Oral succimer decreases the gastrointestinal absorption of lead in juvenile monkeys.

Although succimer (Chemet, meso-2,3-dimercaptosuccinic acid, DMSA) is considered to be a safe and effective chelating agent for the treatment of lead poisoning in humans, there is concern that it may increase the gastrointestinal (GI) absorption and retention of Pb from exposures suffered concurrent with treatment. This concern is justified because the availability of Pb-safe housing during outpatient treatment with oral succimer is limited. We used a juvenile nonhuman primate model of moderate childhood Pb intoxication and a sensitive double stable Pb isotope tracer methodology to determine whether oral succimer chelation affects the GI absorption and whole-body retention of Pb. Infant rhesus monkeys (n = 17) were exposed to Pb daily for 1 year postpartum to reach and maintain a target blood lead (BPb) level of 35-40 microg/dL. Animals were administered succimer (n = 9) or vehicle (n = 8) over two successive 19 day succimer treatment regimens beginning at 53 and 65 weeks of age. The present study was conducted over the second chelation regimen only. Animals received a single intravenous (iv) dose of stable (204)Pb tracer (5 microg, 24.5 nmol) followed by a single oral dose of stable (206)Pb tracer (72.6 microg, 352 nmol) immediately before chelation, in order to specifically evaluate GI Pb absorption and whole-body Pb retention with treatment. We collected complete urine and fecal samples over the first 5 days and whole blood over the first 8 days of treatment for analyses of stable Pb isotopes using magnetic sector inductively-coupled plasma mass spectrometry. Results indicate that succimer significantly reduced the GI absorption of Pb (vehicle, 64.9% +/- 5.5; succimer, 37.0% +/- 5.8; mean +/- SEM). Succimer also significantly increased the urinary excretion of endogenous Pb by approximately 4-fold over the vehicle treatment, while endogenous fecal Pb excretion was decreased by approximately 33%. Finally, although succimer reduced the whole-body retention of endogenous Pb by approximately 10% compared to vehicle, the majority (77%) of the administered internal dose of Pb tracer was retained in the body when assessed after 5 days of treatment. These data do not support the concern that succimer treatment increases GI Pb absorption.

Succimer and the urinary excretion of essential elements in a primate model of childhood lead exposure.

Succimer is considered to be a safe and effective treatment for lead (Pb) poisoning, since it reduces body Pb levels without an apparent diuresis of other essential elements. However, while existing clinical data indicate that succimer does not significantly increase the excretion of non-target elements, those studies have also reported a wide range of outcomes. Therefore, we investigated whether succimer treatment measurably increased the urinary excretion of essential elements in a primate model of childhood Pb exposure. Infant rhesus monkeys (Macaca mulatta) were exposed to Pb from birth through one year of age, and presented blood Pb levels of approximately 40-50 microg/dL at the start of treatment. Subsequently, they were treated with succimer (30 mg/kg/day x 5 days followed by 20 mg/kg/day x 14 days, n = 15) or vehicle (n = 14) for 19 days. Complete urine samples were collected over the first 5 days of treatment, and were analyzed for levels of calcium (Ca), cobalt (Co), copper (Cu), iron (Fe), lead (Pb), magnesium (Mg), manganese (Mn), nickel (Ni), and zinc (Zn), using trace metal-clean techniques and magnetic sector-ICP-MS. Succimer treatment significantly (p < 0.05) reduced blood Pb levels when compared to the vehicle group over the treatment period, and concomitantly produced a significant >4-fold increase in urinary Pb excretion. Succimer treatment also significantly (p < 0.05, multivariate ANOVA) increased the urinary excretion of essential elements, but only when the cumulative total excretion over treatment days 1-5 for all elements were considered. None of these relative increases reached statistical significance for any particular element x day, although increases in Zn (day 3) excretion were only marginally non-significant (0.1 > p > 0.05). Multivariate analyses of a subset of elements (Cu, Fe, Mn, Zn) similarly indicated no significant effect of succimer treatment overall, although the urinary excretion of Mn was significantly increased on day 3 of treatment. Collectively, these data indicate that succimer does contribute to an increase in the urinary excretion of essential elements, although not significantly for any single element considered here. This may be important in Pb-exposed children, who can possess reduced trace element reserves due to nutritional deficiencies.

Efficacy of succimer chelation for reducing brain lead in a primate model of human lead exposure.

