Colitis, independent of macronutrient intake, compromises bone structure and strength in growing piglets.

BACKGROUND: Deterioration in bone health is a concern in managing pediatric inflammatory bowel diseases, but clear understanding of the independent contributions of disease and nutrition is lacking. This study aimed to ascertain whether bone health could be conserved during colitis by maintaining adequate nutritional intake in growing piglets. METHODS: The effect of colitis on bone structure and strength was determined in piglets with dextran sulphate sodium-induced colitis. Piglets received either 100% macro/micronutrient requirements or 50% macro/100% micronutrient requirements. Femurs were analyzed for dual-energy x-ray absorptiometry, peripheral quantitative computed tomography, microcomputed tomography, and 3-point bending tests. RESULTS: Colitis, regardless of a well-nourished or malnourished diet, compromised areal bone mineral density (-17%) and volumetric bone mineral density (-20%) in cortical and trabecular bone. Structural integrity at mid-diaphysis was maintained during colitis; however, lower cortical area, trabecular area, and bone mineral content resulted in lower energy to break. CONCLUSION: Colitis compromises both bone structure and strength of long bones in piglets, independent of macronutrient intakes. Although confirmation of these findings in pediatric cohorts is needed, these data identify aspects of bone health that may be affected by inflammatory bowel disease.

Experimental colitis and malnutrition differentially affect the metabolism of glutathione and related sulfhydryl metabolites in different tissues.

PURPOSE: Inflammatory bowel diseases (IBD) are characterized by severe inflammation within the gastrointestinal (GI) tract. This inflammation is known to drive the catabolism of protein in the affected tissue and modulate systemic protein metabolism. Yet despite the established increase in oxidative stress and changes in protein catabolism, little is known as to the effects of IBD on metabolism of glutathione (GSH) and related metabolites. The aim of this study was to conduct a comprehensive analysis of the response of GSH and related sulfhydryl metabolites to malnutrition and GI inflammation. We hypothesized that the inflammatory stress of colitis would decrease the concentration and the synthesis of GSH in various tissues of well-nourished piglets. Additionally, the superimposition of malnutrition on colitis would further decrease glutathione status. METHODS: Healthy, well-nourished piglets were compared to those receiving dextran sulphate sodium-induced, a macronutrient-restricted diet or both. The synthesis of GSH was determined by primed constant infusion of [(15)N,(13)C2]glycine and tandem mass spectrometry analysis. Additionally, the concentrations of GSH and related sulfhydryl metabolites were also determined by UHPLC-tandem mass spectrometry-a novel analytic technique. RESULTS: In healthy piglets, GSH synthesis was highest in the liver, along with the concentrations of both cysteine and γ-glutamylcysteine. Piglets with colitis had decreased synthesis of GSH and decreased concentrations of GSH, cysteine and γ-glutamylcysteine in the distal colon compared to healthy controls. Additionally, there was no change with superimposition of malnutrition on colitis in the distal colon. CONCLUSION: Synthesis and metabolism of GSH are uniquely regulated in each tissue. Colitis, independent of nutrition, compromises GSH status and the concentration of cysteine in the distal colon of piglets with GI inflammation. The techniques developed in this study have translational applications and can be scaled for use in clinical investigation of GI inflammation.

Maintaining adequate nutrition, not probiotic administration, prevents growth stunting and maintains skeletal muscle protein synthesis rates in a piglet model of colitis.

Malnutrition and cytokine-induced catabolism are pervasive in children with inflammatory bowel diseases (IBD), however, the benefits of aggressive nutrition support or of probiotics on nutrient and functional deficiencies and growth remain unclear. Piglets with dextran sulfate (DS)-induced colitis consuming a 50% macronutrient restricted diet (C-MR) were compared with those receiving probiotics (C-MRP) or adequate nutrition (C-WN) and with healthy well-nourished controls (REF). C-WN versus REF had reduced growth (-34% chest circumference and -22% snout-to-rump length gain) and a tendency toward lesser weight gain, but no differences in skeletal muscle protein fractional synthesis rates (FSR) or initiation of translation via the mTOR pathway were observed. Compared with C-WN, the C-MR and C-MRP piglets had lower weight gain, growth, and skeletal muscle FSR, and lower phosphorylated p70S6K1 with higher eIF4E*4E-BP1, indicative of reduced initiation of protein translation. Finally, plasma leucine concentrations were positively correlated with weight and phosphorylated p70S6K1, whereas negatively correlated with eIF4E*4E-BP1. In conclusion, reductions in weight gain, growth, protein turnover, skeletal muscle FSR, and initiation of protein translation with moderate macronutrient restriction in colitis are not ameliorated by probiotic supplementation. However, maintaining adequate nutrient intake during colitis preserves whole body protein metabolism, but growth remains compromised.

