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N-Methyl-D-Aspartate Receptor Hypofunction in Meg-01 Cells Reveals a Role for Intracellular Calcium Homeostasis in Balancing Megakaryocytic-Erythroid Differentiation
(Thieme Gruppe, 2020-04-14) Hearn JI; Green TN; Chopra M; Nursalim YNS; Ladvanszky L; Knowlton N; Blenkiron C; Poulsen RC; Singleton DC; Bohlander SK; Kalev-Zylinska ML
The release of calcium ions (Ca2+) from the endoplasmic reticulum (ER) and related store-operated calcium entry (SOCE) regulate maturation of normal megakaryocytes. The N-methyl-D-aspartate (NMDA) receptor (NMDAR) provides an additional mechanism for Ca2+ influx in megakaryocytic cells, but its role remains unclear. We created a model of NMDAR hypofunction in Meg-01 cells using CRISPR-Cas9 mediated knockout of the GRIN1 gene, which encodes an obligate, GluN1 subunit of the NMDAR. We found that compared with unmodified Meg-01 cells, Meg-01-GRIN1 -/- cells underwent atypical differentiation biased toward erythropoiesis, associated with increased basal ER stress and cell death. Resting cytoplasmic Ca2+ levels were higher in Meg-01-GRIN1 -/- cells, but ER Ca2+ release and SOCE were lower after activation. Lysosome-related organelles accumulated including immature dense granules that may have contributed an alternative source of intracellular Ca2+. Microarray analysis revealed that Meg-01-GRIN1 -/- cells had deregulated expression of transcripts involved in Ca2+ metabolism, together with a shift in the pattern of hematopoietic transcription factors toward erythropoiesis. In keeping with the observed pro-cell death phenotype induced by GRIN1 deletion, memantine (NMDAR inhibitor) increased cytotoxic effects of cytarabine in unmodified Meg-01 cells. In conclusion, NMDARs comprise an integral component of the Ca2+ regulatory network in Meg-01 cells that help balance ER stress and megakaryocytic-erythroid differentiation. We also provide the first evidence that megakaryocytic NMDARs regulate biogenesis of lysosome-related organelles, including dense granules. Our results argue that intracellular Ca2+ homeostasis may be more important for normal megakaryocytic and erythroid differentiation than currently recognized; thus, modulation may offer therapeutic opportunities.
Requirement of digestible calcium at different dietary concentrations of digestible phosphorus for broiler chickens 3. Broiler finishers (d 25 to 35 post-hatch)
(y Elsevier Inc (USA), on behalf of Poultry Science Association Inc, 2023-04) David LS; Abdollahi MR; Bedford MR; Ravindran V
An experiment was conducted to determine the digestible calcium (Ca) and digestible phosphorous (P) requirements of 25 to 35-day-old broiler chickens. Fifteen corn-soybean meal-based diets containing 2.0, 2.5, 3.0, 3.5, and 4.0 g/kg standardized ileal digestible (SID) Ca and 2.5, 3.5, and 4.5 g/kg SID P were fed to broilers from d 25 to 35 post-hatch. Each experimental diet was randomly allocated to 6 replicate cages (8 birds per cage). Body weight and feed intake were recorded, and the feed conversion ratio was calculated. On d 35, birds were euthanized to collect the ileal digesta, tibia, and carcass for the determination of ileal Ca, and P digestibility, concentrations of ash, Ca, and P in tibia and the retention of Ca and P in the carcass. Titanium dioxide (5.0 g/kg) was included in all diets as an indigestible indicator for the ileal digestibility measurement. Feed intake and total excreta output were measured during the last 4 d of the experimental period for the measurement of apparent total tract retention of Ca and P. Fixed effects of the experiment were dietary concentrations of SID Ca and SID P and their interaction. If the interaction or main effects were significant (P < 0.05), the parameter estimates for second-order response surface model (RSM) were determined using General Linear Model procedure of SAS. The maximum response was not predicted for most of the parameters (including growth performance and tibia) as the Ca effect was linear which indicated that the highest level of Ca employed in the study may have not been high enough. The requirement of dietary SID Ca for maximization of these parameters, therefore, depends on the dietary SID P concentration when the dietary SID Ca is within 2.0 to 4.0 g/kg. However, based on the factorial analysis, the highest weight gain was observed at 3.5 g/kg SID P and 3.5 g/kg SID Ca concentrations. Tibia ash was higher in birds fed 4.5 g/kg SID P and was unaffected by dietary SID Ca concentrations. However, based on overall findings, a combination of 3.5 g/kg SID P and 3.0-3.5 g/kg SID Ca may be recommended for the optimum tibia ash. The recommended SID Ca requirements (at 3.5 g/kg SID P) for weight gain (3.5 g/kg or 6.4 g/kg total Ca) and tibia ash (3.0-3.5 g/kg or 5.5-6.4 g/kg total Ca) are lower than the current Ca recommendations (7.8 g/kg total Ca equivalent to 4.25 g/kg SID Ca; Ross, 2019) for broiler finishers, suggesting possible excess of Ca in diets formulated based on the current recommendation.