The extent to which succimer (meso-2,3-dimercaptosuccinic acid [DMSA], Chemet) reduces brain lead (Pb) levels may be a primary consideration in evaluating its efficacy for reducing neurotoxicity. Clinical research in this area has been hampered by the need to use blood Pb levels as the index of treatment efficacy, despite the fact that brain Pb level is the exposure parameter of greater relevance to cognitive outcomes. Here, a nonhuman primate model of human Pb exposure was used to determine: (1) The efficacy of oral succimer for reducing brain Pb derived from chronic or recent exposures, and (2) The extent to which blood Pb levels reflect brain Pb prior to and following chelation. Adult rhesus monkeys were chronically exposed to Pb orally for 5 weeks to reach and maintain a target blood Pb level of 35-40 microg/dL. Chelation of Pb from recent exposures was assessed using a stable (204)Pb isotope tracer administered over 4 days prior to treatment. Immediately prior to chelation, a prefrontal cortex (PFC) biopsy was collected to determine pretreatment brain Pb levels. Subsequently, monkeys were assigned to vehicle (n = 5) or succimer (n = 6, 30 mg/kg/day x 5 days followed by 20 mg/kg/day x 14 days) groups. Blood and brain PFC, frontal lobe (FL), hippocampus (H), and striatum (S) were analyzed for total Pb and (204)Pb tracer concentrations by magnetic sector inductively coupled plasma-mass spectrometry. There were no measurable differences in brain Pb concentrations between the succimer and vehicle groups, indicating that succimer treatment was not efficacious in reducing brain Pb levels. In contrast, the cessation of Pb exposure significantly reduced brain (PFC) Pb ( approximately 34%) when compared to pretreatment levels (succimer and vehicle groups). Pb concentrations also varied among brain regions (PFC > FL approximately H > S). Finally, pretreatment PFC Pb concentrations were significantly correlated with the integrated blood Pb level (AUC) over the Pb exposure period, but not with the single pretreatment blood Pb collected concurrently with the PFC biopsy. Following treatment, blood Pb levels correlated only with Pb in the PFC, and not the other brain regions measured (FL, H, S). These data indicate that, under the conditions of this study, succimer treatment did not reduce brain Pb levels beyond the cessation of Pb exposure alone. Moreover, a single blood Pb measurement may be a poor predictor of brain Pb levels, reflecting limitations in the use of blood Pb level as an indicator of treatment efficacy.

Performance and digestion by steers grazing tall fescue and supplemented with energy and protein.

We studied the effects of different levels of protein and energy supplementation on BW gains and sites of nutrient digestion in steers grazing the primary growth of endophyte-infected tall fescue (Festuca arundinacea Schreb cv. Kentucky 31). Angus steers (n = 168; mean BW = 246.8 +/- 15.0 kg) grazed tall fescue without supplementation (C) or were supplemented with 1.4 kg/d of cracked corn (CC1), 1.4 kg/d of corn gluten feed (CGF1), 2.8 kg/d of CC (CC2), 2.8 kg/d of CGF (CGF2), or .7 kg/d of cornstarch and .7 kg/d of corn gluten meal (CS-CGM) during an 85-d period in a randomized complete block design. Supplemented steers (S) had a higher (P = .03) ADG than the control steers (.64 vs .74 kg/d). There was an interaction (P = .02) between supplement type and level. Supplementation during the grazing period had no effect on subsequent feedlot ADG (average 1.3 kg/d). In a digestion study, Angus x Simmental steers (n = 4; 412 +/- 20.4 kg) fitted with cannulas in the esophagus, rumen, and duodenum grazed tall fescue (C), C + 3.1 kg/d CGF, C + 3.1 kg/d of cracked corn (CC), or C + 1.4 kg/d CS-CGM over four experimental periods in a Latin square design. The ruminal ammonia nitrogen concentration tended to be higher (P < .09) in C than in CGF and in CC (21.9 vs 19.2 mg/dL), but ruminal pH and total VFA concentration did not differ among treatments. Forage OM intakes were lower (P < .05) in steers supplemented with CC or CGF (an average of 7,570 g/d) than in C (9,658 g/d). Total OM intakes and digestibilities did not differ (P > .10) among treatments. Duodenal N flows and efficiencies of net ruminal microbial protein synthesis were not different (P > .10) among treatments. Supplementation of steers grazing primary growth of tall fescue improved animal gain but did not have a great effect on ruminal fermentation, OM digestion, or net ruminal microbial protein synthesis.

Glycolysis is a source of pyruvate for transamination of glutamine amino nitrogen in jejunal epithelial cells.

