK cells. In the present study, AFB1-treated cells responded to PTH; however, AFB1 exposure decreased Na(+)-P(i) uptake such that additional decreases in Na(+)-P(i) uptake in response to PTH were minimal. In the presence of insulin, AFB1-treated cells were only able to increase Na(+)-P(i) uptake to levels 30% below that of control cells. The net result was that the range of the proximal renal epithelium to adjust Na(+)-P(i) co-transport in response to hormonal modulation was reduced by AFB1 exposure. Sodium-dependent L-alanine uptake was measured and was found not to be affected by the highest concentration of AFB1; thus, indicating that AFB1 exposure may have specific effects on Na(+)-P(i) uptake and does not generally inhibit Na(+)-dependent transport. These observations are evidence that AFB1 exposure may alter key elements of renal function. Such effects raise concern that AFB1 exposure may have broad physiological impact in addition to its known carcinogenic properties.

We tested the hypothesis that dopamine (DA) acts as an autocrine/paracrine regulator of Na(+)-Pi symport in proximal tubules, using opossum kidney (OK) cells as an in vivo model. Both DA and parathyroid hormone (PTH) increased adenosine 3',5'-cyclic monophosphate (cAMP) and inhibited Na(+)-gradient-dependent uptake of 32P but not that of L-[3H]-alanine. Incubation of OK cells with L-dopa, a DA precursor, resulted in accumulation of DA (7.4 nM), a ninefold increase of cAMP in the medium, and an inhibition (-10%) of Na(+)-Pi uptake. Carbidopa, an inhibitor of aromatic-L-amino acid decarboxylase, prevented the formation of DA from L-dopa, the increase in cAMP, and the inhibition of Na(+)-Pi cotransport. Pi-replete OK cells produced more DA (+15%) from L-dopa than Pi-deprived cells; however, the endogenous DA inhibited Na(+)-Pi cotransport both in Pi-deprived and in Pi-replete cells. Thus OK cells can synthesize DA from L-dopa in a quantity sufficient to elicit both the maximum DA-stimulated cAMP accumulation and inhibition of Na(+)-Pi cotransport in the same cell population. Our data, obtained on an in vitro system, support the hypothesis proposing that DA generated in proximal tubular cells can modulate, via cAMP, the Na(+)-Pi symport in the same or adjacent cells. If present in the kidney, this pathway might represent an autocrine/paracrine system that can contribute to regulation of renal Pi homeostasis.

Systemic hypovolemia and hypotension increase plasma renin activity (PRA) in fowl, but it is not clear whether this response is mediated directly by reduced renal arterial perfusion pressure (RAPP) or indirectly via renal nerves activated when systemic baroreceptors detect hypotension. To evaluate the influence of RAPP on renin release, arterial and renal venous blood samples were collected as RAPP was reduced step-wise from 108 mm Hg (control) to 71 and 47 mm Hg. PRA in systemic arterial (aPRA) and renal venous (vPRA) plasma was measured as the rate of fowl angiotensin I (ANG I) generation. Basal vPRA (2.47 +/- 0.6 ng x ml-1 x min-1) tended to be higher than aPRA (1.24 +/- 0.3 ng x ml-1 x min-1). When RAPP was reduced to 47 mm Hg both vPRA (4.35 +/- 0.6 ng x ml-1 x min-1) and aPRA (1.91 +/- 0.3 ng x ml-1 x min-1) increased significantly. Significant negative slopes (P = 0.01) were obtained when changes in aPRA or vPRA were regressed on RAPP. Mean systemic arterial pressure did not change during reductions in RAPP, nor did angiotensinogen concentrations differ when systemic arterial (472 +/- 30 ng/ml) and renal venous (460 +/- 27 ng/ml) plasma values were compared. Renal plasma flow was fully autoregulated as RAPP was reduced from 108 to 47 mm Hg; consequently, RAPP-induced increases in vPRA cannot be attributed to hemoconcentration of secreted renin. These results demonstrate that reductions in RAPP directly stimulate renin release from domestic fowl kidneys.