pH-dependent sedimentation and protein interactions in ultra-high-temperature-treated sheep skim milk
(Elsevier Inc and Fass Inc on behalf of the American Dairy Science Association, 2023-03) Pan Z; Ye A; Dave A; Fraser K; Singh H
Sheep milk is considered unstable to UHT processing, but the instability mechanism has not been investigated. This study assessed the effect of UHT treatment (140°C/5 s) and milk pH values from 6.6 to 7.0 on the physical properties of sheep skim milk (SSM), including heat coagulation time, particle size, sedimentation, ionic calcium level, and changes in protein composition. Significant amounts of sediment were found in UHT-treated SSM at the natural pH (∼6.6) and pH 7.0, whereas lower amounts of sediment were observed at pH values of 6.7 to 6.9. The proteins in the sediment were mainly κ-casein (CN)-depleted casein micelles with low levels of whey proteins regardless of the pH. Both the pH and the ionic calcium level of the SSM at all pH values decreased after UHT treatment. The dissociation levels of κ-, β-, and αS2-CN increased with increasing pH of the SSM before and after heating. The protein content, ionic calcium level, and dissociation level of κ-CN were higher in the SSM than values reported previously in cow skim milk. These differences may contribute to the high amounts of sediment in the UHT-treated SSM at natural pH (∼6.6). Significantly higher levels of κ-, β-, and αS2-CN were detected in the serum phase after heating the SSM at pH 7.0, suggesting that less κ-CN was attached to the casein micelles and that more internal structures of the casein micelles may have been exposed during heating. This could, in turn, have destabilized the casein micelles, resulting in the formation of protein aggregates and high amounts of sediment after UHT treatment of the SSM at pH 7.0.
Requirement of digestible calcium at different dietary concentrations of digestible phosphorus for broiler chickens. 2. Broiler growers (d 11 to 24 post-hatch)
(Elsevier Inc. on behalf of Poultry Science Association Inc., 2022-11) David LS; Abdollahi MR; Bedford MR; Ravindran V
An experiment was conducted to determine the digestible calcium (Ca) and digestible phosphorous (P) requirements of 11 to 24 d old broiler chickens. Eighteen corn-soybean meal-based diets containing 1.80, 2.35, 2.90, 3.45, 4.00, and 4.55 g/kg standardized ileal digestible (SID) Ca and 3.5, 4.5, and 5.5 g/kg SID P were fed to broilers from d 11 to 24. Each experimental diet was randomly allocated to six replicate cages (8 birds per cage). Body weight and feed amount were recorded at the start and end of the experiment and the feed conversion ratio was calculated. On d 24, birds were euthanized to collect ileal digesta, tibia, and carcass for the determination of digestible Ca and P, the concentration of ash, Ca and P in tibia and the retention of Ca and P in the carcass, respectively. Titanium dioxide (5 g/kg) was included in all diets as an indigestible indicator for apparent ileal digestibility measurement. Total excreta output was measured during the last 4 d of the experimental period for the measurement of apparent total tract retention of Ca and P. Fixed effects of the experiment were dietary concentrations of SID Ca and SID P and their interaction. If the interaction or main effect was significant (P < 0.05), the parameter estimate for second-order response surface model was determined using General Linear Model procedure of SAS. The weight gain of broiler growers was optimized at the SID P concentration of 3.5 g/kg and SID Ca concentrations between 2.35 and 4.00 g/kg. At 3.5 g/kg SID P concentration, the required SID Ca for maximum weight gain was determined to be 3.05 g/kg, which corresponded to SID Ca to SID P ratios of 0.87. The concentration of SID Ca that maximized tibia ash at 3.5 g/kg SID P was 3.69 g/kg, which corresponded to SID Ca to SID P ratio of 1.05. Maximizing bone ash requires more Ca than maximizing weight gain. Carcass Ca and P retention were reflective of total tract Ca and P retention values. The estimated SID Ca requirements (at 3.5 g/kg SID P) for both maximized weight gain (3.05 g/kg or 6.11 g/kg total Ca) and bone ash (3.69 g/kg or 7.28 g/kg total Ca) are lower than the current Ca recommendation (8.70 g/kg total Ca equivalent to 4.03 g/kg SID Ca; Ross, 2019) for broiler growers, indicating possible oversupply of Ca in diets formulated based on the current recommendation.
