Adherence to Vitamin D Supplementation Recommendations for Breastfed Infants and Young Children: An Analysis of Canadian Community Health Survey Data Cycles From 2015 to 2018.

BACKGROUND: In Canada, nutrition policy, as outlined in the Nutrition for Healthy Term Infants recommendations, includes a daily vitamin D supplement of 10 µg (400 IU) for breastfed infants and young children to support adequate vitamin D status. OBJECTIVES: This study aimed to report on adherence to vitamin D supplementation recommendations for breastfed infants (≤12 months); and for children breastfed >12 mo. METHODS: Canadian Community Health Survey (paired-cycles 2015/2016 and 2017/2018) maternal experiences data for infants born 2012-2018 who received any breastmilk formed the sample (n = 7079). Whether the infant was given a vitamin D supplement (yes/no) and the frequency (daily/almost every day, 1-2/wk, or <1/wk) were surveyed. Weighted data (95% CI) were summarized according to breastfeeding history (exclusive to 6 mo and continuing; partial to 6 mo and continuing; and stopped ≤6 mo). Correlates of supplement adherence were explored using logistic regression. RESULTS: Overall, 87.1% (95% CI: 85.9%, 88.3%) of participants reported giving their infant (≤12 mo) a vitamin D supplement, and of these, 83.3% (95% CI: 81.9%, 84.7%) did so daily/almost every day, 12.4% (95% CI: 11.1%, 13.7%) did so 1-2/wk, and 4.3% (95% CI: 3.6%, 5.0%) did so <1/wk. Lower adjusted odds of adherence were observed among participants reporting: stopped breastfeeding ≤6 mo, lower education or income, recent immigration, and overweight prepregnancy body mass index; higher odds of adherence were observed in the western provinces. Regarding mothers of children >12 mo and breastfed (n = 2312), 58.0% (95% CI: 54.9%, 61.1%) gave a vitamin D supplement daily/almost every day. CONCLUSIONS: Adherence to providing a vitamin D supplement to breastfed infants is high in Canada. Nonetheless, we estimate that ∼27% of mothers are nonadherent to daily/almost every day administration of a vitamin D supplement and that adherence declines in children breastfed >12 mo. Further promotion to support uptake of the current guidance may be necessary, particularly for parents of recent immigration or lower socioeconomic status.

Population Iron Status in Canada: Results from the Canadian Health Measures Survey 2012-2019.

BACKGROUND: In Canada, population iron status estimates are dated (2009-2011) and did not consider the presence of inflammation. OBJECTIVES: This study aimed to update iron status estimates in Canada using serum ferrin (SF) and evaluate different correction methods for inflammation based on c-reactive protein (CRP). METHODS: Data from the Canadian Health Measures Survey cycles 3-6 (2012-2019) formed a multiyear, cross-sectional, nationally representative sample (3-79 y) (n = 21,453). WHO cutoffs for SF and hemoglobin were used to estimate iron deficiency (ID), iron deficiency anemia (IDA), anemia, and elevated iron stores. ID was first estimated without considering inflammation. Correction approaches evaluated were excluding individuals with CRP >5 mg/L, using modified SF cutoffs, and regression correction. RESULTS: Total population uncorrected prevalence estimates were 7% (95% CI: 6.2, 7.9) ID, 6.1% (95% CI: 5.2, 7.0) anemia, and 2.0% (95% CI: 1.6, 2.4) IDA. The uncorrected prevalence of ID was the highest among females of reproductive age with 21.3% (95% CI: 17.6, 25.0) and 18.2% (95% CI: 15.4, 21.1) in 14-18 y and 19-50 y, respectively. Corrected ID estimates were higher than the uncorrected values, independent of the correction approach. Regression correction led to a moderate increase in the prevalence to 10.5% for the total population, whereas applying the higher modified SF cutoffs (70 µg/L for those older than 5 y) led to the largest increases in the prevalence, to 12.6%. Applying modified cutoffs led to implausibly high ID estimates among those with inflammation. Elevated iron stores were identified in 17.2% (95% CI: 16.2, 18.2) of the population, mostly in adult males. CONCLUSIONS: Correction methods for estimating population iron status need further research. Considering the fundamental drawbacks of each method, uncorrected and regression-corrected estimates provide a reasonable range for ID in the Canadian population. Important sex-based differences in iron status and a public health ID problem of moderate magnitude among females of reproductive age are evident in Canada.