Domestic fowl kidneys autoregulate total renal blood flow and glomerular filtration rate (GFR) over a wide range of renal arterial perfusion pressure (RAPP). Sustained (approximately 2-4 h) restriction of renal portal blood flow attenuates the autoregulatory responses. The present study was designed to assess the effects of acute (approximately 10 min) alterations of renal portal blood flow on renal function, and to dissociate the renal responses to altered renal portal blood flow from the renal responses to reductions in RAPP. The thermal pulse decay (TPD) technique and p-aminohippuric acid clearance (CPAH) were used to measure blood flow. During acute increases and decreases in renal portal blood flow, regional renal blood flow as measured by the TPD system (RBFTPD) was significantly positively correlated with total kidney blood flow represented by CPAH (RBFPAH). These results indicate that changes in total kidney blood flow induced by alteration of portal perfusion were reflected in the regional measurement of renal blood flow. Changes in renal portal blood flow did not affect the urine flow rate (UFR), GFR, or fractional excretion of sodium (FENa). Reducing RAPP from 120 to 50 mmHg significantly reduced UFR, GFR, and FENa. Overall, these results indicate that large acute changes in renal portal blood flow can significantly alter total renal blood flow without significantly affecting parameters (UFR, GFR, and FENa) primarily influenced by the renal arterial vasculature.

Dopamine (DA) is natriuretic and phosphaturic. However, whether the effect of DA on Pi reabsorption is a consequence of its effect on sodium transport is not known. Therefore, this study was performed to determine the effect of DA on the maximal transport of phosphate (TmPi), and upon the capacity of renal proximal brush border membrane (BBM) for (Naextra-vesicular greater than Naintravesicular)-gradient-dependent transport of Pi, as compared with the transport of other solutes. Graded infusions of Pi (0, 1, 2, and 3 mumols/min) were given to thyroparathyroidectomized male Sprague-Dawley rats in the presence of vehicle (0.9% NaCl; N = 5), DA 15 micrograms/kg/min; N = 6), or parathyroid hormone ((PTH); 1 U/kg/min; N = 5). The TmPi for rats infused with DA (3.3 +/- 0.3 mumol/mL) was significantly less than the TmPi for saline control rats (4.4 +/- 0.2 mumol/mL). Rats infused with PTH exhibited the lowest TmPi (1.8 +/- 0.3 mumol/mL). No differences in sodium excretion were observed among any of the groups. Na-dependent Pi transport was studied in BBM vesicles (BBMV) prepared from rats fed a low-phosphate diet for 2 days that were anesthetized, acutely thyroparathyroidectomized, and systemically infused with DA (350 micrograms bolus, plus 35 micrograms/kg/min; N = 8), PTH (33 U/kg bolus, followed by a continuous infusion of 1 U/kg/min; N = 6), or vehicle (1 mL/kg bolus, plus 2 mL/h constant infusion of 0.9% NaCl; N = 8) for 90 min. DA significantly inhibited the Na cotransport of Pi by 22.4 +/- 4.1% (P less than 0.01) as compared with the control group.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The effect of aflatoxin on plasma aspartate aminotransferase (AST), protein, and hepatic glutathione (GSH) and hepatic blood flow (perfusion), were determined in 3-week-old male chickens. 2. Daily aflatoxin gavage (2 mg/kg body wt, in corn oil) for 5 and 10 days elevated plasma AST and hepatic GSH, and depressed plasma protein and hepatic perfusion. Also, renal GSH was elevated after 10 days of aflatoxin treatment. 3. Birds given aflatoxin for 10 days followed by a 10-day recovery period exhibited tissue GSH, plasma AST and protein levels that were not different from control, but hepatic perfusion remained depressed.