50 Years of the steric-blocking mechanism in vertebrate skeletal muscle: a retrospective
(Springer Nature Switzerland AG, 2023-09) Parry DAD
Fifty years have now passed since Parry and Squire proposed a detailed structural model that explained how tropomyosin, mediated by troponin, played a steric-blocking role in the regulation of vertebrate skeletal muscle. In this Special Issue dedicated to the memory of John Squire it is an opportune time to look back on this research and to appreciate John’s key contributions. A review is also presented of a selection of the developments and insights into muscle regulation that have occurred in the years since this proposal was formulated.
The effect of calcium and milk formulations on biofilm formation of Geobacillus stearothermophilus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Microbiology, the School of Food and Advanced Technology, Massey University, Palmerston North, New Zealand
(Massey University, 2021) Wang, Tianyang
G. stearothermophilus contaminates milk powder products from bacteria spores released from biofilms on product contact surfaces in dairy manufacturing plants. The dairy industry has observed that calcium-reduced milk protein powder is associated with reduced spore contamination of thermophilic bacteria during milk protein powder manufacture. Calcium, as a major component of milk minerals, was previously found to affect G. stearothermophilus biofilms grown on stainless steel exposed to milk formulations. Additionally, G. stearothermophilus cells cultured with additional calcium showed an increased attachment to stainless steel. The current study investigated the effect of calcium and milk formulations on cell attachment, biofilm formation, spore production and spore heat resistance of G. stearothermophilus to pinpoint the potential factors contributing to the reduced thermophile contamination in the calcium-reduced milk protein powder. The effect of calcium on biofilm formation of G. stearothermophilus dairy isolates A1, P3, and one reference strain 7953 in modified TSB media was studied to gain more insights into the role of calcium in biofilm formation of G. stearothermophilus. The presence of calcium increased biofilm cell numbers of strain A1, but reduced biofilm cell numbers of the reference strain and showed minimal effect on strain P3. Extracellular polymeric substances (EPS) quantity, in particular extracellular protein, varied between strains. Unlike the consistent biofilm promotive effect of calcium in milk formulations found in a previous study, the current findings suggest that a strain-to-strain difference exists in biofilm formation of G. stearothermophilus species in the presence of calcium. Calcium plays an important role in cell attachment by changing cell surface properties, cell physiology and metabolism, modifying cell surface structure and bridging between bacteria and substrata. In the dairy industry, milk formulations with different cation profiles may affect the cell attachment of a common contaminant G. stearothermophilus. The current study investigated the effect of calcium on cell attachment of G. stearothermophilus strains A1, P3 and 7953 to stainless steel and polystyrene substrata and characterized the cell surface charge, hydrophobicity and cell surface polymers. In addition, cell attachment in milk formulations on stainless steel was characterized. The presence of calcium increased the cell attachment of dairy isolates on polystyrene but not the reference strain, while calcium did not affect cell attachment of all strains on stainless steel. The presence of calcium changed the amount of cell surface polymers produced but not hydrophobicity. Although calcium affected the zeta potential, this did not correlate with the trend in cell attachment. It is assumed that the cell attachment of G. stearothermophilus is affected by the substrata, strain specific cell surface polymers, as well as calcium induced changes in cell surface polymers. Milk formulations (MF1 and MF2) with different cation profiles showed little effect on cell attachment of G. stearothermophilus on stainless steel, indicating a minimal effect of cell attachment to contamination levels of different cation-modified milk protein powder products in dairy manufacturing plant. The effect of total calcium concentration, total sodium concentration