Iodine Status of Mother-Infant Dyads from Montréal, Canada: Secondary Analyses of a Vitamin D Supplementation Trial in Breastfed Infants.

BACKGROUND: Most pregnant or lactating women in Canada will not meet iodine requirements without iodine supplementation. OBJECTIVES: To assess the iodine status of 132 mother-infant pairs based on secondary analyses of a vitamin D supplementation trial in breastfed infants from Montréal, Canada. METHODS: Maternal iodine status was assessed using the breastmilk iodine concentration (BMIC). Singleton, term-born infants were studied from 1-36 months of age. Usual (adjusted for within-person variation) iodine intakes were estimated from urinary iodine and creatinine concentrations. Iodine status was assessed using median urinary iodine concentrations (UICs) and by estimating inadequate intakes by the cut-point method using a proposed Estimated Average Requirement for infants 0-6 months of age (72 µg/d). RESULTS: At 1, 3, and 6 months of age, 70%, 63%, and 3% of infants, respectively, were exclusively breastfed. From 1-36 months of age (n = 82-129), the median UICs were ≥100 µg/L (range, 246-403 µg/L), which is the cutoff for adequate intakes set by the WHO for children <2 years. Almost all (98%-99%) infants at 1 and 2 months, 2 and 3 months, and 3 and 6 months of age had usual creatinine-adjusted iodine intakes ≥ 72 µg/d. The median BMIC was higher (P < 0.001) at 1 month compared to 6 months of lactation [1 month, 198 µg/kg (IQR, 124-274; n = 105) and 6 months, 109 µg/kg (IQR, 67-168; n = 78)]. At 1 and 6 months, 96% and 79% of mothers, respectively, had a BMIC ≥ 60 µg/kg, the lower limit of a normal reference range. The percentages of mothers that used a multivitamin-mineral (MVM) supplement containing iodine were 90% in pregnancy and 79% and 59% at 1 and 6 months of lactation, respectively. CONCLUSIONS: The iodine status of infants was adequate throughout infancy. These results support a recommendation that all women who could become pregnant, who are pregnant, or who are breastfeeding take a daily MVM supplement containing iodine.

Iodine Status of Canadian Children, Adolescents, and Women of Childbearing Age.

BACKGROUND: Adequate iodine intake is important for children and women of childbearing age because iodine is vital for fetal brain development and early life. OBJECTIVE: Iodine status of children (n = 1875), adolescents (n = 557), and women of childbearing age (n = 567) was assessed using urinary iodine concentrations (UIC) from duplicate spot samples collected in the Canadian Health Measures Survey, cycle 5 (2016-2017). METHODS: Daily iodine intakes were estimated from urinary iodine and creatinine concentrations using a formula based on iodine absorption and predicted 24-h creatinine excretion. Usual UIC and iodine intakes, adjusted for within-person variation, were estimated using the National Cancer Institute method. Iodine status was assessed by 1) comparing median UIC with WHO/UNICEF/ICCIDD reference ranges and 2) estimating the prevalence of inadequate and excessive intakes using the estimated average requirement (EAR) and tolerable upper intake level (UL) cut-point method, respectively. RESULTS: Median UIC for males and females 6-11 or 12-19 y old were ≥100 µg/L, the lower cutoff for adequate intakes. For women 20-39 y old, the median UIC of an unadjusted sample was 81 µg/L (95% CI: 67, 95) and for the usual UIC was 108 µg/L (95% CI: 84, 131). The percentage of children 3 y old with iodine intake ≥EAR was 82% (95% CI: 75, 89). The corresponding estimates for males 4-8, 9-13, and 14-18 y old were 93% (95% CI: 88, 97), 91% (95% CI: 87, 96), and 84% (95% CI: 76, 91), respectively. Estimates for females 4-8, 9-13, 14-18, and 19-39 y old were 86% (95% CI: 83, 89), 87% (95% CI: 80, 95), 68% (95% CI: 55, 80), and 68% (95% CI: 59, 76), respectively. For all sex-age groups, 91-100% had iodine intakes ≤UL. CONCLUSIONS: Iodine intakes may be insufficient for some women of childbearing age. Public health policies and programs should continue to recommend that all women who could become pregnant, or women who are pregnant or breastfeeding, take a daily multivitamin-mineral supplement containing iodine.

