The Source Matters–Effects of High Phosphate Intake from Eight Different Sources in Dogs

SIMPLE SUMMARY: The vast majority of pet food products on the market contain the major mineral phosphorus in amounts exceeding the recommended daily allowance. A considerable amount of phosphorus in such products is highly water-soluble and bioavailable. Even though a sufficient supply of phosphorus is important for the body, an excessive intake can be harmful, especially in renal patients but also healthy individuals. This study investigated the effects of an excessive intake of organic phosphate sources (e.g., meat and bone meal) and inorganic phosphate salts compared with a balanced control diet without inorganic phosphates on factors of the body’s phosphorus regulatory system in healthy, adult beagle dogs. Most inorganic phosphates but not the organic phosphorus sources caused significant changes in these regulatory factors compared with the control diet. We conclude that the use of these inorganic phosphates in pet food is potentially harmful and should be restricted. ABSTRACT: Elevated serum phosphate concentrations are an established risk factor for cardiovascular disease and mortality in chronic kidney disease in various species. Independent associations of other parameters of phosphorus metabolism, such as phosphorus intake from different sources and serum concentrations of phosphorus, as well as parameters involved in the regulation, such as parathyroid hormone (PTH) or markers of bone turnover, have been studied in less detail. Therefore, the serum kinetics of phosphate, PTH, and the bone resorption marker bone-specific alkaline phosphatase (BAP) were investigated after 18 days of feeding a control diet and diets supplemented with eight different organic and inorganic phosphate sources aiming at 1.8% phosphorus per dry matter and calcium to phosphorus ratio between 1.3 and 1.7 to 1. Eight healthy beagle dogs (f/m, 2–4 years, 12.9 ± 1.4 kg body weight) were available for the trial. Highly significant differences in the serum kinetics of phosphorus, PTH, and BAP with the highest postprandial levels after feeding highly water-soluble sodium and potassium phosphates were found. We conclude that the use of certain inorganic phosphates in pet food is potentially harmful and should be restricted

Engineering yield and rate of reductive biotransformation in Escherichia coli by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport

Optimization of yields and productivities in reductive whole-cell biotransformations is an important issue for the industrial application of such processes. In a recent study with Escherichia coli, we analyzed the reduction of the prochiral β-ketoester methyl acetoacetate by an R-specific alcohol dehydrogenase (ADH) to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate (MHB) using glucose as substrate for the generation of NADPH. Deletion of the phosphofructokinase gene pfkA almost doubled the yield to 4.8 mol MHB per mole of glucose, and it was assumed that this effect was due to a partial cyclization of the pentose phosphate pathway (PPP). Here, this partial cyclization was confirmed by 13C metabolic flux analysis, which revealed a negative net flux from glucose 6-phosphate to fructose 6-phosphate catalyzed by phosphoglucose isomerase. For further process optimization, the genes encoding the glucose facilitator (glf) and glucokinase (glk) of Zymomonas mobilis were overexpressed in recombinant E. coli strains carrying ADH and deletions of either pgi (phosphoglucose isomerase), or pfkA, or pfkA plus pfkB. In all cases, the glucose uptake rate was increased (30–47%), and for strains Δpgi and ΔpfkA also, the specific MHB production rate was increased by 15% and 20%, respectively. The yield of the latter two strains slightly dropped by 11% and 6%, but was still 73% and 132% higher compared to the reference strain with intact pgi and pfkA genes and expressing glf and glk. Thus, metabolic engineering strategies are presented for improving yield and rate of reductive redox biocatalysis by partial cyclization of the PPP and by increasing glucose uptake, respectively

NADPH-dependent reductive biotransformation with Escherichia coli and its pfkA deletion mutant: Influence on global gene expression and role of oxygen supply

An Escherichia coli ΔpfkA mutant lacking the major phosphofructokinase possesses a partially cyclized pentose phosphate pathway leading to an increased NADPH per glucose ratio. This effect decreases the amount of glucose required for NADPH regeneration in reductive biotransformations, such as the conversion of methyl acetoacetate (MAA) to (R)-methyl 3-hydroxybutyrate (MHB) by an alcohol dehydrogenase from Lactobacillus brevis. Here, global transcriptional analyses were performed to study regulatory responses during reductive biotransformation. DNA microarray analysis revealed amongst other things increased expression of soxS, supporting previous results indicating that a high NADPH demand contributes to the activation of SoxR, the transcriptional activator of soxS. Furthermore, several target genes of the ArcAB two-component system showed a lower mRNA level in the reference strain than in the ΔpfkA mutant, pointing to an increased QH2/Q ratio in the reference strain. This prompted us to analyze yields and productivities of MAA reduction to MHB under different oxygen regimes in a bioreactor. Under anaerobic conditions, the specific MHB production rates of both strains were comparable (7.4 ± 0.2 mmolMHB h−1 gcdw−1) and lower than under conditions of 15% dissolved oxygen, where those of the reference strain (12.8 mmol h−1 gcdw−1) and of the ΔpfkA mutant (11.0 mmol h−1 gcdw−1) were 73% and 49% higher. While the oxygen transfer rate (OTR) of the reference strain increased after the addition of MAA, presumably due to the oxidation of the acetate accumulated before MAA addition, the OTR of the ΔpfkA strain strongly decreased, indicating a very low respiration rate despite sufficient oxygen supply. The latter effect can likely be attributed to a restricted conversion of NADPH into NADH via the soluble transhydrogenase SthA, as the enzyme is outcompeted in the presence of MAA by the recombinant NADPH-dependent alcohol dehydrogenase. The differences in respiration rates can explain the suggested higher ArcAB activity in the reference strai