Using a simplified avian kidney model, renal arterial perfusion pressure (RAPP) was reduced from 120 (control) to 70 mmHg (near the glomerular filtration rate autoregulatory limit) and then to 46 mmHg (below the glomerular filtration rate autoregulatory range) in kidneys with ambient or partially restricted renal portal flow. Renal blood flow (RBF) was measured with a thermal pulse decay (TPD) system, using TPD thermistor probes inserted at three locations to evaluate regional differences in RBF. The clearance (CPAH) and extraction of p-aminohippuric acid were used to calculate renal plasma flow (RPF). CPAH, RPF, and RBF values were consistently lower for kidneys with restricted portal flow than for kidneys with ambient portal flow. Reducing RAPP to 46 mmHg did not significantly reduce CPAH, RPF, or RBF in the ambient group but did significantly reduce CPAH and RPF (regressed on RAPP) in the restricted group. RBF was not significantly affected when RAPP was reduced in the restricted group, although significant regional differences in blood flow were recorded. Renal vascular resistance decreased significantly as RAPP was reduced to 46 mmHg in the ambient group, confirming the renal autoregulatory response. In separate validation studies, significant reductions in RBF were detected by the TPD system during acute obstructions of portal and/or arterial flow. Overall, the results support previous evidence that avian RBF remains constant over a wide range of RAPPs. Observations of nonuniform intrarenal distributions of portal blood flow suggest that the portal system maintains the constancy of RBF in regions with proportionately high portal-to-arterial flow ratios.

Experiments were designed to determine the effects of aflatoxicosis on avian renal function, calcium (CA), inorganic phosphorous (Pi), and vitamin D metabolism, and to determine if the effects of aflatoxin are reversible upon discontinuation of toxin administration. Three-week-old male broiler chickens (n = 12 per treatment) received aflatoxin (AF; 2 mg/kg po) or an equal volume of corn oil, the AF carrier vehicle, for 10 consecutive days. After 10 d of treatment, half of the birds from each treatment group were anesthetized and prepared for renal function analysis, which included a 2-h phosphate loading period. Ten days after discontinuation of AF treatment, the remaining birds in each treatment group were anesthetized and prepared for renal function analysis. AF decreased plasma 25-hydroxy vitamin D [25(OH)D] and 1,25-dihydroxy vitamin D [1,25(OH)2D] levels after 5 d of treatment. After 10 d of treatment, urine flow rate (V), fractional sodium excretion (FENa), and fractional potassium excretion (FEK) were lower in AF-treated birds. In addition, total plasma Ca tended to be lower (p = .10) and fractional Ca excretion (FECa) tended to be higher (p = .10) in the AF-treated birds. Intravenous phosphate loading produced a sharp increase in urine hydrogen ion concentration ([H+]) in the AF-treated birds. Glomerular filtration rate (GFR) was reduced and plasma osmolality was increased in AF-treated birds 10 d after discontinuation of toxin administration. The results indicate that AF directly or indirectly affects Ca and Pi metabolism in avians. At the present time, the effects may be related to altered vitamin D and parathyroid hormone (PTH) metabolism. Aflatoxicosis may decrease endogenous PTH synthesis and the renal sensitivity to PTH. The AF-related increase in urine [H+] during phosphate loading is probably due to increased Na+/H+ counterport, suggesting that AF stimulates sodium reabsorption. Also, the decrease in GFR exhibited 10 d after toxin removal indicates that AF may cause prolonged alteration in renal function.

Experiments were conducted to determine the effects of aflatoxicosis on acid-base balance, urine flow rate (V), glomerular filtration rate (GFR), clearance of para-aminohippuric acid (CPAH), plasma osmolality, and the renal handling of Na, K, Ca, and P. Three-week-old broilers were gavaged with aflatoxin at a dose of 2 mg/kg of BW per day for 10 consecutive days. Control birds received an equal volume of corn oil, the aflatoxin carrier vehicle. On the eleventh day, the birds were anesthetized and prepared for renal function analysis. A solution containing inulin, para-aminohippuric acid, and mannitol was infused at a low infusion rate (.1 mL/kg of BW per min) and a high infusion rate (.4 mL/kg of BW per min) to determine if aflatoxin affects the renal response to an acute volume load. Aflatoxicosis decreased the fractional excretion of phosphorous (FEP) and plasma Ca concentration but did not significantly alter any other renal function or acid-base variables. The decrease in FEP and plasma Ca may be a direct result of renal tubular damage, decreased Ca absorption from the gut, or a result of altered circulating levels of parathyroid hormone (PTH), and possibly decreased renal sensitivity to PTH.