Dietary supplementation with L-lysine affects body weight and blood hematological and biochemical parameters in rats.

L-Lysine (Lys) is a popular additive in foods, but the physiological effects of excess Lys supplementation are poorly understood and upper limits of safe intake have not been established. The objectives of this study were to examine the effects of dietary supplementation with increasing amounts of Lys on body weight (BW), food intake, and various blood hematological and biochemical parameters in rats. Male Sprague-Dawley rats at 10 weeks of age were assigned to ten diet groups (eight rats/group) and fed diets containing either 7% or 20% casein and supplemented with either 0% (Control), 1.5%, 3%, 6% Lys, or 6% Lys + 3% arginine for 1 week. Rats fed 7% casein with ≥ 1.5% Lys supplementation had lower serum albumin and leptin and higher LDL cholesterol (LDLC), ratios of total cholesterol (TC):HDL cholesterol (HDLC) and LDLC:HDLC than those fed 7% casein Control diet (P < 0.05). Rats fed 7% casein diet supplemented with 3% Lys diet had lower BW gain, food intake, serum alkaline phosphatase activity, and increased mean corpuscular hemoglobin concentration, blood urea nitrogen and serum pancreatic polypeptide compared to rats fed the Control diet (P < 0.05). Addition of 6% Lys in 7% casein caused significant BW loss (P < 0.001) and altered additional parameters. Addition of 6% Lys in a 20% casein diet reduced BW gain and food intake and altered numerous parameters. Arg supplementation normalized many of the endpoints changed by Lys. Collectively, these results show that Lys supplementation affects BW, food intake and a number of hematological and biochemical parameters. These effects of Lys supplementation were confined primarily in diets with lower levels of dietary protein. In the context of a low protein diet (7% casein), levels of Lys supplementation ≥ 1.5% may exert adverse health effects in rats.

Diet-induced obese rats have higher iron requirements and are more vulnerable to iron deficiency.

PURPOSE: Since obesity is associated with poorer iron status, the effects of diet-induced obesity on iron status and iron-regulatory pathways were examined. METHODS: Weanling male diet-induced obese sensitive (n = 12/diet group) and resistant (n = 12/diet group) rats were fed one of four high-fat, high-energy diets supplemented with 5 (5Fe, low), 15 (15Fe, marginal), 35 (35Fe, normal) or 70 (70Fe, high) mg iron/kg diet for 12 weeks. At the end of the study, rats in each diet group were categorised as obese (>19 %) or lean (<17 %) based on percentage body fat. RESULTS: Obese rats gained more weight, had larger total lean mass, consumed more food and showed greater feed efficiency compared with lean rats. Obese rats fed the 5Fe and 15Fe diets had poorer iron status than lean rats fed the same diet. Obese 5Fe rats had lower serum iron and more severe iron-deficiency anaemia. Obese 15Fe rats had lower mean corpuscular haemoglobin and liver iron concentrations. Hepcidin mRNA expression in liver and adipose tissue was similar for obese and lean rats. Iron concentration and content of the iron transporters divalent metal transporter 1 and ferroportin 1 in duodenal mucosa were also similar. CONCLUSIONS: Obese rats that were larger, regardless of adiposity, had higher iron requirements compared with lean rats that appeared independent of hepcidin, inflammation and intestinal iron absorption. Higher iron requirements may have resulted from larger accretion of body mass and blood volume. Greater food consumption did not compensate for the higher iron needs, indicating increased susceptibility to iron deficiency.

