Preparation of CO2 from blood and protein-bound amino acid carboxyl groups for quantification and13C-isotope measurements

Read, W. W. C.; Read, M.; Rennie, Michael J.; Griggs, Robert C.; Halliday, D.

doi:10.1002/bms.1200110706

Cited by 64 publications

(23 citation statements)

References 10 publications

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“…Blood samples were taken in disposable syringes containing lithium heparin (Starstedt Canada Inc., St-Laurent, QC) as anticoagulant and placed immediately on ice. An aliquot of each sample (0.75 mL) was injected into 10-mL evacuated tubes (Vacutainer, Becton Dickinson Vacutainer Systems, Rutherford, NJ) containing 1 mL of frozen lactic acid and anti-foam reagent (Read et al 1984) and stored at -20°C until assayed for the isotopic enrichment of CO 2 . The remaining blood was centrifuged at 1042 × g for 20 min and the plasma stored at -20°C until analysis.…”

Section: Methodsmentioning

confidence: 99%

Whole-body protein synthesis in growing barrows: diurnal and day-to-day variation and the effect of site of tracer infusion and sampling

Roy¹,

Lapierre²,

Estrada³

et al. 1998

Can. J. Anim. Sci.

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. 1998. Whole-body protein synthesis in growing barrows: diurnal and dayto-day variation and the effect of site of tracer infusion and sampling. Can. J. Anim. Sci. 78: 575-585. In the first experiment, two catheters were placed in one of three sites for infusion and sampling (vena cava and vena cava with 7 cm between catheter tips, VC/VC, n = 5; right ventricle and atrium, RV/RA, n = 5; anterior aorta and left ventricle, AA/LV, n = 4) to determine the effect of site on the measurement of CO 2 and leucine kinetics. Pigs received a 6-h continuous i.v. infusion of NaH 13 CO 3 (1.45 µmol h -1 kg -1 , preceded by a priming dose of 2.05 µmol kg -1 ) and of L[1-13 C]leucine (9.7 µmol h -1 kg -1 , preceded by a priming dose of 9.7 µmol kg -1 ), the following day. No effect (P > 0.10) of site was observed on NaH 13 CO 3 recovery rate, leucine irreversible loss rate (leucine ILR) and leucine used for protein synthesis (LS). A tendency (P < 0.10) for increased leucine oxidation (LO) was observed with the VC/VC compared to the RV/RA sites. In the second experiment, six growing barrows with catheters in the VC/VC site were infused as earlier with NaH 13 CO 3 and L[1-13 C]leucine for three different 6h periods (12:00 to 18:00 h, first day; 20:00 to 02:00 h, night; 12:00 to 18:00 h, second day) to determine the diurnal and day-to-day variation of CO 2 and leucine kinetics. No effect (P > 0.10) of period was observed on NaH 13 CO 3 recovery rate, leucine ILR and LS. The LO was lower (P < 0.05) on the second day compared with the first day (day-to-day variation) while similar (P > 0.10) LO was observed between the first day and night (diurnal variation). Together, these results indicate that 1) the VC/VC site can be used for L[1-13 C]leucine and NaH 13 CO 3 infusions, 2) NaH 13 CO 3 recovery studies can be performed either the day preceding or following the measurement of substrate and 3) protein synthesis estimated from a 6-h infusion period can be extrapolated to the 24 h periods under conditions of frequent feeding.Key words: Pigs, leucine, protein synthesis, tracer techniques, diurnal variation Roy, N., Lapierre, H., Estrada, R. et Bernier, J. F. 1998. Synthèse protéique de l'organisme entier chez les porcs en croissance: variation diurne, quotidienne et l'effet de sites d'infusion et de prélèvement du traceur. Can. J. Anim. Sci. 78: 575-585. Dans la première expérience, deux cathéters ont été placés dans un des sites sélectionnés (veine cave et veine cave avec 7 cm entre les extrémités des cathéters, VC/VC, n = 5; ventricule et oreillette droits, RV/RA, n = 5; aorte antérieure et ventricule gauche, AA/LV, n = 4) pour vérifier l'effet du site de perfusion et de prélèvement sur les cinétiques du CO 2 et de la leucine. Les porcs ont été perfusés i.v. durant 6 h avec du NaH 13 CO 3 (1,45 µmol h -1 kg -1 , précédé d'une dose d'amorçage de 2,05 µmol kg -1 ) et avec de la L-[1-13 C] leucine (9,7 µmol h -1 kg -1 , précédé d'une dose d'amorçage de 9,7 µmol kg -1 ), le lendemain. Aucun effet (P > 0,10) des sites n'a été observé sur l...

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Section: Methodsmentioning

confidence: 99%

Whole-body protein synthesis in growing barrows: diurnal and day-to-day variation and the effect of site of tracer infusion and sampling

Roy¹,

Lapierre²,

Estrada³

et al. 1998

Can. J. Anim. Sci.

