James D. House

Regulation of homocysteine metabolism.

We have used a combination of in vivo and in vitro techniques to measure factors regulating homocysteine metabolism and the plasma concentration of this atherogenic amino acid. The germane findings include: 1. Homocysteine metabolism in rat kidney proceeds predominantly through the transsulfuration pathway, whose enzymes are enriched within the proximal cells of kidney tubules. Furthermore, the rat kidney possesses significant reserve capacity to handle both acute and chronic elevations in plasma homocysteine concentrations. 2. Plasma homocysteine concentrations are lower in diabetic rats. Insulin administration corrects this perturbation. Therefore, insulin and/or one of its counter-regulatory hormones affects homocysteine metabolism, possibly through an increased flux in the hepatic transsulfuration pathway. In support of these data, glucagon administration to rats produced similar results. Further support was provided by studies with isolated rat hepatocytes, from which homocysteine export was reduced when incubated in the presence of glucagon.

Performance and welfare of laying hens in conventional and enriched cages

Concerns regarding the welfare of laying hens raised in battery cages have led to the development of enriched cages that allow hens to perform natural behaviors including nesting, roosting, and scratching. This study was conducted to compare indices of production and welfare in birds housed in 2 different caging systems. Shaver White hens were housed from 21 to 61 wk in either conventional battery cages (n = 500; 10 cages; 5 hens/cage; floor space = 561.9 cm(2)/hen) or enriched cages (n = 480; 2 cages; 24 hens/cage; floor space = 642.6 cm(2)/hen) and were replicated 10 times. Enriched cages provided hens with a curtained nesting area, scratch pad, and perches. Production parameters and egg quality measures were recorded throughout the experiment. Plumage condition was evaluated at 37 and 61 wk. Bone quality traits and immunological response parameters were measured at 61 wk, and 59 and 61 wk, respectively. Hen-day egg production, feed consumption, egg weight, and percentage of cumulative mortality of laying hens were not affected by the cage designs. Specific gravity and the percentage of cracked and soft-shelled eggs were also similar between the 2 housing systems. The incidence of dirty eggs was, however, significantly higher (P < 0.0001) in enriched cages than in conventional cages. Feather scores were similar between birds except for the wing region, which was higher (P < 0.05) for hens housed in conventional cages. Bone quality measures tended to be higher for hens housed in enriched cages compared with hens in conventional cages. However, the increase was significant only for bone mineral density. Immunological response parameters did not reveal statistically significant differences. Overall, laying performance, exterior egg quality measures, plumage condition, and immunological response parameters appear to be similar for hens housed in the 2 cage systems tested. Enrichment of laying hen cages resulted in better bone quality, which could have resulted from increased activity.

Evaluating the quality of protein from hemp seed (Cannabis sativa L.) products through the use of the protein digestibility-corrected amino acid score method.

The macronutrient composition and the quality of protein of hemp seed and products derived from hemp seed grown in Western Canada were determined. Thirty samples of hemp products (minimum 500 g), including whole hemp seed, hemp seed meal from cold-press expelling, dehulled, or shelled, hemp seed and hemp seed hulls, were obtained from commercial sources. Proximate analysis, including crude protein (% CP), crude fat (% fat) and fiber, as well as full amino acid profiles, were determined for all samples. Protein digestibility-corrected amino acid score (PDCAAS) measurements, using a rat bioassay for protein digestibility and the FAO/WHO amino acid requirement of children (2-5 years of age) as reference, were conducted on subsets of hemp products. Mean (±SD) percentage CP and fat were 24.0(2.1) and 30.4(2.7) for whole hemp seed, 40.7(8.8) and 10.2(2.1) for hemp seed meal, and 35.9(3.6) and 46.7(5.0) for dehulled hemp seed. The percentage protein digestibility and PDCAAS values were 84.1-86.2 and 49-53% for whole hemp seed, 90.8-97.5 and 46-51% for hemp seed meal, and 83.5-92.1 and 63-66% for dehulled hemp seed. Lysine was the first limiting amino acid in all products. Removal of the hull fraction improved protein digestibility and the resultant PDCAAS value. The current results provide reference data in support of protein claims for hemp seed products and provide evidence that hemp proteins have a PDCAAS equal to or greater than certain grains, nuts, and some pulses.