Zinc supplementation does not alter sensitive biomarkers of copper status in healthy boys.

The tolerable upper intake levels (UL) for zinc for children were based on limited data and there is concern that the UL may be set too low. The first effect of excessive zinc intake is a reduction in copper status. The primary objective of this study was to examine the effect of zinc supplementation on copper status in children. Healthy, 6- to 8-y-old boys from Ontario, Canada were assigned to take a placebo (n = 10) or 5 mg (n = 10), 10 mg (n = 9), or 15 mg (n = 8) of zinc supplement daily for 4 mo in a double-blinded, placebo-controlled, randomized trial. Biochemical measures were evaluated at baseline and after 2 and 4 mo of supplementation. Food records were completed near the baseline and 4-mo visits. Age and anthropometric measurements did not differ (P > 0.05) between treatment groups at baseline. Mean zinc intakes from food alone (10.9-14.8 mg zinc/d) approached or exceeded the UL of 12 mg/d. Compared with the placebo group, the zinc groups had a greater change in the urine zinc:creatinine ratio at 4 mo (P = 0.02). Traditional (plasma copper and ceruloplasmin activity) and more sensitive biomarkers of copper status, including erythrocyte SOD1 activity and the erythrocyte CCS:SOD1 protein ratio, were unchanged in zinc-supplemented boys, demonstrating that copper status was not depressed. Serum lipid measures and hemoglobin concentrations were also unaffected and gastrointestinal symptoms were not reported. These data provide evidence in support of the need for reexamining the current UL for zinc for children.

Mineral concentrations in bottled water products: implications for Canadians' mineral intakes.

PURPOSE: The popularity of bottled water products (BWPs) is growing in Canada. Concentrations of minerals with important implications for health were compared in different types of BWPs. METHODS: One sample of each brand and type of plain BWP (purified, remineralized, spring, mineral, and artesian), flavoured BWP, and nutrient-enriched BWP sold in major stores in Ottawa, Ontario, was purchased to allow determination of mineral concentrations by flame atomic absorption or emission spectroscopy. A total of 124 BWPs representing 37 brands were analyzed. RESULTS: In general, spring and mineral water contained higher amounts of magnesium and calcium than did purified, remineralized, artesian, flavoured, or nutrient-enriched water. Most plain BWPs contained little sodium and potassium, whereas 15% to 35% of flavoured and nutrient-enriched products had considerably higher concentrations. Only magnesium and calcium concentrations were highly correlated (r=0.76, p<0.001). Calculation of the percentage of Dietary Reference Intakes that could be supplied by each product revealed that, if they are consumed habitually, many products can contribute substantially to recommended intakes of these minerals. CONCLUSIONS: Mineral concentrations in most types of BWP varied, but distinct differences between types of products were identified. Consumers should be aware of the mineral content of BWPs because some could influence intakes of certain minerals significantly.

A low chromium diet increases body fat, energy intake and circulating triglycerides and insulin in male and female rats fed a moderately high-fat, high-sucrose diet from peripuberty to young adult age.

