Calcium competes with zinc for a channel mechanism on the brush border membrane of piglet intestine.

Interactions between Ca(+2) and Zn(+2) at the intestinal brush border membrane occur via unclear mechanisms. We hypothesized that Zn(+2) and Ca(+2) are transported across the brush border membrane via a multidivalent metal channel. Using brush border membrane vesicles (BBMV) prepared from intestines of 8 sow-fed piglets, we sought to determine whether Ca(+2) competes with Zn(+2) for uptake. Extravesicular Zn(+2) was removed with ethylenediamine-tetraacetic acid. Time curves of Zn(+2) and Ca(+2) uptake by BBMV were conducted with increasing concentrations of Ca(+2) and Zn(+2), respectively. Saturation curves compared kinetic parameters of Zn(+2) uptake with and without Ca(+2). In addition, Zn(+2) uptake was measured in the presence of various classical Ca(+2) channel modulators. Over 20 min, a 0.4x concentration of Zn(+2) lowered Ca(+2) uptake by vesicles, whereas a 30x concentration of Ca(+2) was necessary to lower Zn(+2) uptake. These data suggest that Ca(+2) has lower affinity than Zn(+2) for a brush border membrane transport protein. Kinetic parameters showed higher K(m) values with 4 or 15 mM Ca(+2) but unchanged J(max), suggesting competitive inhibition. The Ca(+2) channel blocking agents, La(+3), Ba(+2), verapamil, and diltiazem, inhibited Zn(+2) uptake, whereas calcitriol, trans 1,2 cyclohexanediol, cis/trans 1,3 cyclohexanediol, and the L-type Ca(+2) channel agonist, Bay K8644, induced Zn(+2) uptake. These data were consistent with competition for a common transport mechanism on the brush border membrane, possibly a novel multimetal channel. Copyright 2001 Elsevier Science Inc.

Divalent metals inhibit and lactose stimulates zinc transport across brush border membrane vesicles from piglets.

Interactions between metals of similar coordination chemistry are of relevance to infant nutrition due to the highly variable metal:metal ratios found in formulas. Using ratios similar to those found in infant formulas, our objectives were to determine the effects of metals and of lactose and other saccharides on Zn(+2) transport across intestinal brush border membranes. Brush border membrane vesicles prepared from intestines of 5 preweaned piglets were used to determine whether Ca(+2), Mg(+2), Fe(+2), Cu(+2), Cd(+2), or Mn(+2) would antagonize Zn(+2) uptake. (65)Zn(+2) uptake by brush border membrane vesicles was measured over 20 min with metal concentrations constant, and at 1 min with increasing metal concentrations. Zn(+2) bound to the external surface of vesicles was removed with ethylenediamine-tetraacetic acid. Lactose induced Zn(+2) uptake to a greater extent than glucose polymer, whereas maltose, galactose, or galactose/glucose had no effect. Over 20 min, a 10:1 concentration of Fe(+2), Cd(+2), Cu(+2), and Mn(+2) lowered Zn(+2) uptake significantly (P < 0.05). Higher concentrations of divalent cation significantly lowered Zn(+2) (0.2 or 0.1 mM) uptake for all metals tested (P < 0.05), except for Mn(+2) (0.1 mM Zn(+2)). Inhibition constant determination quantified relative competitive potential with Mg(+2) < Ca(+2) << Mn(+2) < Fe(+2) < Zn(+2) << Cu(+2). Relative amounts of Ca(+2), Mg(+2), and Fe(+2) similar to those found in infant formulas reduced Zn(+2) uptake by at least 40%. Our data demonstrate that dietary minerals compete during brush border membrane transport, and may help explain antagonistic mineral interactions observed in vivo. Divalent metal concentrations and lactose content of milk affect zinc absorption in neonates and must be carefully considered in formula design.

The effect of a Tropaeolum speciosum oil supplement on the nervonic acid content of sphingomyelin in rat tissues.

The lipids of Tropaeolum speciosum (T. speciosum) are a rich source of naturally occurring nervonic acid (24:1n-9). We report that adding a T. speciosum oil supplement to a semi-purified diet significantly increased the amount of 24:1n-9 in liver and heart, but not brain, sphingomyelin (SM) of young rats. The bioavailability of 24:1n-9 from the lipids of T. speciosum was similar to that of 24:1n-9 ethyl ester in this rat bioassay.

Fatty acid analysis of blood plasma of patients with Alzheimer's disease, other types of dementia, and cognitive impairment.