and bacteria growth history were investigated on biofilm formation of G. stearothermophilus in MF1 and MF2. The numbers of culturable biofilm cells of G. stearothermophilus strain A1, P3 and 7953 were compared in MF1 and MF2 over 18 h. MF1 and MF2 have similar protein, lactose and fat content except for cation profiles, where MF2 has relatively high sodium and low calcium concentrations. Biofilm formation of all three strains was lower in MF2 than in MF1, but the inhibition of MF2 was conditional and was highly dependent on the growth history of bacterial inocula. The inhibition of MF2 on dairy isolates A1 and P3 were further investigated by cation supplementation. Supplementation of MF1 with sodium decreased biofilm formation at 18 h for A1 and P3. Supplementation of MF2 with either 2 or 26 mM calcium increased biofilm formation of A1 at 18 h, and P3 at 10 h. High sodium and low calcium concentrations in the milk formulation seem to be required to inhibit biofilm formation of G. stearothermophilus. However, it is not known if the inhibitory effect of MF2 was due to the direct effect of cations on biofilms or the change of milk protein structure on biofilms due to calcium removal. Cations such as calcium and sodium were shown to affect sporulation and spore heat resistance of G. stearothermophilus. MF1 and 2 have distinctive cation profiles and therefore the sporulation and spore heat resistance of G. stearothermophilus A1, P3 and 7953 in milk formulations were investigated. MF2 effectively reduced the total biofilm spore numbers of A1 and P3 over the 18 h culture period compared to MF1, but the effect was not observed in 7953. The sporulation percentage of A1 was higher in MF1 than MF2 at 14 and 18 h, and a similar effect was observed for P3 at 10 and 14 h. Supplementation of calcium to MF2 increased the sporulation percentage of A1 at 14 and 18 h, as well as P3 at 14 h. Supplementation of sodium to MF1 decreased the sporulation percentage of A1 at 18 h. No significant difference in spore heat resistance of A1, P3 and 7953 was observed between spores produced from MF1 and MF2. Overall, the reduced biofilm formation and sporulation percentage of G. stearothermophilus in MF2 provided evidence to the reduced thermophile contamination in the calcium-reduced milk protein powder.
Sensing and signalling intercalary growth in Epichloë festucae : a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand
(Massey University, 2019) Ozturk, Aslinur
Epichloë festucae is a seed-transmitted symbiont that colonises the aerial parts of grasses and provides protection from biotic and abiotic stress. Although fungal hyphae normally extend at apical tips, exceptions to polar growth characterise the ecology of many important species. Recently, E. festucae has been shown to undergo intercalary growth during host colonization, where hyphae elongate and new compartments are created between existing compartments. Intercalary growth enables the synchronized growth of E. festucae hyphae and plant cells, and rapid hyphal elongation in plant intercellular tissue. Intercalary growth in E. festucae in vitro has been shown to be stimulated by mechanical stretch, mimicking the forces thought to be imposed on hyphae in plants due to their attachment to growing host cells. This research also showed that the High Affinity Calcium Uptake (HAC) and Cell Wall Integrity (CWI) systems influence intercalary growth, however, the mechanisms that regulate cell wall plasticity and compartmentalization are still largely unknown. The aim of this study was to identify the global gene responses to mechanical stress in E. festucae and to further investigate the roles of HACS and CWI in cell wall plasticity and intercalary growth. First, the role of E. festucae MidA, a homolog of the S. cerevisiae Mid1 stress-activated calcium influx channel complex, was addressed to better understand its involvement in intercalary growth and host colonisation. E. festucae MidA had been partly characterized previously and found to regulate vegetative growth, cell wall morphology, calcium influx and colonisation of the intercalary growth zone of ryegrass leaves. In this study, L. perenne seedlings were inoculated with E. festucae wild type, ΔmidA, and midA complementation strains. The effects of midA deletion on rates of plant colonisation, and the phenotype of