Citrinin is a nephrotoxic mycotoxin produced by common molds. Previous research has shown that citrinin causes increased urine flow, increased free water clearance, and increased sodium, potassium, and inorganic phosphate excretion. The present study was conducted to evaluate the dose-response effects of citrinin and to further evaluate previously reported phosphaturic effects of citrinin. Ureteral urine was collected from anesthetized domestic fowl during a control period (30 min) and during unilateral renal portal infusion (90 min) of citrinin carrier vehicle, 200 ppm citrinin, or 400 ppm citrinin. Comparisons of the portal infused vs. uninfused kidneys were used to evaluate the direct effects of citrinin. Citrinin caused acute (unilateral) dose-related increases in urine flow, free water clearance, and fractional sodium excretion and dose-related decreases in urine osmolality. Citrinin had no direct effect on glomerular filtration rates, fractional potassium excretion, or fractional inorganic phosphate excretion. An additional group of birds received systemic intravenous infusions of parathyroid hormone (PTH) beginning 40 min after the start of unilateral renal portal infusion of citrinin. The citrinin and PTH infusion were continued for 60 min. Fractional inorganic phosphate excretion increased bilaterally during the citrinin-PTH infusion period but citrinin had no direct phosphaturic effect. Previously reported phosphaturic effects of citrinin were not confirmed in the present study.

Eleven healthy, normotensive males undergoing routine restorative dental care were evaluated during treatment for possible hemodynamic alterations in response to potential sympathetic nervous system stimulation. It is concluded that once acceptable local anesthesia is achieved, the hemodynamic and sympathetic responses to routine restorative dental care are minimal. These findings serve as a baseline for further studies evaluating the response of medically and pharmacologically compromised patients to similar dental procedures.

Experiments were designed to determine if Gray strain infectious bronchitis virus (IBV) infection increases the incidence of urolithiasis type kidney damage when the urine is already high in Ca and relatively alkaline due to a high Ca-low available P diet (i.e., layer ration). In addition, experiments were conducted to determine the effects of Gray strain IBV on pullet renal function of 6 and 14-wk-old pullets at 2, 5, and 10 days postinoculation (PI). Blood gas parameters were measured to determine the mechanism by which layer ration decreases hydrogen ion concentration [( H+]). Urine flow rate, glomerular filtration rate, electrolyte excretion (Na, K, Ca, P), free water clearance, urine osmolality, urine [H+], and renal plasma flow (para-aminohippuric clearance) were measured to assess renal function. Gray strain IBV increased urine [H+] and decreased renal plasma flow in 6-wk-old pullets, and induced a diuresis in 14-wk-old pullets between 5 and 10 days PI. The layer ration increased Ca excretion and induced a metabolic alkalosis, thus decreasing urine [H+] and causing urolith formation. Feeding layer ration followed by Gray strain IBV infection had an additive effect on the incidence of urolithiasis and gross kidney damage. Gray strain IBV infection 8 wk prior to feeding layer ration did not induce urolithiasis. The results suggest that the additive effect of Gray strain IBV on the incidence of urolithiasis is probably due to tubular damage rather than direct changes in renal function parameters.