Probiotics stimulate liver and plasma protein synthesis in piglets with dextran sulfate-induced colitis and macronutrient restriction.

Adequate nutrition and probiotics have each been shown to reduce the severity of colitis, but their impact on hepatic and gastrointestinal protein metabolism has not been studied. Our objective was to determine whether maintaining adequate nutrition compared with administering probiotics affected protein synthesis, colon histopathology, and oxidative stress in our macronutrient-restricted piglet model of colitis. Piglets (n = 8/group) receiving dextran sulfate to induce colitis were randomized to 3 treatment groups: macronutrient restricted (MR); macronutrient restricted with VSL #3 probiotics (MRP), or well nourished (WNC). An additional 8 piglets served as healthy references for comparative purposes given the unique nature of the experimental model. A primed, constant infusion of the tracer L-[ring-(2)H(5)]phenylalanine was performed in colitis piglets after 14 d to determine the fractional synthesis rates of proteins in small intestinal mucosa, colon, and liver and of plasma proteins (total protein, fibrinogen, albumin). Colon histopathology and oxidative stress were also assessed. Compared with MR piglets, both WNC and MRP piglets had higher protein synthesis rates in liver and plasma protein pools. However, only adequate nutrition increased protein synthesis in the colon and decreased colitis severity. Whereas probiotics did not stimulate gastrointestinal protein synthesis or reduce colitis severity, a still-unidentified signaling mechanism between the gut and liver seems to be responsible for the probiotic-induced increase in liver protein and plasma protein synthesis. These data underscore the importance of maintaining nutrient intake in pediatric patients with gastrointestinal disease. A strategy for correcting compromised nutrition seems to be more beneficial for reducing damage during colitis than using probiotics only.

Methionine-adequate cysteine-free diet does not limit erythrocyte glutathione synthesis in young healthy adult men.

Most methods of determining amino acid (AA) requirements are based on endpoints that determine adequacy for protein synthesis. However, the sulfur AA (SAA) cysteine is believed to be the rate-limiting substrate for synthesis of the most abundant intracellular antioxidant, glutathione (GSH). Our objectives were to determine whether supplementation of cysteine in a diet containing adequate SAA for protein synthesis, as methionine, increased GSH synthesis by measuring the fractional and absolute synthesis rates, and if concentration of GSH changed in response to feeding 5 graded intakes of cysteine (0, 10, 20, 30, and 40 mg x kg(-1) x d(-1)) in a random order with a fixed methionine intake of 14 mg x kg(-1) x d(-1) and a protein intake of 1 g x kg(-1) x d(-1). Each subject received a multivitamin and choline supplement during the study. Four healthy adult men each underwent 5 isotope infusion studies of 7-h duration after a 2-d adaptation to the level of cysteine intake being studied on the isotope infusion day. The isotope used was [U-(13)C(2)-(15)N]glycine. Analyses included erythrocyte GSH synthesis rates and concentration and urinary sulfate excretion. The GSH synthesis rates and concentration, measured at a methionine intake of 14 mg x kg(-1) x d(-1), did not change with increasing intakes of cysteine. Urinary sulfate excretion showed a significant positive relationship with cysteine intake (r = 0.92; P < 0.01). In conclusion, this study provides preliminary evidence that consumption of SAA adequate to meet the requirement for protein synthesis does not limit GSH synthesis in healthy adult men receiving an otherwise adequate diet.

Orchidectomy-induced alterations in volumetric bone density, cortical porosity and strength of femur are attenuated by dietary conjugated linoleic acid in aged guinea pigs.