infected plants was determined, and found to be the same as the wild type, although hyphae were more difficult to detect. The biomass of the different strains in host tissues, enriched either for the shoot apex (including the meristem) or surrounding leaf tissues, was quantified. The results revealed that E. festucae ΔmidA colonization in both tissue types was reduced compared to wild type (WT), but the effect was most convincing during growth in leaf tissues. These findings suggests that MidA function is required for host colonization, particularly in the leaf expansion zone where intercalary growth occurs. The role of MidA in cell wall plasticity and hyphal growth responses to mechanical stretch was next addressed. E. festucae WT and ΔmidA strains were grown on Potato Dextrose Agar (PDA), with or without 50 mM CaCl2 supplementation in a custom stretching device. When grown on PDA without calcium supplementation, the cell walls of WT and midA-complemented deletion strains were able to withstand mechanical stretching equivalent to 6.5% of their hyphal length (applied over approximately 15 min). However, when grown in the presence of 50 mM CaCl2, wild type hyphae and midA-complemented deletion strains were able to withstand 26% of mechanical stretch without visible evidence of cell wall fracture. In contract, ΔmidA strains grown on PDA alone were damaged after 2% of mechanical stress. Supplemental calcium was able to partly rescue this defect, and the ΔmidA strains were able to undergo 8.9% of stretch in PDA plus 50 mM CaCl2. These findings showed that supplemental calcium increases the resilience of E. festucae cell walls to mechanical stretch, and that midA is required for this, presumably by facilitating calcium influx and cell wall plasticity. Next, a transcriptomics study was conducted on E. festucae cultures undergoing various degrees of stretch. Hyphae were grown in vitro on silicon membranes and stretching forces applied to induce intercalary compartment extension and division, as observed in developing leaves. In cultures harvested 5 min after stretch, 105 genes were differentially expressed, whereas after 3 h, that number increased to 403. Analysis of these genes suggested that reprogramming of primary metabolism and plasma membrane organisation occurs almost immediately in response to mechanical stress, and mobilisation of cell wall enzymes and hyphal growth occurs over a longer time period. Finally, previous research as shown that deletion of E. festucae WscA, a homologue of the S. cerevisiae mechano-sensor Wsc1, induced cell wall and hyphal growth defects during growth in culture, however deletion strains were able to colonise ryegrass plants similarly to the wild type. To further elucidate the role of the CWI pathway in intercalary growth, a comprehensive bioinformatics study was co nducted to identify additional E. festucae Wsc proteins which may function upstream of the CWI pathway. A putative E. festucae WscB homolog was identified, plus a new putative cell wall protein with a unique domain. Phylogenetic analysis showed similar proteins in 17 other Epichloë species and entomopathogenic fungi, suggesting the presence of an E. festucae sensor protein that has been evolutionarily conserved. Vectors to delete these genes were constructed and E. festucae antibiotic-resistant colonies recovered. The putative deletion mutants of both strains were very small and compact compared to wild type growth in culture. Efforts to confirm the deletion loci and functionally characterise the mutants will be part of future research. In conclusion, a transcriptomics study has revealed that mechanical stretching induces metabolic changes during early and late responses in E. festucae, promoting early induction of primary metabolism and later changes associated with hyphal growth and cell wall remodelling. Moreover, further investigation of MidA revealed its importance for cell wall plasticity during intercalary expansion, and indicated that calcium is an essential requirement for hyphal resilience to mechanical stretch. Finally a new protein was discovered that responded to mechanical stress and could be a potential mechanosensor protein. This PhD project attempted to broaden our understanding of intercalary growth in E. festucae and pave the way for future studies on mechanical stress response in fungi.