Fatty acid differences, including docosahexaenoic acid (DHA; 22:6n-3) have been shown in the brains of Alzheimer's patients (AD) as compared with normal age-matched individuals. Furthermore, low serum DHA is a significant risk factor for the development of AD. The relative concentration of DHA and other fatty acids, however, in the plasma of AD patients compared with patients with other kinds of dementias (other dementias; OD), patients who are cognitively impaired but nondemented (CIND), or normal patients is not known. In this study we analyzed the total phospholipid, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and lysophosphatidylcholine (lysoPC) fractions of plasma from patients diagnosed with AD, OD, or CIND and compared them with a group of elderly control subjects with normal cognitive functioning. Plasma phospholipid and PC levels of 20:5n-3, DHA, total n-3 fatty acids, and the n-3/n-6 ratio were lower in the AD, OD, and CIND groups. Plasma phospholipid 24:0 was lower in the AD, OD, and CIND groups as compared with the group of control patients, and total n-6 fatty acid levels were higher in the AD and CIND groups only. In the plasma PE fraction, levels of 20:5n-3, DHA, and the total n-3 fatty acid levels were significantly lower in the AD, OD, and CIND groups. DHA levels were lower in the lysoPC fraction of CIND individuals only. There were no other differences in the fatty acid compositions of the different phospholipid fractions. Therefore, in AD, OD, and CIND individuals, low levels of n-3 fatty acids in the plasma may be a risk factor for cognitive impairment and/or dementia. Interestingly, a decreased level of plasma DHA was not limited to the AD patients but appears to be common in cognitive impairment with aging.

The occurrence of Iso 24:0 (22-methyltricosanoic acid) fatty acid in sphingomyelin of rat tissues.

The occurrence of an unusual fatty acid, iso 24:0 (22-methyltricosanoic acid), has been shown to be present in the sphingomyelin from rat liver by gas liquid chromatography-mass spectrometry. The fatty acid is also present in the sphingomyelin of rat tissues tested, except for brain and fetal serum. Supplementation of diets with L-valine elevated the content of iso 24:0 in rat liver, heart, and kidney sphingomyelin. The fatty acid appears to be present in sphingomyelin of the sera of mice, dogs, and cats, but not humans.

Dietary very long chain fatty acids directly influence the ratio of tetracosenoic (24:1) to tetracosanoic (24:0) acids of sphingomyelin in rat liver.

Twenty-one groups of weanling male Wistar rats were fed semipurified diets containing 5% (w/w) of different dietary fats. After 2 wk, liver sphingomyelin (SM) fatty acid composition was determined. The ratio of 24:1 to 24:0 in liver SM varied over a tenfold range in response to dietary fat type. Step-wise multiple regression analysis indicated that dietary 24:1, 24:0, and 22:1 were the most significant factors in predicting the 24:1/24:0 ratio of liver SM. The mathematical relation between the dietary fatty acid composition and liver SM 24:1/24:0 was y = 1.88 (24:1) -1.49 (24:0) +0.21 (22:1) +0.01 (18:1) +0.26, r2 = 0.95, P < 0.0001. These results were confirmed by a second experiment in which the rats were fed olive oil-based diets supplemented with various fatty acid ethyl esters.

Mechanism of recovery in esophageal epithelia of rats with severe zinc deficiency.

Forty-six rats fed a Zn-deficient diet for 18 days, were divided into three groups and treated with Zn-deficient diet (GI), a normal (Zn-adequate) diet (GII) or a pharmacological Zn therapy diet (GIII) for 3 days. For light and electron microscopy, samples were taken at times 0, 8, 12, 24 and 72 h after treatment. In treatment GI, at all times, all rats had esophageal parakeratosis. With treatment GII, there was a variable progression toward normalization of the epithelium at 12 and 24 h. At 72 h there was almost complete recovery of normal epithelium. In treatment GIII at 8 h, large, light cells in the basal layer were shown to be present between dark cylindrical cells, a finding which was transitionary and disappeared at 12 h. Also, a thin keratinized layer was observed above the granular layer at 12 h. Recovery had progressed at 24 h and was complete after 72 h. The results are discussed in terms of a potential role of Zn in the sequence of cytochemical events in epithelial differentiation.

Zinc and selenium, site-specific versus general antioxidation.