Previous research has shown that glucose increases transamination of glutamine amino nitrogen with pyruvate. It is unclear whether glucose or glutamine provides the pyruvate used for transamination. In the current study, it was hypothesized that glucose provides pyruvate for transamination of glutamine amino nitrogen. This hypothesis was tested by tracing the metabolism of [2-(13)C]glucose in these cells incubated in the presence of [2-(13)C] glucose or [2-(13)C] glucose and glutamine using (13)C nuclear magnetic resonance. Glutamine supplementation increased alanine production but did not affect lactate production. The 1-(13)C, 2-(13)C, 3-(13)C, 1,2-(13)C, and 2,3-(13)C isotopomers of alanine and lactate were produced when glutamine was supplemented. Glutamine supplementation increased production of 2-(13)C, 1,2-(13)C, and 2,3-(13)C isotopomers of alanine but did not affect the production of isotopomers of lactate. The ratio of production of [2-(13)C]alanine to [3-(13)C]alanine was 37:1 when glutamine was present. The predominance of production of [2-(13)C]alanine vs. all other isotopomers demonstrates that a large proportion of the pyruvate used for transamination of glutamine amino nitrogen was derived from glycolysis.

Production, digestion, and hepatic lipid metabolism of dairy cows fed increased energy from fat or concentrate.

Eight Holstein cows (mean = 52 DIM) were used in a replicated Latin square design (28-d periods) to determine the effects of source and amount of dietary energy on hepatic lipid metabolism. Diets were 1) low concentrate without supplemental fat (1.6 Mcal of NEI/kg), 2) low concentrate with supplemental fat (1.7 Mcal/kg), 3) high concentrate without supplemental fat (1.7 Mcal/kg), and 4) high concentrate with supplemental fat (1.8 Mcal/kg). Intakes of DM and digestible energy were greatest for diets 2 and 3. Milk production, efficiency, and milk CP production were not different among diets. Milk fat percentage and production were increased by supplemental fat and decreased by high concentrate. Contents of CP, true protein, and casein in milk were decreased by supplemental fat and increased by high concentrate. Digestibility of fatty acids was decreased by either supplemental fat or high concentrate. Concentrations of NEFA and somatotropin in plasma were increased, and concentration of IGF-I was decreased, by supplemental fat; NEFA, BHBA, and IGF-I were decreased by high concentrate. In vitro rates of peroxisomal beta-oxidation and esterification of palmitate and concentrations of total lipid and triglycerides in liver were not different among diets, but glycogen content was increased by high concentrate. The rate of total beta-oxidation of palmitate in liver slices tended to be increased by supplemental fat; total metabolism of palmitate was increased by supplemental fat and tended to be increased by high concentrate. Supplemental fat may affect hepatic lipid metabolism in dairy cows; effects of dietary fat during the periparturient period deserve investigation.

Effects of glycine and bovine serum albumin on inhibition of propionate metabolism in ovine hepatocytes caused by reduced phenolic monomers.

Hepatocytes isolated from sheep were incubated in the presence of reduced phenolics and glycine to determine the effects of these compounds on hepatic propionate metabolism in vitro. 3-Phenyl-propionic (PPA) or t-cinnamic (CA) acids, but not benzoic (BA) or 3-(4-hydroxyphenyl)propionic (4OHPPA) acids, decreased conversion of propionate to glucose at .05 mM in the absence of supplemental glycine. At 1.2 mM, all reduced phenolics decreased conversion of propionate to glucose in the absence of supplemental glycine. Addition of glycine to the incubation medium linearly alleviated the inhibition by BA, PPA, or CA, suggesting that physiological glycine concentrations limited alleviation of inhibition of propionate metabolism. Hippuric acid production increased as glycine concentration increased in the presence of PPA, CA, or 4OHPPA. Bovine serum albumin did not alleviate inhibition of conversion of propionate to glucose caused by BA, PPA, or CA and slightly alleviated inhibition caused by 4OHPPA (.4 mM). Of the reduced phenolics tested, PPA is the most likely to inhibit gluconeogenesis from propionate in ovine liver in vivo.

Prenatal androgenization of lambs: II. Metabolism in adipose tissue and liver.