Adult female chickens are known to have higher heart rates than adult males. Previous evidence suggested that sex-related differences in heart rates exist in embryos. The objective of the present study was to determine if differences in heart rate can be used for sexing chickens during embryonic development. The effects of hyper and hypothermia also were evaluated. On Day 14 of incubation, Single Comb White Leghorn embryos were candled and three surface electrodes were placed on the eggshell. The electrodes were attached to an electrocardiograph. Heart rates were recorded from Days 15 to 20 of incubation. Female embryos exhibited higher mean heart rates than males (2 to 4 beats/min) for Days 15 to 19 of incubation. Average heart rates of male and female embryos decreased and were significantly different when exposed to hypothermia (26.1 C for 2 h) on Day 15 of incubation. Hyperthermia (39.4 C for 45 min) on Day 15 of incubation increased embryonic heart rates, but male and female heart rates did not differ significantly. Extensive overlap between male and female heart rates under normal and hypothermic conditions makes this technique an industrially impractical method for determining embryonic sex.

The present study was designed to evaluate the acute effects of ochratoxin A (OA) on pullet renal function, and to determine if the diuretic effects of citrinin are altered by acute ochratoxicosis. Birds were injected intramuscularly with a 1-mg/mL solution of OA at a dose of 5 mg/kg body weight for 2 consecutive days. Control birds received an equal volume injection of the OA carrier vehicle (100% ethanol). On the 3rd day, birds were anesthetized and prepared for renal function studies. Following 30 min of control urine and plasma collection, an intravenous infusion containing 400 ppm citrinin was initiated, and urine and plasma were collected for an additional 70 min. The OA alone caused an increase in manure moisture and increased hematocrits (hemoconcentration), but direct effects on glomerular filtration rate (GFR), urine flow rate/GFR, para-aminohippuric acid clearance (CPAH), free water clearance (FWC), and electrolye excretion (Na, K, Ca, P) were not consistently demonstrated. The OA may cause hemoconcentration by causing a subtle increase in urine flow (diuresis), or by increasing intestinal water loss (diarrhea). Citrinin infusion caused increased urine flow rate, increased urine flow rate/GFR, increased FWC, increased Na excretion, and decreased urine osmolality. Pretreatment with OA attenuated these effects of citrinin, presumably due to renal compensation for the OA-induced hemoconcentration. Citrinin and OA do not appear to have additive diuretic effects during the first 48 h of toxin exposure.

The objectives of the present study were to examine the effects of repeated or chronic ochratoxin A (OA) and citrinin exposure, and to determine if severe ochratoxicosis permanently alters renal function in pullets. The OA-treated birds were injected intramuscularly (breast muscle) with a 1 mg/mL solution of OA at a dose of .25 mg/kg BW or .5 mg/kg BW. Citrinin-treated birds were injected with a 6 mg/mL solution of citrinin at a dose of 6 mg/kg BW. Control birds received an equal volume injection of the citrinin and OA solvent, 100% ethanol, at a dose of 1 mL/kg BW. Kidney function was evaluated after 10 consecutive days of OA, citrinin, or ethanol injection, and 2 wk following the final injections. Venous blood gas measurements were taken after the seventh day of injection. The OA increased urine flow rate, decreased urine osmolality, increased ion excretion (Na, K, Ca, P), increased water consumption, increased manure moisture, and caused a relative alkalosis when measured immediately after 10 days of OA injection. These effects of OA were not detected 2 wk later. Citrinin increased manure moisture, decreased plasma P, increased the clearance of para-aminohippuric acid, and had no consistent effect on blood acid-base parameters. The results suggest that OA may cause an osmotic diuresis by inhibiting tubular reabsorption of electrolytes. The data also suggests that the effects of OA may be reversible simply by discontinuing toxin administration. Although the citrinin-induced increase in manure moisture indicates that citrinin had an effect on renal function, renal function analysis suggests that even repeated exposure to high doses of citrinin may only have a short duration of diuretic action on the kidneys.