The essential nutrients zinc (Zn) and selenium (Se) provide an antioxidant function to animal cells by very different mechanisms. Se is an integral part of Se-dependent glutathione peroxidases, a group of water-soluble enzymes that catalyze the destruction of water-soluble and, in some cases, membrane-bound hydroperoxides. In dietary Se deficiency, Se-dependent glutathione peroxidase activities are decreased; at Se intakes above that which is required for optimal growth, there is a slight to moderate increase in Se-dependent glutathione peroxidase activities. Because of the enzymatic nature of the major role of Se as an antioxidant, Se can be categorized as having a general antioxidant function, controlling peroxide levels in cells by degrading hydroperoxides. On the other hand, Zn functions as an antioxidant only at specific sites, and is not a required cofactor for an antioxidant enzyme. Although Zn plays a structural role in the enzyme Cu, Zn superoxide dismutase, the activity of this enzyme is not decreased in Zn deficiency and its activity is usually depressed at high Zn intakes. Zn may function as a site-specific antioxidant by two mechanisms. Firstly, it competes with Fe and Cu for binding to cell membranes and some proteins, displacing these redox-active metals and making them more available for binding to ferritin and metallothionein, respectively. Secondly, Zn binds the sulfhydryl groups in proteins, protecting them from oxidation. Zn status does not directly control tissue peroxide levels but can protect specific molecules against oxidative and peroxidative damage.

Investigations of the gallbladder pathology associated with dietary exposure to disodium arsenate heptahydrate in juvenile rainbow trout (Oncorhynchus mykiss).

Juvenile rainbow trout were fed semi-purified diets with (58 micrograms As/g diet) or without arsenic, added as disodium arsenate heptahydrate (DSA), under standard laboratory conditions for up to 12 weeks, to determine the time-course of development of gallbladder pathology in response to dietary DSA exposure, to correlate this pathology with levels of total arsenic and specific arsenic metabolites in the hepatobiliary system and thereby to attempt to gain some insight regarding the mechanism(s) by which the pathological changes develop. Gallbladder lesions associated with this level of dietary arsenic exposure to juvenile rainbow trout include acute inflammation with oedema of the submucosal tissues and sloughing of the epithelium within the first day of exposure, developing to chronic inflammation with fibrosis of the gallbladder wall. These changes may result from the toxic influence of arsenite absorbed into the epithelial cells. The arsenic content of washed gallbladder tissue is a sensitive indicator of recent dietary DSA exposure, while the chronic inflammatory lesion with extensive fibrosis of the gallbladder wall may provide a longer-term indicator of exposure to toxic levels of DSA in the diet of rainbow trout.

Zinc deficiency in the rat alters the lipid composition of the erythrocyte membrane Triton shell.

The effect of dietary zinc deficiency on the lipid composition of the erythrocyte membrane Triton shell was determined. Weanling male Wistar rats were fed an egg white-based diet containing < 1.0 mg Zn/kg diet ad libitum. Control rats were either pair-fed or ad libitum-fed the basal diet supplemented with 100 mg Zn/kg diet. A Zn refed group was fed the -Zn diet until day 18 and then pair-fed the +Zn diet until day 21. Dietary Zn deficiency caused an increased cholesterol/phospholipid ratio in Triton shells compared to those from pair-fed controls. Zn deficiency caused a decreased double bond index of fatty acids in phosphatidylinositol (PI) and phosphatidylcholine (PC); there was a decreased proportion of 18:2n-6 and 22:4n-6 in PC and 20:4n-6 in PI as compared to that found in pair-fed controls. All glycerophospholipids that were retained in the shell had a lower double bond index and increased content of 16:0 and/or 18:0 relative to the phospholipid in the intact membrane.

Erythrocytes, erythrocyte membranes, neutrophils and platelets as biopsy materials for the assessment of zinc status in humans.

During a controlled zinc depletion-repletion study, fifteen men aged 25.3 (SD 3.3) years were fed on a low-Zn diet with high phytate:Zn and phytate x calcium:Zn molar ratios for 7 weeks, followed by a 2 week repletion period when 30 mg supplemental Zn/d was given. Changes in plasma, urine, and hair Zn concentrations, taste acuity, and cellular immune response confirmed the development of mild Zn deficiency. Zn concentrations in neutrophils, platelets, erythrocytes and erythrocyte membranes, mean platelet volume, and activities of alkaline phosphatase (EC 3.1.3.1) and alpha-D-mannosidase (EC 3.2.1.24) in neutrophils did not respond to changes in Zn status. In contrast, alkaline phosphatase activity in erythrocyte membranes showed a significant decline which was consistent in all subjects (nmol product formed/min per mg protein; baseline v. 7-week Zn depletion, 0.656 (SD 0.279) v. 0.506 (SD 0.230), at 7 weeks; P < 0.05); neutral phosphatase activity remained unchanged. Alkaline phosphatase activity in erythrocyte membranes may be a potential index of Zn status in humans.

The relationship between the rate of chelator-induced zinc efflux from erythrocytes and zinc status.