Age-related osteoporosis and sarcopenia are ascribed in part to reductions in anabolic hormones. Dietary conjugated linoleic acid (CLA) improves lean and bone mass, but its impact during androgen deficiency is not known. This study tested if CLA would attenuate the effects of orchidectomy (ORX)-induced losses of bone and lean tissue. Male guinea pigs (n=40; 70-72 weeks), were randomized into four groups: (1) SHAM+Control diet, (2) SHAM+CLA diet, (3) ORX+Control diet, (4) ORX+CLA diet. Baseline blood sampling and dual-energy X-ray absorptiometry (DXA) scans were conducted, followed by surgery 4 days later with the test diets started 7 days after baseline sampling. Serial blood sampling and DXA scans were repeated 2, 4, 8 and 16 weeks on the test diets. Body composition and areal BMD (aBMD) of whole body, lumbar spine, femur and tibia were measured using DXA. At week 16, muscle protein fractional synthesis rate (FSR), volumetric BMD (vBMD), microarchitecture and bone strength were assessed. Body weight declined after SHAM and ORX surgery, with slower recovery in the ORX group. Dietary CLA did not affect weight or lean mass, but attenuated gains in fat mass. Lean mass was stable in SHAM and reduced in ORX by 2 weeks with whole body and femur bone mineral content (BMC) reduced by 4 weeks; CLA did not alter BMC. By week 16 ORX groups had lower free testosterone and myofibrillar FSR, yet higher cortisol, osteocalcin and ionized calcium with no alterations due to CLA. ORX+Control had higher prostaglandin E2 (PGE2) and total alkaline phosphatase compared to SHAM+Control whereas ORX+CLA were not different from SHAM groups. Femur metaphyseal vBMD was reduced in ORX+CTRL with the reduction attenuated by CLA. Femur cortical thickness (Ct.Th.) and biomechanical strength were reduced and cortical porosity (Ct.Po.) elevated by ORX and attenuated by CLA. This androgen deficient model with a sarcopenic-osteoporotic phenotype similar to aging men responded to dietary CLA with significant benefits to femur density and strength.

Protein anabolic responses to a fed steady state in healthy aging.

BACKGROUND: Protein anabolism in response to feeding may be impaired with aging. To determine if this could contribute to muscle loss, we studied fed-state metabolic responses in healthy, non-sarcopenic elderly women. METHODS: Whole-body [(3)H]glucose and protein ([(13)C]leucine) kinetics were measured, and muscle protein fractional synthesis rate ([(2)H(5)]phenylalanine) and signaling events were assessed from vastus lateralis biopsies in eight elderly (73 ± 3 years) and eight young women (24 ± 1 years), using a simulated fed steady-state clamp. RESULTS: Both groups had similar muscle and lean body mass indices and activity level. During insulin, glucose (8 mmol/L), and amino acid (AA; 2× fasting) infusions, serum insulin was lower in the elderly women and C-peptide increased less. Glucose uptake was stimulated, and production suppressed similarly. Suppression of whole-body protein breakdown was less in the elderly women, leading to lower AA infusion rates, oxidation, and net positive protein balance, but differences were not present when adjusted for serum insulin. Whole-body protein synthesis and muscle protein fractional synthesis rate increased similarly. Similar increases in phosphorylated Akt(Ser473), PRAS40(Thr246), FoxO3a(Thr32), and rpS6(Ser240/244) indicated no alterations in insulin/nutrient signaling with aging. CONCLUSIONS: Both whole-body and muscle fed-state protein anabolic responses were preserved, as was insulin sensitivity of glucose metabolism, in active, healthy elderly women. This is consistent with other factors such as sedentarity, low protein intake, and concurrent diseases, being responsible for the sarcopenia of aging.

Effect of orthotopic liver transplantation (OLT) on branched-chain amino acid requirement.

Little is known regarding the impact of liver transplantation on amino acid requirements in children. Since plasma levels of the branched-chain amino acids (BCAA) are decreased in the presence of normal levels of the aromatic amino acids after liver transplantation, normalization of hepatic function may not fully correct changes in BCAA metabolism that occur in the pretransplant period. The goal of the present study was to determine total BCAA requirements of children following liver transplantation. The requirement of total BCAA was determined using indicator amino acid oxidation (IAAO) in five clinically stable children (5.7 +/- 3.5 y, mean +/- SD) 1-8 y post liver transplantation. Children received in random order 6 graded intakes of total BCAA. Individual BCAA in the test diet were provided in the same proportions as present in egg protein to minimize the potential interactive effects of individual BCAA on assessment of requirement. Total BCAA requirement was determined by measuring the oxidation of L-[1-13C] phenylalanine to 13CO2 [F13CO2 in micromol/kg/h], after a primed, continuous infusion of the tracer and using a two-phase linear regression crossover regression analysis. The estimated average requirement and the upper limit of the 95% CI for total BCAA in children who have undergone liver transplantation were 172 and 206 mg/kg/d), respectively. Total BCAA requirement in children who have undergone orthotopic liver transplantation (OLT) remain increased in the post-liver transplant period when compared with healthy school aged children, but is decreased when compared with children with mild-moderate chronic cholestatic (MCC) liver disease.