Urinary acidification previously was shown to be an effective treatment for calcium-induced urolithiasis in domestic fowl, but diuresis caused by the acidifying agent (ammonium chloride) was an undesirable side effect. Because supplemental dietary methionine reportedly acidifies mammalian urine, an experiment was conducted to evaluate the efficacy of the free acid form of methionine hydroxy analog (MHA) as an acidifying agent for treating avian urolithiasis. From 5 to 17 wk of age, immature Single Comb White Leghorns were fed diets containing normal calcium (1%) or high calcium (3.5%). Diets were supplemented with 0, 0.3 or 0.6% MHA. Relative to birds fed the normal calcium diets, birds fed the high calcium diet without added MHA were in a state of metabolic alkalosis and excreted more alkaline urine containing high levels of calcium. Birds fed the high calcium diet without MHA also had significantly higher kidney asymmetry ratios, a higher incidence of gross kidney damage, and a higher incidence of urolith formation when compared with birds fed normal calcium diets. When compared with the high calcium diet without MHA, the high calcium diet supplemented with 0.6% MHA significantly acidified the urine without causing detectable metabolic acidosis, significantly reduced kidney asymmetry and gross kidney damage, and reduced the incidence of urolith formation without increasing water consumption or urine flow. These data demonstrate that MHA effectively prevents calcium-induced kidney damage in domestic fowl without causing undesirable side effects. MHA did increase both fractional and absolute calcium excretion during calcium loading.

Experiments were designed to evaluate the effects of Gray strain infectious bronchitis virus (IBV) and high dietary calcium (Ca), alone and in combination, on renal function in pullets. Eight hundred female Single Comb White Leghorn chicks were raised on starter ration. Five hundred chicks were inoculated intravenously with Gray strain IBV at 4 wk of age; the remaining chicks were not exposed to IBV. At 6 wk of age, IBV-inoculated and uninoculated chicks were randomly divided into two diet treatment groups. Half the chicks were fed commercial grower ration (approximately 1.0% Ca, .6% available P) and half were fed commercial layer ration (approximately 3.25% Ca, .5% available P). Birds remained on their respective diets until 18 wk of age. Kidney function studies were conducted on anesthetized birds at 6 wk of age prior to initiation of the diet treatments, at 10 wk of age, and at 18 wk of age. The layer ration increased Ca excretion, decreased inorganic phosphate excretion, and decreased urine hydrogen ion concentration in 10-wk-old pullets in comparison with the grower ration. These diet effects on kidney function were attenuated when the pullets reached 18 wk of age. The layer ration also caused an 11.5% incidence of urolithiasis, and significantly increased kidney asymmetry in 18-wk-old pullets relative to the effects of the grower ration. Gray strain IBV exposure significantly increased kidney asymmetry in 18-wk-old pullets, but had no gross effect on kidney function clearly related to the etiology of urolithiasis. Gray strain IBV did not enhance the incidence of urolithiasis in any of the age groups.

One of the major causes of mortality in poultry is urolithiasis. Although two of the major causes of urolithiasis have been defined, active and preventative treatments for this disease have not been extensively field tested. Previous research demonstrated that a diet acidified with NH4Cl was effective in preventing the formation of uroliths in 16-wk-old pullets. In the present study, experiments were designed to evaluate the effects of dietary acidification and alkalinization on preformed uroliths in mature laying hens. Urolithiasis was induced by feeding excess dietary Ca (in commercial layer ration) during pullet growout (6 to 18 wk of age) and during the early laying period (18 to 32 wk of age). At 32 wk of age birds were randomly divided into three diet treatment groups: a normal layer ration group, an acidified layer ration group (1% NH4Cl added); and an alkalinized layer ration group (1% NaHCO3 added). Each group received the respective diets until 52 wk of age, when the experiment was terminated. Blood gas analysis at 44 wk of age demonstrated that hens fed the acidified diet had significantly higher blood H+ concentrations, lower blood bicarbonate concentrations, and lower total blood carbon dioxide levels than hens fed the alkalinized layer ration. At 52 wk of age, hens fed the acidified layer ration had higher urinary H+ concentrations, higher blood H+ concentrations, and lower blood HCO3 and total CO2 concentrations than hens fed the normal and alkalinized layer rations. The acidified layer ration group had no uroliths present in their ureters (0% urolithiasis), whereas the normal and alkalinized layer ration groups had 8.3 and 13.1% incidences of urolithiasis.(ABSTRACT TRUNCATED AT 250 WORDS)