In vitro measurements of metabolism were made in subcutaneous and perirenal adipose tissue (AT) and liver from prenatally androgenized ewe lambs (TE), control ewe lambs (CE), and control wether lambs (CW). In adipose tissue slices, release of glycerol or fatty acids into the medium was not different among treatments, but glycerol release was greater (P < .01) from subcutaneous AT than from perirenal AT. Basal fatty acid release and the free fatty acid pool were greater (P < .05) for perirenal AT than for subcutaneous AT; fatty acid release and the fatty acid response (increased NEFA in media and tissue) were increased more by lipolytic stimuli in subcutaneous AT than in perirenal AT. Adipose tissue from CW had the greatest (P < .05) fatty acid response under conditions of near-maximal stimulation; rates from TE were intermediate to those from CW and CE. Incorporation of glucose into fatty acids and glycerol in subcutaneous AT was lowest (P < .05) for TE. Oxidation of glucose and acetate to CO2 and incorporation of acetate into fatty acids or glycerol in subcutaneous AT, glucose and acetate metabolism in perirenal AT, and cellularity measurements for both AT did not differ among treatments. In liver slices, oxidation of [1-14C]propionate to CO2 was greater (P < .05) for CE than for TE or CW, and gluconeogenic capacity from [1-14C]propionate tended to be greater (P < .10) for CE than for TE. Glucose and CO2 production from [2-14C]propionate, [U-14C]alanine, or [U-14C]glycerol and total and peroxisomal first cycle of beta-oxidation of [1-14C]palmitate were not altered by prenatal androgenization or sex. There were no effects (P > .1) of prenatal exposure to testosterone on mitochondrial protein content of liver, rates of mitochondrial state 3 or state 4 respiration, the ratio of ADP:oxygen in the presence of respiratory substrates, or hepatic contents of lipid, triglyceride, or glycogen. Protein content of liver was greater (P < .05) for CW than for CE; TE were intermediate. Collectively, there were minimal modifications of in vitro metabolism in AT or liver attributable to prenatal androgenization or sex that would directly influence ADG and carcass composition.

Portal and hepatic fluxes in sheep and concentrations in cattle ruminal fluid of 3-(4-hydroxyphenyl)propionic, benzoic, 3-phenylpropionic, and trans-cinnamic acids.

Extraction methods and HPLC procedures were developed for analysis of potential ruminal metabolites of dietary phenolics (reduced phenolics). Hepatic portal venous blood from wethers fed bromegrass, bermudagrass, ryegrass-wheat, and alfalfa hays also was analyzed for hippuric (HA), 3-(4-hydroxyphenyl)propionic (4OHPPA), benzoic (BA), 3-phenylpropionic (PPA), and t-cinnamic (CA) acids. Additionally, mesenteric arterial and hepatic venous blood was analyzed and, in conjunction with blood flow measurements, fluxes for portal-drained viscera (PDV) and liver were calculated. Ruminal fluid from four steers fed two levels of forage and two forage particle sizes in a Latin square design was analyzed for PPA and CA. 3-Phenylpropionic and benzoic acids were the most concentrated reduced phenolics identified in hepatic portal venous blood. Concentrations of PPA in ruminal fluid varied with ruminal disappearance of p-coumaric and ferulic acids. Additionally, hepatic portal venous concentrations of PPA were correlated (P < .05) with p-coumaric acid (r = .57) and ferulic acid (r = .67) intakes. Net release of PPA from PDV was observed, suggesting absorption of PPA from the gut. The liver removed PPA and BA with less efficiency. Given the relatively high concentrations of PPA in blood of ruminants, specific effects of this reduced phenolic on liver metabolism of ruminants should be assessed.

Effect of roasting on site and extent of digestion of soybean meal by sheep: I. Digestion of nitrogen and amino acids.

Six mature wethers (BW 72 kg) with ruminal, duodenal, and ileal cannulas were fed a control (no added soybean meal [SBM]) diet or diets containing unheated SBM or SBM roasted at 165 degrees C for 75, 150, 180, or 210 min in a 6 x 6 Latin square experiment. Concentrations of indicators of heat exposure (ADIN, ADF, NDF) in SBM increased with increasing roasting time. Duodenal flows of total N, non-bacterial N, and SBM N increased (P < .05) linearly with increased roasting time. Small intestinal (SI) digestibility (percentage entering SI) of total N and SBM N was influenced quadratically (P < .05) by roasting time; SI digestibilities were modestly increased by heating SBM to 150 min, then declined dramatically when SBM was heated for 180 and 210 min. These responses resulted in a quadratic (P < .05) increase in quantity of total and SBM N disappearing in the SI; quantities increased as SBM was heated to 150 min and remained unchanged when SBM was heated for 180 and 210 min. These responses resulted in a quadratic (P < .05) increase in quantity of total and SBM N disappearing in the SI; quantities increased as SBM was heated to 150 min and remained unchanged when SBM was heated for 180 and 210 min. Duodenal flows and SI digestibilities of total and non-bacterial amino acids (AA) and of most individual AA followed patterns similar to those observed for N. Maximum quantities of total and individual AA disappeared from the SI when wethers were fed SBM roasted at 165 degrees C for 150 min. Evaluation of the effects of heat treatment on the nutritive value of a protein source for ruminants should include considerations for both ruminal protein escape and SI availability of escaped protein and for the nature of the AA supply absorbed from the SI.