Mild-to-moderate chronic cholestatic liver disease increases leucine oxidation in children.

Malnutrition is prevalent in children with chronic cholestatic liver disease. Using the noninvasive indicator amino acid oxidation (IAAO) technique, we recently determined that mild-to-moderate chronic cholestatic (MCC) liver disease increases the need for branched-chain amino acids (BCAA) in children. To examine the underlying mechanisms responsible for this increased need for BCAA in liver disease, we measured L-[1-(13C)]-leucine oxidation in the postabsorptive and fed states in 10 children with MCC liver disease (8.8 +/- 3.5 y) and in 11 healthy children (9.4 +/- 2.2 y). The oxidation of L-[1-(13C)]-leucine to 13CO2 [F13CO2 in micromol/(kg.h)] was determined after a primed, continuous oral administration of the tracer. Total BCAA in diet was provided at 300 mg/(kg.d) to ensure that leucine oxidation was measured when leucine intake was in excess of requirements. In the postabsorptive state, the rate of release of 13CO2 from 13C-leucine oxidation (F13CO2) and whole-body leucine oxidation were significantly higher in children with MCC liver disease (P < 0.05). However, F13CO2 and whole-body leucine oxidation did not differ in the fed state. We conclude that the increased need for dietary BCAA in MCC liver disease is mediated in part by increased leucine oxidation in the postabsorptive state.

Branched-chain amino acid needs in children with mild-to-moderate chronic cholestatic liver disease.

Protein-energy malnutrition is prevalent in children with chronic cholestatic liver disease. Supplementation of branched-chain amino acids (BCAA) in infants and children with chronic liver disease has been associated with significant improvement in growth and nitrogen balance, suggesting that BCAA requirements are increased in chronic liver disease. The goal of the present study was to determine the total BCAA requirement in children with mild-to-moderate chronic cholestatic (MCC) liver disease using indicator amino acid oxidation (IAAO). Total BCAA requirements were determined in 6 children (6.3 +/- 3.7 y, mean +/- SD) with MCC liver disease. Children were randomly assigned to receive 7 graded intakes of total BCAA. Individual BCAA in the test diet were provided in the same proportions as those present in egg protein to minimize the potential interactive effects of individual BCAA on assessment of requirement. The total BCAA requirement was determined by measuring the oxidation of l-[1-13C] Phe to 13CO2 [F13CO2 in micromol/(kg x h)], after a primed, continuous oral administration of the tracer and using a 2-phase linear regression crossover regression analysis. The estimated mean requirement and the upper limit of the 95% CI for total BCAA establishing using the IAAO in children with MCC liver disease were 209 and 272 mg/(kg x d), respectively. Total BCAA estimated average requirements using the IAAO were significantly higher than mean requirements established previously for healthy children (P < 0.05).

Leucine is not a good choice as an indicator amino acid for determining amino acid requirements in men.

Leucine tracer has been widely used for examining whole-body protein turnover in humans, but has not been evaluated as an indicator to be used in the indicator amino acid oxidation (IAAO) method. The goal of this study was to determine whether the L-[1-(13C)]leucine isotope is an acceptable indicator by comparing it with an established tracer, L-[1-(13C)]lysine. Healthy men (n = 7; 29.9 +/- 4.8 y old) were fed in random order a diet with 7 graded intakes of phenylalanine without tyrosine. In the first study (n = 5), subjects were administered an excess leucine intake of 65 mg/(kg.d), and in the second study (n = 5), they were given the mean requirement of 45 mg/(kg.d) to determine whether leucine intake affected the pattern of response. Previous IAAO studies using lysine and phenylalanine demonstrated a clear pattern in 13CO2 production, i.e., increasing test amino acid intake resulted in a linear decrease to plateau, with a readily discernable breakpoint indicating the requirement. This pattern of production of 13CO2, indicates clear partitioning of the indicator amino acid between oxidation and protein synthesis. This was not observed with leucine at an intake of 65 mg/(kg.d). Conversely, at the lower leucine intake of 45 mg/(kg.d), a breakpoint was seen and a total aromatic amino acid requirement estimate that did not differ from that obtain using lysine as the indicator was obtained. In conclusion, leucine may be used as the indicator in the IAAO technique only when the daily intake leucine is given at its mean requirement level and the potential metabolic effects of other variables are taken into consideration.