Folic acid supplementation inhibits NADPH oxidase-mediated superoxide anion production in the kidney

Hyperhomocysteinemia, a condition of elevated blood homocysteine (Hcy) levels, is a metabolic disease. It is a common clinical finding in patients with chronic kidney diseases and occurs almost uniformly in patients with end-stage renal disease. Hyperhomocysteinemia is also a risk factor for cardiovascular disease. Our recent studies indicate that hyperhomocysteinemia can lead to renal injury by inducing oxidative stress. Oxidative stress is one of the important mechanisms contributing to Hcy-induced tissue injury. Folic acid supplementation is regarded as a promising approach for prevention and treatment of cardiovascular disease associated with hyperhomocysteinemia due to its Hcy-lowering effect. However, its effect on the kidney is not clear. The aim of this study was to examine the effect of folic acid supplementation on Hcy-induced superoxide anion production via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the kidney during hyperhomocysteinemia. Hyperhomocysteinemia was induced in male Sprague-Dawley rats fed a high-methionine diet for 12 wk with or without folic acid supplementation. A group of rats fed a regular diet was used as control. There was a significant increase in levels of superoxide anions and lipid peroxides in kidneys isolated from hyperhomocysteinemic rats. Activation of NADPH oxidase was responsible for hyperhomocysteinemia-induced oxidative stress in the kidney. Folic acid supplementation effectively antagonized hyperhomocysteinemia-induced oxidative stress via its Hcy-lowering and Hcy-independent effect. In vitro study also showed that 5-methyltetrahydrofolate, an active form of folate, effectively reduced Hcy-induced superoxide anion production via NADPH oxidase. Xanthine oxidase activity was increased and superoxide dismutase (SOD) activity was decreased in the kidney of hyperhomocysteinemic rats, which might also contribute to an elevation of superoxide anion level in the kidney. Folic acid supplementation attenuated xanthine oxidase activity and restored SOD activity in the kidney of hyperhomocysteinemic rats. These results suggest that folic acid supplementation may offer renal protective effect against oxidative stress.

Innate immune response to yeast-derived carbohydrates in broiler chickens fed organic diets and challenged with Clostridium perfringens

Necrotic enteritis (NE) caused by Clostridium perfringens is a reemerging disease of economic importance in areas of the world where antibiotic growth promoters have been banned. The effect of mannan-oligosaccharide (MOS) supplementation in organic diets of broilers challenged with C. perfringens on performance, gut morphology, and innate immunity was investigated. Three hundred Ross-308 broilers were fed antibiotic-free certified organic starter and grower diets. On d 14, birds were orally challenged with 1 mL of C. perfringens culture at 3 × 10(10) cfu/bird. Treatments consisted of a control no-challenge (CO; 0 g/kg of MOS in the basal diet), control challenge (COC, 0 g/kg of MOS in the basal diet), and MOS challenge (2 g/kg of MOS in the basal diet). Challenge of birds resulted in decreased feed intake and BW gain (P = 0.048 and P = 0.026, respectively). Even though supplementation of diet with MOS improved feed intake (P = 0.985), BW gain and G:F were not improved compared with those of the CO group (P = 0.026 and P = <0.001, respectively). There was no significant difference among treatments in jejunal and ileal villus height, crypt depth, and goblet cells/mm(2) (P > 0.05). Quantitative real-time PCR showed that, in the ileum, the MOS diet resulted in an upregulation of toll-like receptor (TLR)2b, TLR4, interleukin (IL)-12p35, and interferon (IFN)-γ compared with CO (P = 0.003, P = 0.018, and P = 0.024, respectively). In the cecal tonsil, challenging birds with C. perfringens resulted in an upregulation of TLR2b compared with CO (P = 0.036), and MOS resulted in an upregulation of TLR4 (P = 0.018). In conclusion, feeding a MOS-supplemented diet to C. perfringens-challenged broiler chickens did not improve performance and gut morphology-associated responses. However, MOS was capable of altering TLR and cytokine profiles, where dual TLR2 and TLR4 pathways were associated with MOS supplementation with subsequent upregulation of ileal IL-12p35 and IFN-γ, implying that MOS supplementation in C. perfringens-challenged chickens supports a proinflammatory effect via T-helper cell-1 associated pathways.