Trivalent chromium (Cr) may function to potentiate the action of insulin, but the effects of chromium intakes on metabolic parameters are unclear. Cr is listed as a potentially beneficial element for rodents based on studies that show feeding low quantities affect glucose metabolism. Cr is recommended at 1 mg per kg in rodent diets. This study examined the effects of different levels of dietary Cr on body weight, body composition, energy intake, food efficiency and metabolic parameters of lipid and glucose metabolism in male and female rats when fed from peripuberty to young adult age in the background of a moderately high-fat, high-sucrose diet. Sprague-Dawley CD rats (n = 10 males and 10 females/group) at 35 days of age were assigned by weight to the low (LCr, 0.33 ± 0.06 mg/kg), normal (NCr, 1.20 ± 0.11 mg/kg) or high (HCr, 9.15 ± 0.65 mg/kg) Cr diets. Diets were fed ad libitum for 12 weeks (83 days). At baseline, body weights and composition were similar (p≥0.05) among diet groups. Compared to the NCr group, the LCr group weighed more (p<0.01) and consumed more energy (food) from Day 56 onwards, but food efficiency was unaffected. Following an oral glucose challenge (Day 77), dietary chromium levels did not affect plasma glucose, but fasting plasma insulin and insulin at 30 and 60 min after dosing were higher in the LCr group compared to the NCr group. At the end of the study, whole-body fat, accrued body fat from baseline and fasting serum triglycerides were higher in the LCr group compared to the NCr group. Effects were similar in both sexes and not observed in the HCr group. These data show that low dietary Cr affects metabolic parameters common in chronic diseases underscoring the need for clinical trials to define the nutritional and/or pharmacological effects of Cr.

L-Lysine supplementation affects dietary protein quality and growth and serum amino acid concentrations in rats.

Single amino acid (AA) supplementations in foods are increasing, however their potential nutritional and physiological impacts are not fully understood. This study examined the effects of L-lysine (Lys) supplementation on protein quality of diets, serum AA concentrations and associations between the ratio of supplemental Lys to dietary protein (X) with body weight gain (BWG) in Sprague-Dawley male rats. Rats were fed one of 10 diets containing either 7% or 20% casein and supplemented with 0% (Control), 1.5%, 3%, 6% Lys or 6% Lys + 3% L-arginine (Arg) (8 rats/diet group) for 1 week. Lys supplementation reduced the protein quality of the casein-based diets (p < 0.01). BWG was reduced by supplemental Lys when X > 0.18. Free Lys supplementation dose-dependently increased serum Lys levels (p < 0.01), while increased protein-bound Lys (1.4% vs 0.52%) had little effect on serum Lys (p > 0.05). In the 7% casein diets, ≥ 1.5% supplemental Lys reduced serum alanine, asparagine, glycine, isoleucine, leucine, serine, tyrosine, valine, carnitine, ornithine, and increased urea. Supplementation of ≥ 3% Lys additionally reduced tryptophan and increased histidine, methionine and α-aminoadipic acid (α-AAA) compared to the Control (p < 0.05). In the 20% casein diets, addition of ≥ 1.5% Lys reduced serum asparagine and threonine, and ≥ 3% Lys reduced leucine, proline, tryptophan, valine, and ornithine, and 6% Lys reduced carnitine, and increased histidine, methionine, and α-AAA. Overall, this study showed that free Lys supplementation in a Lys-sufficient diet reduced the protein quality of the diets and modified the serum concentrations of many amino acids. Excess free Lys intake adversely affected growth and utilization of nutrients due to AA imbalance or antagonism. Overall lower protein intake increases susceptibility to the adverse effects of Lys supplementation.

Iodine nutrition: Disorders, monitoring and policies.

Iodine is an essential mineral nutrient and an integral component of thyroid hormones. Iodine deficiency is typically associated with goiter, but can have more serious health implications. Adequate iodine status is important for normal brain development. Iodine deficiency in utero or in early life can cause severe neurological and cognitive impairment. Over the last three decades, global efforts have reduced the prevalence of iodine deficiency disorders (IDD) in many areas of the world with implementation of nutrition policies and programs such as "salt" iodization. However, in a number of areas iodine deficiency is still widespread. Iodine deficiency in remote regions with high poverty will be more difficult to eradicate. Efforts to eliminate IDD in affected areas and sustaining successful iodine programs will be a priority given the substantial public health and economic benefits. A key component will be periodic monitoring of population iodine status to ensure sufficient intakes and the absence of excessive intakes. Median urinary iodine concentration (UIC), a validated biomarker for assessing population iodine status, will facilitate monitoring. Research validating "usual" UIC for use in combination with the Estimated Average Requirement cut-point method will expand its utility and allow accurate determination of the prevalence of inadequate intakes in populations. Further research on the development of biomarkers for assessment of individual iodine status for routine patient care will be important.