Insulin-like growth factor II and binding proteins 1 and 3 from second trimester human amniotic fluid are associated with infant birth weight.

The developing fetus begins to swallow amniotic fluid (AF) early in gestation, a process that results in ingestion of numerous growth factors. Our objectives were 2-fold: 1) to assess the concentration and distribution of insulin-like growth factor II (IGF II) and its binding proteins (BP) 1 and 3 in 2nd trimester amniotic fluid using ELISA, and 2) to establish whether concentrations of AF IGF II and its binding proteins IGF BP1 and 3, measured early in pregnancy, were associated with and predictive of infant birth weight. Birth weights were categorized using recently developed birth-weight-for-gestational-age percentiles for fetal growth in which infants < 10% were classified as SGA (small-for-gestational-age) and those > 90% as LGA (large-for-gestational-age). AF samples were collected after routine genetic testing (15.1 +/- 0.04 wk, range 12-20 wk) from 543 mother-infant pairs in Montreal, QC, Canada. Maternal and fetal characteristics were obtained from questionnaires and medical chart review. Multivariate regression analysis that controlled for maternal height, prepregnancy weight, smoking behavior, infant gender, gestational age, parity, as well as amniocentesis week showed that higher AF IGF BP1 was associated with lower birth weight (partial r2 = 0.0062). Regression analyses revealed that AF IGF BP3 was positively associated with birth weight within LGA and macrosomia subpopulations (partial r2 = 0.0283 and 0.0404, respectively). These results show that 2nd trimester AF IGF BP1, BP3, and IGF II may emerge as early indicators of fetal growth.

Colitis increases albumin synthesis at the expense of muscle protein synthesis in macronutrient-restricted piglets.

Our aim was to examine the effect of acute inflammation localized in the colon and early macronutrient restriction on protein synthesis in a piglet model. In a 2 x 2 factorial design, piglets (n = 32) were fed an adequate or macronutrient-restricted diet with or without dextran sulfate-induced colitis for 7 d. The stable isotope tracer L-[5,5,5-(2)H(3)]leucine was infused to determine protein kinetics at the whole-body level and synthesis of tissue and plasma proteins. In the well-nourished state, colitis did not affect weight gain or protein kinetics except for an increase in albumin synthesis (P < 0.05). Macronutrient restriction alone caused a general slowing of protein metabolism including decreased weight gain (P < 0.0004), whole-body protein turnover (P < 0.0001), and liver (P < 0.01) and plasma protein (P < 0.03) synthesis. However, in the presence of macronutrient restriction, colitis compromised weight gain further (P < 0.02) and decreased muscle protein synthesis (P < 0.05) due to a redistribution of protein metabolism that supported enhanced synthesis of plasma proteins. The increased contribution of plasma protein synthesis to whole-body protein turnover was attributable mainly to increased synthesis of albumin (P < 0.006). Concentrations of plasma proteins were unaffected despite dramatic changes in their synthesis rates, thereby underestimating the effects of malnutrition and colitis on protein metabolism. Increased synthesis of plasma proteins, particularly the negative acute phase reactant albumin, compromises weight gain and muscle protein synthesis only when macronutrient intake is inadequate, underscoring the role of adequate nutrition in preventing growth impairment and muscle wasting in acute inflammation. These results suggest that the hypoalbuminemia of inflammatory bowel disease should not be attributed to decreased synthesis.

Urinary sulfur excretion and the nitrogen/sulfur balance ratio reveal nonprotein sulfur amino acid retention in piglets.

We evaluated the use of urinary sulfur (S) excretion as a measure of sulfur amino acid (SAA) catabolism and the nitrogen/sulfur (N/S) molar balance ratio as an indicator of nonprotein SAA storage in growing piglets. After confirming that an intravenous dose of sulfate is fully recovered in urinary sulfate, we measured urinary S recovery after an intravenous dose of methionine in 6 piglets fed an adequate protein (AP) diet and 6 piglets fed a low protein (LP) diet with normal energy provision. As measured over 48 h, recoveries of the methionine load as urinary total S was 106% in the AP group but only 69% in the LP group (P < 0.05). On the baseline diets the N/S balance ratio in the AP group was 36, whereas that in the LP group was 30 (P < 0.05); immediately after the methionine load, this ratio remained constant in the AP group but decreased further, to 26 (P < 0.05) in the LP group. These results indicate that protein-deficient piglets accumulate relatively more S than N from their diet, and under these conditions a significant portion of the S derived from a methionine load is retained in nonprotein compounds. Urinary S excretion, a simple nontracer measurement, can provide an accurate measure of SAA catabolism, and the N/S balance ratio is a potentially useful indicator of changes in nonprotein SAA stores of growing piglets.