A study on the mechanisms of calcium-induced gelation in skim milk : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Albany, New Zealand
(Massey University, 2019) Lin, Liangjue
The destabilisation and aggregation of milk proteins is the first step towards the gelation of milk. The addition of calcium to milk is known to destabilise milk proteins and may result in gelation on heating. However, the mechanisms involved in gelation induced by heating calcium-added milk was not well understood. Therefore, this project aimed to determine the fundamental mechanisms involved in the development of a calciuminduced skim milk gel. Skim milk was selected as the model system and gelation was induced in-situ by heating the calcium-added skim milk at the rheometer. The changes in the storage modulus, G′, were monitored to study the development of the gel network. This project examined the impact of the following factors on the rheological properties of a calciuminduced skim milk gel: the type of soluble calcium salt added (calcium chloride, calcium lactate, calcium gluconate, calcium lactobionate and calcium iodide), pH, holding temperature during gelation (70°C to 90°C), preheat treatment, ionic strength (by addition of sodium chloride) and the contribution of casein and whey proteins. A higher calcium ion activity (aCa 2+), which indicated a higher calcium ion (Ca2+) concentration, and a lower pH favoured the formation of a stronger gel. An increase in ionic strength by addition of sodium chloride decreased the final G′ of the calciuminduced skim milk gel due to reduced calcium bridging and increased hydration repulsion. A higher heating temperature also resulted in gels with higher final G′ due to more frequent particle collisions. Casein micelles and whey proteins were both responsible for the structure of the gel network. The contribution of whey proteins towards the gel network was dependent on if they were denatured prior to heating, on the concentration of calcium ions available, and on the ratio between the casein and whey proteins present. At lower added calcium concentrations (10 mmol L-1) where the available calcium ions were limited, interactions and aggregation amongst denatured whey proteins via hydrophobic and disulphide bonds may have resulted in the formation of a stronger gel. However, at higher added calcium concentrations (20 and 40 mmol L-1), where sufficient calcium ions may be available for binding, interactions between casein and calcium dominated over the self-aggregating effect of denatured whey proteins. In conclusion, the results demonstrated that the final gel properties of a calcium-induced skim milk gel were dependent on the net effect of all the factors involved in the stability and interactions of the milk proteins, including the calcium salt concentration, pH, preheat treatment, ionic strength, and the protein composition in solution. These findings provide alternative methods for texture modification in milk.
The effect of dietary calcium and other nutritionally relevant divalent cations on fatty acid-soap formation : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Sciences at Massey University, Manawatū, New Zealand
(Massey University, 2016) Stroebinger, Natascha
A growing amount of scientific evidence appears to support a relationship between dietary calcium (Ca) and body weight where increased dietary Ca intake leads to weight reduction and the faecal excretion of several fatty acids. One possible mechanism, explaining the effect of dietary Ca on body weight and faecal fatty acid excretion, is the formation of indigestible Ca-fatty acid soaps within the gastrointestinal tract, leading to reduced fat and therefore reduced energy absorption. The objectives of this research were 1) to confirm that dietary Ca reduces fatty acid absorption and that the effect is via the formation of fatty acid soaps, 2) to explore the potential of cations other than Ca to form fatty acid soaps and 3) to investigate where in the gastrointestinal tract Ca-fatty acid soap formation occurs. In order to investigate the presence of fatty acid-soaps in the gastrointestinal tract, an assay was developed to determine fatty acid-soaps in digesta and faeces. Faecal fatty acid-soap excretion, apparent faecal fatty acid digestibility