Effects of reduced phenolic acids on metabolism of propionate and palmitate in bovine liver tissue in vitro.

Benzoic acid, 3-phenylpropionic acid, trans-cinnamic acid, and 3-(4-hydroxyphenyl)propionic acid in ruminal fluid are presumed to be the products of chemical reduction of dietary phenolic monomers by ruminal microorganisms. Effects of reduced phenolics on metabolism in bovine liver tissue were evaluated by measurement of 1) conversion of propionate to glucose and CO2, 2) conversion of palmitate to oxidized products, and 3) leakage of lactate dehydrogenase from liver slices in vitro. In Experiment 1, .4 mM benzoic, 3-phenylpropionic, trans-cinnamic, or 3-(4-hydroxyphenyl)propionic acids decreased conversion of propionate to glucose and decreased conversion of palmitate to total oxidation products. At .2 mM, 3-(4-hydroxyphenyl)propionic acid did not inhibit conversion of propionate to glucose compared with that of controls, but the other reduced phenolics did. In Experiment 2, the same reduced phenolics inhibited conversion of propionate to glucose. Of the reduced phenolics tested, cinnamic acid inhibited conversion of propionate to glucose at the lowest concentration, .1 mM. Additionally, when present at > or = .4, .1, or .005 mM, benzoic, 3-phenylpropionic, or trans-cinnamic acids, respectively, increased leakage of lactate dehydrogenase from liver tissue. The reduced phenolics tested, which are representative of those in ruminal fluid, inhibited metabolism of bovine liver tissue in vitro at supraphysiological concentrations. Data at physiological concentrations were inconclusive.

Effect of fiber source on short-chain fatty acid production and on the growth and toxin production by Clostridium difficile.

BACKGROUND: Fermentable fiber promotes the growth of resident gut microbes, which modify the environment of the gastrointestinal tract and thus prevent colonization by Clostridium difficile. METHODS: An in vitro system with pigs as fecal inoculum donors was used to estimate fiber fermentability and changes in intestinal microbiota. RESULTS: Acetate and propionate production (mumol/mg substrate fermented/day) was greatest for gum arabic (1013.4 and 704.1, respectively); butyrate production was greatest for xylo-oligosaccharide (345.6). Growth of total anaerobes and clostridia was greatest for gum arabic (21.2 and 16.2 x 10(8) counts/ml, respectively) and xylo-oligosaccharides (21.0 and 19.6 x 10(8) respectively); growth of acidogenic bacteria was greatest with fructo-oligosaccharide (6.7 x 10(8) counts/ml). No culturable counts of C. difficile were obtained, nor was toxin A detected. CONCLUSIONS: Fermentable fibers support the growth of indigenous intestinal bacteria, particularly acidogenic bacteria, and yield large amounts of short-chain fatty acids with decreased gut pH. These factors contribute to the prevention of growth and toxin elaboration by C. difficile.

Digestion criteria in nursing beef calves supplemented with limited levels of protein and energy.

This study was conducted with grazing nursing calves (197 kg) to determine the effects of 1) limiting creep feed intake and 2) increasing the concentration of ruminal escape CP in creep feed at a limited level of creep feed intake on fescue and milk intake, ruminal NDF digestion, and total tract digestibility in calves fed high-quality, freshly harvested fescue. The treatments were 1) control (no creep feed), 2) limited intake of creep feed (.60 kg/d) having a moderate concentration of CP (13%), 3) limited intake of creep feed (.60 kg/d) having a high concentration of CP (35%), and 4) unlimited (high) intake (1.62 kg/d) of the same creep feed fed in Treatment 2. Forage OM intake was negatively correlated (r = -.995, P less than .05) with level of creep feed OM intake, whereas milk OM intake was not affected by level of creep feed intake. Decreases in ruminal fiber digestion and total tract NDF digestion caused by unlimited creep feeding were partially avoided by limiting creep feed intake. Digestible OM intake increased by .47 kg per kilogram of creep feed OM intake.