Valine may be the first limiting branched-chain amino acid in egg protein in men.

Recently, we defined an estimate for total branched-chain amino acids (BCAA) using the indicator amino acid oxidation technique in men fed the three BCAA (leucine, isoleucine and valine) in the proportion present in egg protein. Although egg protein is regarded as a high quality dietary protein source, it is not known whether the proportions of the three BCAA are optimal. Five men with known total BCAA requirements were restudied. Each men was studied with isoleucine, leucine or valine held constant at that individual's requirement level while the intake of the other two BCAA was reduced; one BCAA was held constant and the intake of the other two was reduced by 10 and 20% in random order. The label appearance from the oxidation of L-[13C]-phenylalanine to 13CO2 (F13CO2) in breath was monitored in response to the change in amino acid intake. When either isoleucine or leucine was held constant, and the other two BCAA reduced by 20% (valine and leucine, or valine and isoleucine, respectively) F13CO2 increased (P = 0.007, P = 0.038, respectively). We conclude that valine may be the first limiting BCAA in egg protein.

Branched-chain amino acid requirements in school-aged children determined by indicator amino acid oxidation (IAAO).

The current WHO/FAO/UNU recommendations for BCAA requirements in school-aged children are based on nitrogen balance studies that have tended to produce lower estimates of amino acid requirements that those determined using stable isotope methodologies. The new dietary reference intake (DRI) recommendations for total BCAA requirements in children were determined using a factorial approach that included adult BCAA requirements plus the additional needs for growth. The purpose of this study was to determine directly total BCAA requirements in school children aged 6-10 y using indicator amino acid oxidation (IAAO). Five children (8.5 +/- 1.2 y) were assigned randomly to receive 7 graded intakes of total BCAA. Individual BCAA in the test diet were provided in the same proportions as those present in egg protein to minimize the potential interactive effects of individual BCAA on assessment of requirement. Total BCAA requirement was determined by measuring the oxidation of L-[1-13C] phenylalanine to 13CO2 [F13CO2 in micromol/(kg x h)], after a primed, continuous infusion of the labeled tracer and using a two-phase linear crossover regression analysis. The mean requirement and the population-safe intake level (upper limit of the 95% CI) of the total BCAA in healthy school aged children were 147 and 192 mg/(kg x d), respectively. The estimated mean requirements of the total BCAA as determined by IAAO is approximately 48% higher than the current DRI recommendations, suggesting that these recommendations may be too low to meet the needs of school-aged children.

Oral and intravenous tracer protocols of the indicator amino acid oxidation method provide the same estimate of the lysine requirement in healthy men.

To investigate whether splanchnic uptake of the indicator amino acid ([1-(13)C] phenylalanine) during the fed state alters the estimate of lysine requirement, five healthy men were studied at graded levels of lysine intake, with either an oral or intravenous (IV) tracer protocol, in a randomized, crossover design. Splanchnic extraction of the oral tracer was expressed as the difference between the ratio of the enrichments in urinary phenylalanine between tracer protocols. The rate of release of (13)CO(2) from (13)C-phenylalanine oxidation (F(13)CO(2)) was measured and a two-phase linear regression crossover model was applied to determine the lysine requirement. Mean splanchnic extraction of the oral tracer was approximately 19%. Although actual F(13)CO(2) was higher during oral tracer infusion (P < 0.001), the breakpoint was not different from that determined with IV infusion (P = 0.98), with both yielding a mean lysine requirement of 36.6 mg/(kg. d). The upper 95% confidence intervals were 52.5 and 53.3 mg/(kg. d) for the oral and IV isotope infusions, respectively. These results demonstrate that routes of isotope administration using the indicator amino acid oxidation technique do not affect the estimated amino acid requirement. Therefore, the indicator amino acid oxidation method using the oral route, which is less invasive and allows for studies in vulnerable groups such as infants and children, should be the preferred method for studying amino acid requirements.

Long-term effects of histidine depletion on whole-body protein metabolism in healthy adults.