The rate of zinc (Zn) release from rat erythrocytes incubated in buffers containing a variety of chelators was measured. Only o-phenanthroline, 8-hydroxyquinoline-5-sulfonate, and EDTA caused detectable Zn release. The relationship between the rate of this release in the presence of o-phenanthroline and Zn status was determined in rats. Rats were fed one of the following: a modified AIN-76 diet providing 46 mumol (3 mg) Zn per kg of diet, a pair-fed diet providing 459 mumol (30 mg)/kg, or the previous diet fed ad lib. Animals were sacrificed at 2-wk intervals for 12 wk, and the Zn efflux rate, plasma, liver, and femur Zn concentrations were determined. The efflux rate was lower in erythrocytes taken from the rats fed the low-Zn diet. The efflux rate was also well correlated with femur Zn (r = 0.509, n = 98, p < 0.0001). A poorer correlation was observed with plasma Zn in the rats. Correlations also were determined between efflux rates and plasma Zn levels in human subjects. There was a significant correlation only in the males. In was concluded that the Zn efflux rate from erythrocytes incubated in the presence of o-phenanthroline is related to Zn status but is not sensitive enough to be a useful index of this status.

Indices of iron and copper status during experimentally induced, marginal zinc deficiency in humans.

This study examined the effect of diet-induced, marginal zinc deficiency for 7 wks in 15 men (aged 25.3 +/- 3.3 yrs; mean +/- SD) on selected indices of iron and copper status. The regimen involved low-zinc diets based on egg albumin and soy protein with added phytate and calcium such that mean [phytate]/[Zn] and [phytate] X [Ca]/[Zn] molar ratios were 209 and 4116, respectively, for 1 wk, followed by 70 and 2000, respectively, for 6 wks. Subjects were then repleted with 30 mg Zn/d for 2 wks. Plasma copper, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activity in plasma and red blood cells (RBC), hemoglobin, hematocrit, and serum ferritin were determined weekly on fasting blood samples. Significant reductions (p less than 0.05) after 7 wks in RBC Cu,Zn-superoxide dismutase (49.5 +/- 7.2 vs 33.6 +/- 6.3 U/mg Hb) and serum ferritin (69.2 +/- 38.7 vs 53.8 +/- 33.7 micrograms/L) occurred; no comparable decline was noted for plasma Cu, hemoglobin, or hematocrit. Significant (p less than 0.05) but less consistent changes were also observed in plasma superoxide dismutase activity. None of the changes were associated with the decreases in plasma, urinary and hair zinc concentrations, and alkaline phosphatase activity in RBC membranes. Results indicate that the biochemical iron and copper status of the subjects was marginally impaired, probably from the dietary regimen that induced marginal zinc deficiency.

Zinc deficiency alters the protein composition of the membrane skeleton but not the extractability or oligomeric form of spectrin in rat erythrocyte membranes.

We examined the effect of dietary Zn deficiency on the composition and structure of the rat erythrocyte membrane skeleton. Weanling rats were given free access to egg white-based diets (less than 1.0 mg Zn/kg) for 3 wk. Controls were fed diets (pair-fed or ad libitum) supplemented with 100 mg Zn/kg. Membrane skeleton proteins were extracted from isolated erythrocyte membranes in a low ionic strength buffer. Dietary Zn deficiency did not alter the content of spectrin, the major membrane skeleton protein, in the intact membranes or the percentage of spectrin extracted after 24 or 96 h. Zinc deficiency did not alter the oligomeric form of spectrin in the extracts that were analyzed in the presence or absence of EDTA. However, Zn deficiency resulted in a significant reduction in the relative content of protein R5 in the membrane skeleton extracts. The food restriction associated with dietary Zn deficiency was the major factor in the significant reduction in the relative content of RA (adducin) and R4 (protein 4.1) in the membrane skeleton extracts. Dietary Zn deficiency altered membrane skeleton protein composition but had no effect on the extractability or oligomeric form of spectrin.

Chronic toxicity of dietary disodium arsenate heptahydrate to juvenile rainbow trout (Oncorhynchus mykiss).

Juvenile rainbow trout were fed semi-purified diets containing graded levels of disodium arsenate heptahydrate (DSA) for 12-24 weeks under standard laboratory conditions to define the maximum acceptable toxicant concentration (MATC) and to correlate signs of toxicity with diet and tissue arsenic concentrations. The MATC for DSA was between 13 and 33 micrograms As/g diet or 0.281-0.525 mg As/kg body weight/day. The most sensitive and reliable indicator of chronic dietary DSA toxicity in rainbow trout was chronic inflammation of the gallbladder wall. Chronic inflammatory changes in the sub-epithelial tissues of the gallbladder wall were evident in 71% of rainbow trout exposed to 33 micrograms As/g diet for 24 weeks, and 100% of rainbow trout exposed to 65 micrograms As/g diet for 24 weeks or 49 micrograms As/g diet for 12 weeks. No fish exposed to 13 micrograms As/g diet or less for up to 24 weeks showed any demonstrable gallbladder lesions or any other ill effect of arsenic exposure. Other signs of chronic dietary DSA toxicity to rainbow trout included decreased growth rate, mild to moderate anemia, and, at higher levels of exposure, active feed refusal leading to decreased feed consumption. Mild nephrocalcinosis was noted in one experiment where kidney arsenic residues exceeded 14 micrograms As/g tissue dry weight.