Measurement of homocysteine and related metabolites in human plasma and urine by liquid chromatography electrospray tandem mass spectrometry

The sulfur amino acids, methionine and cysteine play crucial roles in cells as a substrate for protein synthesis, as a methyl donor, and for the synthesis of sulfur-containing compounds, including the key intracellular tripeptide, glutathione. Homocysteine is an intermediary metabolite formed during the metabolism of methionine to cysteine. Dysregulation of homocysteine metabolism is implicated in adverse clinical outcomes such as increased risk of cardiovascular disease, stroke, Alzheimers disease dementia and osteoporosis. While hyperhomocysteinemia is commonly observed in those conditions, the impact on other related metabolites is condition-specific. Therefore, there exists a need to establish precise and sensitive analytical techniques that allow for the simultaneous measurement of homocysteine and related metabolites in biological samples. The current review outlines the development and use of liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) to simultaneously measure metabolites involved in sulfur amino acid metabolism. Additionally, extensions of the technique in relation to the measurement of sulfur amino acid and one-carbon kinetics in vivo are discussed. The LC-MS/MS technique has the capacity for unambiguous analyte identification and confirmation, due to its high specificity and sensitivity. It has the greatest potential of being accepted and utilized as a dedicated homocysteine and its related metabolite Standard reference method (SRM).

Folate Status of Young Canadian Women after Folic Acid Fortification of Grain Products

Women of childbearing age are advised to consume folic acid-containing supplements. Whether this remains necessary after folic acid fortification of the food supply in North America has yet to be determined. The objectives of this study were to assess folate intakes and the contribution of folic acid to the diets of women of childbearing age in the post-folic acid fortification era. Using a cross-sectional study design, fasting blood samples were obtained from 95 women (aged 18 to 25 years), and the samples were analyzed for serum and red blood cell folate, as well for total homocysteine. Dietary and supplemental folate intakes were assessed. The biochemical evidence showed that no women were folate deficient, but only 14% reached red blood cell folate concentrations associated with significant reductions in neural tube defect risk. Mean dietary intake of food folic acid was 96+/-64 microg/day, supplemental folic acid was 94+/-189 microg/day, natural folate was 314+/-134 microg/day, and the total intake, as dietary folate equivalents, was 646+/-368 microg dietary folate equivalents/day. Therefore, intakes of folic acid from fortified foods are within the level originally predicted for the fortification efforts; however, only 17% of participants met the special recommendation for women capable of becoming pregnant (400 microg folic acid daily from supplements, fortified foods, or both in addition to consuming food folate from a varied diet). These data suggest that women of childbearing age are achieving positive folate status in the postfortification era, but it may not be sufficient to achieve red blood cell folate concentrations associated with a significant reduction in neural tube defect risk. Even with food fortification, women of childbearing age should be advised to take a folic acid-containing supplement on a daily basis.

Long-Term High Intake of Whole Proteins Results in Renal Damage in Pigs

Despite evidence of potential antiobesity effects of high-protein (HP) diets, the impact of consuming diets with protein levels at the upper limit of the acceptable macronutrient distribution range (AMDR) on kidney health is unknown. To test whether HP diets affect renal health, whole plant and animal proteins in proportions that mimicked human diets were given to pigs, because their kidneys have a similar anatomy and function to those of humans. Adult female pigs received either normal-protein (NP) or HP (15 or 35% of energy from protein, respectively) isocaloric diets for either 4 or 8 mo. The higher protein in the HP diet was achieved by increasing egg and dairy proteins. Although there were initial differences in body weight and composition, after 8 mo these were similar in pigs consuming the NP and HP diets. The HP compared with NP diet, however, resulted in enlarged kidneys at both 4 and 8 mo. Renal and glomerular volumes were 60-70% higher by the end of the study. These enlarged kidneys had greater evidence of histological damage, with 55% more fibrosis and 30% more glomerulosclerosis. Renal monocyte chemoattractant protein-1 levels also were 22% higher in pigs given the HP diet. Plasma homocysteine levels were higher in the HP pigs at 4 mo and continued to be elevated by 35% at 8 mo of feeding. These findings suggest that long-term intakes of protein at the upper limit of the AMDR from whole protein sources may compromise renal health.