and apparent faecal Ca digestibility were determined in the growing pig for diets containing different sources of fat (tallow, palmolein oil, olive oil and soya bean oil) and increasing concentrations of Ca (0, 2, 4 and 6 g kg-1 diet). Increasing concentrations of dietary Ca resulted in increased faecal fatty acid excretion (P < 0.001), predominantly in the form of fatty acid-soaps (> 80%) for diets containing a fat source rich in saturated fatty acids (tallow and palmolein oil). The fatty acid digestibility of these diets was reduced (P < 0.001) by up to 28% when the dietary Ca intake was increased from 0 g Ca kg-1 diet to 6 g Ca kg-1 diet. Moreover, faecal Ca output of the tallow-based diet, for which the fatty acid soap excretion was the greatest, was statistically higher when compared to the oil containing diets. These results provide evidence that supports the hypothesis that dietary Ca can impair fat absorption via the formation of indigestible Ca-fatty acid soaps but that the effect is largely limited to fat sources rich in saturated fatty acids as evidenced by the reduction in Ca absorption with tallow. Given that Ca appears to react with fatty acids to form soaps, it was decided to investigate whether other nutritionally relevant divalent cations (magnesium (Mg), zinc (Zn), iron (Fe) and copper (Cu)) were able to form fatty acid soaps. To that end, in vitro studies revealed that apart from Ca, other divalent cations such as Zn, Mg, Fe and Cu had the ability to form precipitates in the presence of fatty acids. In general, all the divalent cations examined formed precipitates in the presence of at least some of the fatty acids examined, although the extent to which the divalent cation-fatty acid precipitates (soaps) formed varied depending on the cation and fatty acid present. The precipitation of saturated fatty acids (lauric, myristic, palmitic and stearic acid) when incubated with Zn was comparable with that of Ca. However, the precipitation of unsaturated fatty acids (oleic and linoleic acid) with Zn was greater than that observed for Ca. For Fe and Cu, fatty acid precipitation was less than that observed for Ca. To investigate where in the gastrointestinal tract fatty acid soaps form, growing pigs were fed diets containing either free fatty acids or an intact triacylglyceride (tallow) and calcium carbonate as the Ca source. The amount of insoluble fatty acid-soap present in the gastrointestinal tract was determined at 10 different locations within the tract. The amount of fatty acid-soaps present increased (P < 0.05) at the distal jejunum when the free fatty acid-based diet was fed and at the ileum when pigs received the tallow-based diets, and was correlated with the pH (regardless the diet) of the gastrointestinal tract suggesting that soaps formed as the pH of the gastrointestinal tract increased. Fatty acid-soap formation in the small intestine of pigs receiving the free fatty acids was almost double than for pigs receiving tallow with their diet. There was little soap formation in the hind gut. With the majority of fatty acid soap formation occurring in the distal small intestine (the major absorption site of fatty acids) fatty acid-soap formation has the potential to reduce fatty acid absorption. Feeding a fat-free diet in addition to the two fat containing diets gave insight into mineral absorption in the absence and presence of dietary fat. The apparent digestibility of Ca, Mg, Zn and Fe was lower (P < 0.05) in the presence of dietary fat (free fatty acids or triacylglycerides) suggesting that the formation of divalent cation-fatty acid soaps may have the ability to impair the absorption of divalent cations other than Ca. In conclusion, high dietary Ca intake leads to increased faecal fatty acid excretion in the form of insoluble fatty acid-soaps. Fatty acid-soap formation can impair the digestibility of Ca and other nutritionally relevant divalent cations such as Zn, Mg and Fe. Moreover, fatty acid-soaps appear to form mainly in the distal small intestine and appear to be associated with gastrointestinal pH. These results contribute to the knowledge of where fatty acid soap formation occurs and provide evidence that fatty acid soap formation can reduce fat absorption and thereby possibly contribute to weight loss.