The effect of dietary zinc deficiency on the lipid composition of the rat erythrocyte membrane.

The effect of dietary zinc deficiency in the rat on the lipid composition of the erythrocyte membrane was determined. Weanling male Wistar rats were fed an egg white-based diet containing less than 1.0 mg Zn/kg diet ad libitum. Control rats were either pair-fed or ad libitum-fed the basal diet supplemented with 100 mg Zn/kg diet. A zinc refed group was fed the -Zn diet until day 18 and then pair-fed the +Zn diet until day 21. The voluntary feed restriction associated with dietary zinc deficiency resulted in erythrocyte membranes that had depressed phospholipid/protein and elevated cholesterol/phospholipid ratios. Similarly, all feed restricted groups had elevated 22-carbon n-3 polyunsaturated fatty acids (PUFA) and depressed 22-carbon n-6 PUFA concentrations in alkenyl-acyl and diacyl glycerophosphoethanolamine, phosphatidylserine and phosphatidylcholine; they also had depressed 24:2n-6 levels in sphingomyelin. The relative concentrations of phospholipids in the membrane was similar between -Zn and +Zn (ad libitum) groups; however, the -Zn group had significantly less phosphatidyl-serine relative to +Zn (pair-fed) controls.

Development of a dietary model for the study of mild zinc deficiency in humans and evaluation of some biochemical and functional indices of zinc status.

After a 1-wk baseline period, a dietary regimen was developed to induce mild zinc deficiency in 15 males (aged 25.3 +/- 3.3 y, mean +/- SD). The regimen consisted of 1 wk on a liquid diet containing 0.6 mg Zn/d and molar ratios of phytate to zinc (phy:Zn) and of phytate X calcium to zinc [(phy X Ca): Zn] of 209 and 4116, respectively, followed by 6 wk on a diet based on soy protein and egg albumin containing 4 mg Zn/d and with phy:Zn and (Ca X phy):Zn of 70 and 2000, respectively. Subjects were then repleted with 30 mg Zn/d for 2 wk. Fasting blood and urine samples were taken weekly. Changes were observed in mean plasma (mumol/L) and urinary zinc (mumol/d): baseline 97.0 +/- 10.9 and 8.0 +/- 2.7, depletion 80.1 +/- 13.4 and 4.3 +/- 2.3, and repletion 100.8 +/- 13.6 and 8.2 +/- 3.1, respectively (P less than 0.05); taste acuity (0.05 less than P less than 0.10); and cellular immune responses (P less than 0.05). Activities of plasma angiotensin-1-converting enzyme and acidic alpha-D-mannosidase were unchanged. Mild zinc deficiency was induced by the dietary regimen.

Lack of an association between cellular phospholipid triene: tetraene ratio and proliferation of human skin fibroblasts in culture.

Two experiments were performed in an attempt to establish an association between cellular phospholipid triene:tetraene ratio and proliferation of human neonatal skin fibroblasts in culture. In Experiment 1, a low lipid culture medium was developed that caused an accumulation of (n-9) eicosatrienoic acid in the phospholipids of human fibroblasts. This culture medium, when supplemented with a mixture of mitogens, supported growth of human fibroblasts at a level equivalent to that found under conditions of maximal growth using serum supplementation (8% fetal bovine serum). The triene:tetraene ratio of fibroblast phospholipids under the two conditions was 1.88 vs. 0.03, suggesting that the growth of these cells was not adversely affected by a high (greater than 0.4) triene: tetraene ratio. In Experiment 2, cells were cultured in a low lipid, mitogen-supplemented medium with 16:1(n-7), 18:1(n-9), 18:2(n-6) or 20:4(n-6) added as the albumin complex. All the fatty acids permitted an equivalent maximal growth stimulation in the assay system, although having different effects on the phospholipid triene:tetraene ratio. The results suggest that there is a lack of an association between cellular phospholipid triene:tetraene ratio (range, 0.03 to 3.4) and proliferation of human fibroblasts in this culture system.