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“…Arterialized and femoral venous blood samples for subsequent determination of 13 C enrichment of blood CO 2 were also obtained at the same times: 1 ml blood was immediately injected into glass tubes (Vacu-tainer, Becton Dickinson, Rutherford, NJ; previously prepared with 1 ml 6 M orthophosphoric acid containing silicone antifoaming agent [10% by volume]) and degassed (16) to liberate and store the blood CO 2 . Total CO 2 of heparinized blood was measured with a Corning 189 blood gas analyzer (Medfield, MA).…”

Section: Methodsmentioning

confidence: 99%

Effects of Insulin and Amino Acids on Leg Protein Turnover in IDDM Patients

et al. 1991

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To determine whether the responses of muscle protein metabolism to insulin and amino acids in patients with insulin-dependent diabetes mellitus (IDDM) were different from those in nondiabetic subjects, leg tissue kinetics of [15N]phenylalanine and [1-13C]leucine and its metabolites were measured in eight insulin-withdrawn IDDM patients and eight nondiabetic subjects during basal insulinemia and during infusion of insulin (0.29 nmol.min-1.m-2). The diabetic patients were studied in the absence of amino acids, and both groups were studied during infusion of a mixed-amino acid solution (AA). In the diabetic patients, insulin alone and combined with additional AA reduced leg tissue phenylalanine release by 42 and 41%, respectively (both P less than 0.05), but uptake was unchanged. Leg tissue leucine oxidation was unchanged by insulin alone but was increased (P = 0.012) fourfold during insulin infusion with additional AA. In the nondiabetic subjects, insulin with AA infusion increased leg tissue phenylalanine uptake (45.7 +/- 7.5 to 73.1 +/- 7.3 nmol.min-1.100 g-1, P less than 0.01). Insulin-stimulated glucose uptake in the diabetic patients (1.60 +/- 0.28 mumol.min-1.100 g-1, P = 0.04). These results suggest that, in IDDM patients, 1) infusion of insulin fails to stimulate muscle protein synthesis even when combined with a substantially increased provision of AA, and 2) compared with nondiabetic subjects, muscle protein synthesis as well as glucose uptake exhibit blunted responses to insulin.

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“…To determine whole blood C02 enrichment, C02 was first isolated from whole blood by acid liberation and cryogenic distillation (7). The '3C/'2C ratio was then measured on a Finnigan MAT (San Jose, CA) Delta E isotope ratio mass spectrometer.…”

Section: Methodsmentioning

confidence: 99%

Effects of Glucose Infusion on Leucine Transamination and Oxidation in the Ovine Fetus

et al. 1991

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ABSTRACT. During fasting of the ewe, the rate of amino acid oxidation by the ovine fetus increases substantially. We hypothesized that the increase in amino acid oxidation derived mainly from reduced protein synthesis. We further hypothesized that fetal glucose supplementation would result in diminished amino acid oxidation. To test these hypotheses, nine ovine fetuses were infused with [15N,1-'3C]leucine to determine the rates of leucine appearance and disposal. Simultaneously, the fetal uptake of leucine was determined. Animals were studied in the fed and fasted state. After baseline measurements, glucose was infused into the fetal inferior vena cava at a rate estimated to match the fetal glucose uptake. Results of these studies indicate that leucine nitrogen flux, leucine carbon flux and fetal leucine uptake were constant. Leucine oxidation was increased by 50% in the fasted state (6.3 versus 13.4 pmol/ min); glucose infusion resulted in a 25% decline in oxidation (to 10.4 pmol/min) in the fasted state, but had no effect in the fed state. Mean leucine umbilical uptake during fasting was 9.3 pmollmin, 4.1 pmol/min less than leucine oxidation. These data suggest that leucine (and potentially other amino acids) may be in negative balance during maternal fasting, and can be spared by supplementation of the fetus with exogenous glucose. (Pediatr Res 30: [423][424][425][426][427][428][429]1991) Abbreviations KIC, a-ketoisocaproate GC/MS, gas chromatograph/mass spectrometry BCAA, branched chain amino acid ANOVA, analysis of variance in increased fetal oxidation of amino acids, a diminished rate of protein synthesis, and no significant change in the rate of protein degradation. To further test this hypothesis, a labeled amino acid was used as a tracer to allow the quantitative estimates of fetal protein breakdown, protein synthesis, and oxidation. Coupled with measurements of fetal uptake, all parameters of the movement of a given amino acid through fetal plasma could then be observed. Controlled perturbations of the physiologic state of the fetus were then initiated with the aim of determining the effect of substrate supply on protein synthesis, breakdown, and amino acid oxidation.We have chosen leucine as a representative amino acid to study fetal protein kinetics. The fetal uptake of this essential amino acid even under normal conditions is large relative to its carcass accretion rate (4), implying a significant contribution of this amino acid to oxidative or other pathways. In addition, leucine has been used extensively for modeling protein kinetics in postnatal animals.The aim of the present study was to investigate how the ovine fetus adapts its amino acid metabolism to augmentation of glucose intake. We hypothesized that augmentation of glucose supply to the fetus would result in diminished leucine oxidation. To study this question, the net rates of fetal uptake of leucine and rates of nonoxidative leucine disposal, oxidation, and leucine derived from protein breakdown were determined in the fed and fasted ...

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Preparation of CO2 from blood and protein-bound amino acid carboxyl groups for quantification and13C-isotope measurements

Cited by 64 publications

References 10 publications

Whole-body protein synthesis in growing barrows: diurnal and day-to-day variation and the effect of site of tracer infusion and sampling

Whole-body protein synthesis in growing barrows: diurnal and day-to-day variation and the effect of site of tracer infusion and sampling

Effects of Insulin and Amino Acids on Leg Protein Turnover in IDDM Patients

Effects of Glucose Infusion on Leucine Transamination and Oxidation in the Ovine Fetus

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