The relationship between nutritional adequacy and 24-month fracture occurence in Māori and non-Māori of advanced age : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Nutrition and Dietetics, Massey University, Albany, New Zealand
(Massey University, 2015) Towgood, Alice
Abstract Background The life expectancy of both Māori and non-Māori is continually increasing with more New Zealanders expected to live into advanced age. Adults over the age of 80 experience greater health loss than any other age group, with chronic disease and associated disability increasing substantially with age. Osteoporosis and the morbidity associated with fractures, particularly hip fracture, are of critical concern for an ageing population and may diminish quality of life and independence for older people, thus placing an increased burden on health and disability support services. The role of nutrition in the maintenance of bone mineral density (BMD), bone integrity, and subsequent fracture prevention, particularly in octogenarians is unclear. The ability to meet adequate energy requirements decrease with increasing age and may compromise intake of nutrients related to bone health. Nutrients necessary for bone health including: protein, calcium, vitamin D, phosphorous, magnesium and potassium are modifiable factors. Achieving optimal bone nutrient intakes may influence potential for maintenance of good bone health in adults of advanced age. This study aims firstly to investigate food and nutrient intakes of Māori and Non-Māori octogenarians to establish an understanding of nutrient adequacy. Secondly, to investigate the energy and nutrient intakes of participants who experience a fracture compared with those non-fractured to identify nutrient specific risk factors for fracture in adults of advanced age. Method Comprehensive nutritional parameters were collected using two separate 24-hour multi-pass recalls. FOOD files were used to analyse food sources and nutrient intake. Face to face interviews were conducted to ascertain specific social, demographic, health and fracture information. Fracture occurrence was measured over a 24 month period following the 2 x 24-hour Multi Pass Recall’s and included self-reported and hospitalised fracture occurrences. Hospitalisation data was obtained with permission from the participants. National Health Index New Zealand (NHI) numbers were used to identify fractures. Results There were 317 participants (113 Māori and 204 non-Māori), aged 80-90 years in this study. For men and women respectively the median energy intakes were 6,943kJ vs. 5,603kJ for Māori; and 8,203kJ vs. 6,225kJ for non-Māori; protein as a percentage of energy was 15.5% vs. 15.9% for Māori and 15.7% vs. 15.5% for non-Māori. The top foods contributing to energy were bread, butter and margarine for all Maori and non-Maori with beef and veal contributed the most protein for Māori men, bread for Māori women and milk for non-Māori, men and women. Compared to the Estimated Average Requirement (EAR) intakes of calcium, vitamin D, magnesium and potassium were inadequate for all participants. Compared to an EAR of 1100mg for men and women, median calcium intakes were low, 559mg vs. 539mg for Māori and 748mg vs. 672mg for non-Māori, men and women respectively. The primary food groups contributing to calcium were milk, cheese and bread. Compared to the EAR (15 μg/day in men and women) and vitamin D intake from food was low (≤ 4 μg) for all participants. Compared to the EAR (350mg/day men and 265mg/day women), median magnesium intakes were 259 mg/day vs. 204mg/day for Māori and 271 mg/day vs. 238 mg/day for non-Māori, men and women respectively. The primary food groups contributing to magnesium were bread, breakfast cereals and fruit. A total of 18.6% of Māori and 20.6% of non-Māori sustained a fracture over a 24 month period. One in five Māori and non-Māori women sustained fractures. Among non-Māori women those who fractured were 1.1 times more likely to be financially insecure than non-fractured women (p=0.033). For Māori women who were fractured, inability to afford to eat properly was 3.3 times more likely (p=0.012), and previous fractures were 1.5 times (p=0.015) more likely than for non-fractured women. Fractured Māori women consumed significantly less vitamin D (2.0μg vs 3.0μg) (P=0.01) and magnesium (143.0mg vs 211mg) (P=0.033) compared to non-fractured Māori women. Conclusion Energy intakes were low for all participants and may have manifested the suboptimal intakes of calcium, vitamin D, potassium and magnesium prevalent in Māori and non-Māori, men and women. Fractures were more frequent in women than men, and both Māori and non-Māori sustained similar rates of fracture over the 24 month period. Magnesium and vitamin D intakes were significantly related to fracture occurrence in Māori women; this relationship diminished with further regression analysis. Increased intake of energy in adults of advanced age, with a focus on protein rich and nutrient dense foods, particularly calcium and magnesium, should be encouraged through consuming a variety of foods from the major food groups. Greater intakes of calcium can be achieved through higher consumption of milk and dairy products including yoghurt and cheese; and magnesium through increased green leafy vegetables, seafood, dairy, mushrooms, avocado, beans and bananas. Vitamin D intakes were minimal from food; however it is possible participants were receiving supplementary vitamin D and further investigation is warranted. For fractured Māori women, magnesium intake was significantly lower than those with no fractures. Promoting increased intakes of culturally acceptable foods such as vegetables and seafood may be advantageous to increase magnesium intakes.

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