Copper transporter 2 content is lower in liver and heart of copper-deficient rats.

Copper (Cu) transporter 2 (Ctr2) is a transmembrane protein that transports Cu across cell membranes and increases cytosolic Cu levels. Experiments using cell lines have suggested that Ctr2 expression is regulated by Cu status. The importance of changes in Ctr2 expression is underscored by recent studies demonstrating that lower Ctr2 content in cells increases the cellular uptake of platinum-containing cancer drugs and toxicity to the drugs. In this study, we examined whether Ctr2 expression is altered by a nutritional Cu deficiency in vivo. Ctr2 mRNA and protein in liver and heart from rats fed a normal (Cu-N), moderately deficient (Cu-M) or deficient (Cu-D) Cu diet was measured. Rats fed the Cu-deficient diets showed a dose-dependent decrease in liver Ctr2 protein compared to Cu-N rats. Ctr2 protein was 42% and 85% lower in Cu-M and Cu-D rats, respectively. Liver Ctr2 mRNA was 50% lower in Cu-D rats and unaffected in Cu-M rats. In heart, Ctr2 protein was only lower in Cu-D rats (46% lower). These data show that Cu deficiency decreases Ctr2 content in vivo.

Decreased erythrocyte CCS content is a biomarker of copper overload in rats.

Copper (Cu) is an essential trace metal that is toxic in excess. It is therefore important to be able to accurately assess Cu deficiency or overload. Cu chaperone for Cu/Zn superoxide dismutase (CCS) protein expression is elevated in tissues of Cu-deficient animals. Increased CCS content in erythrocytes is particularly sensitive to decreased Cu status. Given the lack of a non-invasive, sensitive and specific biomarker for the assessment of Cu excess, we investigated whether CCS expression in erythrocytes reflects Cu overload. Rats were fed diets containing normal or high levels of Cu for 13 weeks. Diets contained 6.3 +/- 0.6 (Cu-N), 985 +/- 14 (Cu-1000) or 1944 +/- 19 (Cu-2000) mg Cu/kg diet. Rats showed a variable response to the high Cu diets. Some rats showed severe Cu toxicity, while other rats showed no visible signs of toxicity and grew normally. Also, some rats had high levels of Cu in liver, whereas others had liver Cu concentrations within the normal range. Erythrocyte CCS protein expression was 30% lower in Cu-2000 rats compared to Cu-N rats (P < 0.05). Notably, only rats that accumulated high levels of Cu in liver had lower erythrocyte CCS (47% reduction, P < 0.05) compared to rats fed normal levels of Cu. Together, these data indicate that decreased erythrocyte CCS content is associated with Cu overload in rats and should be evaluated further as a potential biomarker for assessing Cu excess in humans.

Calcium exacerbates the inhibitory effects of phytic acid on zinc bioavailability in rats.