The essentiality of histidine in healthy adults is a controversial topic. To study the potential metabolic effects of a lack of exogenous histidine, four healthy adults consumed a histidine-free diet, with adequate energy and 1.0 g/(kg. d) of an L-amino acid mixture for 48 d. Protein metabolism was monitored every 4 d by using indicator amino acid (L-[1-(13)C]phenylalanine) oxidation (in four subjects) and [(15)N]glycine (in one subject). Urine samples (24-h) were collected for measurement of urea, total nitrogen, creatinine, 3-methylhistidine (3-MH), histidine and beta-alanine. Albumin, transferrin and hematologic concentrations were measured on d 0, 24 and 48. Urinary excretion of nitrogen, urea, creatinine and 3-MH were not affected by the histidine-free diet. However, there was a significant (P < 0.001) linear decline (24-28%) in whole-body protein turnover. Significant (P < 0.05) decreases in albumin (12%), transferrin (17%) and hemoglobin (Hb) (11%) concentrations occurred slowly over the histidine depletion period. The urinary excretion of beta-alanine (an index of carnosine catabolism) generally increased in the smallest subject during the consumption of histidine-free diet. This study demonstrates that a lack of histidine in the diet for a prolonged period resulted in an accommodation of protein turnover and phenylalanine oxidation, measured by the (13)C-phenylalanine indicator amino acid. The extensive metabolic accommodation, together with decreases in Hb, albumin and transferrin during histidine depletion, leaves unresolved the issue of whether histidine is a dietary essential amino acid in healthy adults.

The total branched-chain amino acid requirement in young healthy adult men determined by indicator amino acid oxidation by use of L-[1-13C]phenylalanine.

Previous recommendations for branched-chain amino acids (BCAA), based on nitrogen balance studies, were found to be low in a series of stable isotope-labeled amino acid studies. The BCAA requirement was increased in the new dietary reference intake (DRI) report on the basis of a series of stable isotope studies examining the requirement of leucine and valine individually, but not isoleucine. To reduce the possibility of interactions among these amino acids and imbalances in the mixture affecting the estimate of requirements, we decided to determine the requirement for the total BCAA of young healthy adult men, receiving a mixture of BCAA based on the proportion of these amino acids in egg protein, by use of indicator amino acid oxidation. Seven men were assigned to receive nine graded intakes of a BCAA mixture in random order: 34, 50, 66, 80, 100, 120, 140, 160 and 180 mg/(kg. d). The rate of release of (13)CO(2) from the oxidation of L-[1-(13)C]phenylalanine (F (13)CO(2)) was measured and a two-phase linear regression crossover model was applied to determine total BCAA requirement. The mean requirement and population-safe level (upper limit of 95% confidence interval) of the total BCAA were 144 and 210 mg/(kg. d), respectively. Based on the balance of BCAA in egg protein, our estimate for the mean leucine requirement is 55 mg/(kg. d), which is substantially higher than the 34 mg/(kg. d) recommended by the DRI.

Phase of menstrual cycle affects lysine requirement in healthy women.

The aim of this study was to investigate whether the phases of the menstrual cycle affect lysine requirement in healthy adult females, as determined by the indicator amino acid oxidation (IAAO) method. Five healthy females with regular menstrual cycles were studied at seven graded levels of lysine intake, in random order, with an oral [13C]phenylalanine tracer protocol in both the follicular and luteal phases. A total of 14 studies were conducted for each subject. Breath and plasma samples were collected according to the standard IAAO protocol. Serum 17beta-estradiol and progesterone concentrations were measured on each IAAO study day. The rate of release of 13CO2 from [13C]phenylalanine oxidation (F13CO2) was measured, and a two-phase linear regression crossover model was applied to determine lysine requirement. F13CO2 was higher during the luteal phase (P < 0.001) and was positively associated with serum concentrations of 17beta-estradiol and progesterone. The F13CO2 data were adjusted for subjects and sex hormones and used to define breakpoints for lysine requirements. The lysine requirement of healthy females in the luteal phase was 37.7 mg.kg(-1).day(-1) and higher (P = 0.025) than that of females in the follicular phase (35.0 mg.kg(-1).day(-1)). At all lysine intake levels, plasma amino acids were lower and phenylalanine oxidation was higher in the luteal relative to the follicular phase. Therefore, we reason that the higher lysine requirement observed in the luteal phase is probably due to higher amino acid catabolism.