Response of nursery pigs to a synbiotic preparation of starch and an anti-Escherichia coli K88 probiotic.

ABSTRACT Postweaning diarrhea in pigs is frequently caused by enterotoxigenic Escherichia coli K88 (ETEC). The aim of this study was to test the efficacy of E. coli probiotics (PRO) in young pigs challenged with E. coli K88. We also tested the synbiotic interaction with raw potato starch (RPS), which can be used as a prebiotic. Forty 17-day-old weaned piglets were randomly assigned to four treatments: treatment 1, positive-control diet (C), no probiotics or RPS but containing in-feed antibiotics; treatment 2, probiotic (PRO), no feed antibiotics plus a 50:50 mixture of probiotic E. coli strains UM-2 and UM-7; treatment 3, 14% RPS, no antibiotics (RPS); treatment 4, 14% RPS plus a 50:50 mixture of probiotic E. coli strains UM-2 and UM-7, no antibiotics (PRO-RPS). The pigs were challenged with pathogenic E. coli K88 strains on day 7 of the experiment (24-day-old pigs) and euthanized on day 10 of the experiment (35-day-old pigs). Probiotic and pathogenic E. coli strains were enumerated by selective enrichment on antibiotics, and microbial community analysis was conducted using terminal restriction length polymorphism analysis (T-RFLP) of 16S rRNA genes. The combination of raw potato starch and the probiotic had a beneficial effect on piglet growth performance and resulted in a reduction of diarrhea and increased microbial diversity in the gut. We conclude that the use of E. coli probiotic strains against E. coli K88 in the presence of raw potato starch is effective in reducing the negative effects of ETEC in a piglet challenge model.

Development and in vitro evaluation of an Escherichia coli probiotic able to inhibit the growth of pathogenic Escherichia coli K88.

Weaned piglets commonly suffer from gastroenteritis caused by enterotoxigenic Escherichia coli K88. Our aim was to produce E. coli strains that inhibited the growth of E. coli K88 and could be used as a probiotic against postweaning diarrhea. The inclusion criteria for the probiotics were that in addition to being able to inhibit E. coli K88, they also needed to be negative for virulence genes commonly associated with E. coli. A total of 463 E. coli isolates from the cattle rumen, cattle feces, swine feces, and soil were screened against 18 E. coli K88 clinical isolates using an agar diffusion technique. Growth inhibition of the most sensitive K88 indicator strain 2-12 occurred for 121/463 isolates: 96/358 from cattle feces, 0/33 from rumen fluid, 9/35 from swine feces, and 16/37 from soil. Of the 121 positive strains, 71/121 were negative for toxin genes (LT, STa, STb, VT1, and VT2). The 14 most inhibitory strains were screened against a range of substrates to assess the ability to utilize carbohydrates that could be included in the diet to enhance their ability to compete in the gut. Two strains, UM-2 and UM-7, were weak utilizers of starch and inulin. In vitro competition assays between the probiotic strains and E. coli K88 strain 2-12 were conducted with glucose as the only carbon source (minimal medium; MM), MM + 2% starch, or MM + 2% inulin. The UM-2 and UM-7 strains were able to outcompete strain 2-12 when glucose was the only carbon source, indicating that inhibitory activity was produced against 2-12 independent of carbon source. The UM-2 strain outcompeted strain 2-12 in assays in which potato starch or inulin was the only carbon source; the ability of 2-12 to maintain its concentrations in the culture were probably the result of cross feeding of breakdown sugars of starch and inulin that could be utilized by 2-12. In contrast, UM-7 did not grow as well as UM-2 on starch and inulin and 2-12 declined rapidly in successive cultures likely because of the lack of breakdown products of starch and inulin produced by UM-7. We conclude that probiotic E. coli without known toxins and that produce inhibitory activity against E. coli that cause postweaning diarrhea can be produced. In addition, the ability to utilize starch or inulin is an important phenotype because it likely gives the probiotic a competitive advantage in the gut.