BACKGROUND: Complementary feeding of breastfed infants with foods high in bioavailable zinc (Zn) can help meet physiological requirements for Zn. Some infant cereals contain high concentrations of phytic acid (PA) and calcium (Ca) that may reduce absorbable Zn. OBJECTIVES: This study measured PA, Zn and Ca concentrations in selected infant cereals sold in Canada and investigated the effects of dietary PA and Ca at concentrations present in infant cereals on Zn bioavailability in rats. METHODS AND RESULTS: Male Sprague-Dawley rats (36-day old) were fed a control diet containing normal Zn (29.1 mg/kg) and Ca (4.95 g/kg) or six test diets (n = 12/diet group). Test diets were low in Zn (8.91-9.74 mg/kg) and contained low (2.16-2.17 g/kg), normal (5.00-5.11 g/kg) or high (14.6-14.9 g/kg) Ca without or with added PA (8 g/kg). After 2 weeks, rats were killed and Zn status of the rats was assessed. PA, Zn and Ca concentrations in infant cereals (n = 20) differed widely. PA concentrations ranged from undetectable to 16.0 g/kg. Zn and Ca concentrations ranged from 7.0-29.1 mg/kg and 0.8-13.4 g/kg, respectively. The [PA]/[Zn] and [PA × Ca]/[Zn] molar ratios in infants cereals with detectable PA (16 of 20 cereals) ranged from 22-75 and 0.9-14.9 mol/kg, respectively, predicting low Zn bioavailability. Body weight, body composition (lean and fat mass), right femur weight and length measurements and Zn concentrations in serum and femur indicated that diets higher in Ca had a more pronounced negative effect on Zn status of rats fed a PA-supplemented diet. Addition of PA to the diet had a greater negative effect on Zn status when Ca concentration in the diet was higher. CONCLUSION: These results show that, in rats, higher concentrations of dietary Ca and PA interact to potentiate a decrease in bioavailable Zn and may suggest lower Zn bioavailability in infant cereals with higher PA and Ca concentrations.

Increased incorporation of dietary plant sterols and cholesterol correlates with decreased expression of hepatic and intestinal Abcg5 and Abcg8 in diabetic BB rats.

The aim of this study was to determine the impact of dietary plant sterols and stanols on sterol incorporation and sterol-regulatory gene expression in insulin-treated diabetic rats and nondiabetic control rats. Diabetic BioBreeding (BB) and control BB rats were fed a control diet or a diet supplemented with plant sterols or plant stanols (5 g/kg diet) for 4 weeks. Expression of sterol-regulatory genes in the liver and intestine was assessed by real-time quantitative polymerase chain reaction. Diabetic rats demonstrated increased tissue accumulation of cholesterol and plant sterols and stanols compared to control rats. This increase in cholesterol and plant sterols and stanols was associated with a marked decrease in hepatic and intestinal Abcg5 (ATP-binding cassette transporter G5) and Abcg8 (ATP-binding cassette transporter G8) expressions in diabetic rats, as well as decreased mRNA levels of several other genes involved in sterol regulation. Plant sterol or plant stanol supplementation induced the accumulation of plant sterols and stanols in tissues in both rat strains, but induced a greater accumulation of plant sterols and stanols in diabetic rats than in control rats. Surprisingly, only dietary plant sterols decreased cholesterol levels in diabetic rats, whereas dietary plant stanols caused an increase in cholesterol levels in both diabetic and control rats. Therefore, lower expression levels of Abcg5/Abcg8 in diabetic rats may account for the increased accumulation of plant sterols and cholesterol in these rats.

Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells.

Ctr1 (copper transporter 1) mediates high-affinity copper uptake. Ctr2 (copper transporter 2) shares sequence similarity with Ctr1, yet its function in mammalian cells is poorly understood. In African green monkey kidney COS-7 cells and rat tissues, Ctr2 migrated as a predominant band of approximately 70 kDa and was most abundantly expressed in placenta and heart. A transiently expressed hCtr2-GFP (human Ctr2-green fluorescent protein) fusion protein and the endogenous Ctr2 in COS-7 cells were mainly localized to the outer membrane of cytoplasmic vesicles, but were also detected at the plasma membrane. Biotinylation of Ctr2 with the membrane-impermeant reagent sulfo-NHS-SS-biotin [sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate] confirmed localization at the cell surface. Cells expressing hCtr2-GFP hyperaccumulated copper when incubated in medium supplemented with 10 microM CuSO(4), whereas cells depleted of endogenous Ctr2 by siRNAs (small interfering RNAs) accumulated lower levels of copper. hCtr2-GFP expression did not affect copper efflux, suggesting that hCtr2-GFP increased cellular copper concentrations by promoting uptake at the cell surface. Kinetic analyses showed that hCtr2-GFP stimulated saturable copper uptake with a K(m) of 11.0+/-2.5 microM and a K(0.5) of 6.9+/-0.7 microM when data were fitted to a rectangular hyperbola or Hill equation respectively. Competition experiments revealed that silver completely inhibited hCtr2-GFP-dependent copper uptake, whereas zinc, iron and manganese had no effect on uptake. Furthermore, increased copper concentrations in hCtr2-GFP-expressing cells were inversely correlated with copper chaperone for Cu/Zn superoxide dismutase protein expression. Collectively, these results suggest that Ctr2 promotes copper uptake at the plasma membrane and plays a role in regulating copper levels in COS-7 cells.

Considerations in the development of biomarkers of copper status.

Copper (Cu) is an essential nutrient, but a harmful metal in excess. As part of the North American Dietary Reference Intakes, the Recommended Dietary Allowance for Cu was set using a combination of biomarkers of Cu deficiency, including plasma Cu and ceruloplasmin concentrations, erythrocyte Cu/Zn superoxide dismutase activity, and platelet Cu concentration. Liver damage was the sole indicator used in setting the Tolerable Upper Intake Level. Some studies suggest that these conventional biomarkers may not be sensitive enough to detect marginal reductions or excesses of Cu that could pose a health risk. The insensitivity of conventional biomarkers casts uncertainty as to the prevalence of Cu deficiency or overload in the population and in the accuracy of current nutritional reference values for Cu. Numerous biochemical changes have been associated with alterations in Cu status, and many potential biomarkers of Cu nutriture have been proposed; yet, conventional biomarkers are still the most frequently used, underscoring the need for research efforts to substantiate the use of novel biomarkers. In this report, biomarkers of Cu status are reviewed and practical considerations in the development of novel biomarkers are discussed as diagnostic tools for assessing Cu status in humans.

Dietary calcium affects body composition and lipid metabolism in rats.

Calcium (Ca) intakes may affect cardiovascular disease risk by altering body composition (body weight and fat) and serum lipid profile, but results have been inconsistent and the underlying mechanisms are not well understood. The effects of dietary Ca on body composition and lipid metabolism were examined in rats. Male Sprague-Dawley rats were fed high-fat, high-energy diets containing (g/kg) low (0.75Ca, 0.86 ± 0.05; 2Ca, 2.26 ± 0.02), normal (5Ca, 5.55 ± 0.08) or high (10Ca, 11.03 ± 0.17; 20Ca, 21.79 ± 0.15) Ca for 10 weeks. Rats fed the lowest Ca diet (0.75Ca) had lower (p < 0.05) body weight and fat mass compared to other groups. Rats fed the high Ca diets had lower serum total and LDL cholesterol compared to rats fed normal or low Ca. Liver total cholesterol was lower in rats fed high compared to low Ca. In general, liver mRNA expression of genes involved in cholesterol uptake from the circulation (Ldlr), cholesterol synthesis (Hmgcr and Hmgcs1), fatty acid oxidation (Cpt2) and cholesterol esterification (Acat2) were higher in rats fed higher Ca. Apparent digestibility of total trans, saturated, monounsaturated and polyunsaturated fatty acids was lower in rats fed the high compared to the low Ca diets, with the largest effects seen on trans and saturated fatty acids. Fecal excretion of cholesterol and total bile acids was highest in rats fed the highest Ca diet (20Ca). The results suggest little effect of dietary Ca on body composition unless Ca intakes are very low. Decreased bile acid reabsorption and reduced absorption of neutral sterols and saturated and trans fatty acids may contribute to the better serum lipid profile in rats fed higher Ca.