2-pool Model Research Articles

Inactivity and hormonal deregulation increase human skeletal muscle protein loss

We sought to quantify changes in human skeletal muscle protein kinetics during prolonged inactivity with and without accompanying hypercortisolemia. Oral hydrocortisone sodium succinate (~22 μg/dL) was administered to an experimental group (EXP: male, n=6, 28± 2 y; 84±4 kg; 178± 3 cm;) throughout 28 days of bedrest. The control group (CON: male, n=6; 38±8 y; 86±10 kg; 179±3 cm) received no exogenous cortisol (~10 μg/dL). Femoral arterio-venous blood samples and a vastus lateralis muscle biopsy were obtained during a primed constant infusion of L-[ring-2H5] phenylalanine before and after bed rest. Postabsorptive net balance became more negative in the EXP group following bedrest, a change largely facilitated by a ~40% reduction in protein synthesis in the EXP group. Table 1.. 3-pool model data representing protein synthesis (Fom) and breakdown (Fmo) (nmol/min/100mL leg) before and after bedrest. A similar superscript represents a statistical difference (p<0.05). The EXP group experienced a decrease in plasma testosterone (Day 1: 503.5±56.3 vs. Day 28: 409.5±28.6 ng/dL) and sex hormone binding globulin (SHBG) (Day 1: 22.5±2.5 vs. 12.2±1.5 nmol/L), (p<0.05). This was consistent with 3-fold greater loss of lean leg mass (LLM) in the EXP group (EXP: −1.4±0.1 vs. CON: −0.4±0.1 kg), (p<0.05). In conclusion, a combination of inactivity and hypercortisolemia leads to hormonal deregulation which in-turn further exacerbates muscle protein catabolism. Supported by funding from NPFR00205.

Functional Ryanodine Receptor Expression Is Required for NAADP-mediated Local Ca2+ Signaling in T-lymphocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing nucleotide involved in T cell Ca2+ signaling (Berg, I., Potter, B. V. L., Mayr, G. W., and Guse, A. H. (2000) J. Cell Biol. 150, 581-588). The objective of this study was to analyze whether the first subcellular Ca2+ signals obtained upon NAADP stimulation of T-lymphocytes depend on the functional expression of ryanodine receptors. Using combined microinjection and high resolution confocal calcium imaging, we demonstrate here that subcellular Ca2+ signals, characterized by amplitudes between approximately 30 and 100 nM and diameters of approximately 0.5 microM, preceded global Ca2+ signals. Co-injection of the ryanodine receptor antagonists ruthenium red and ryanodine together with NAADP abolished the effects of NAADP, whereas the D-myo-inositol 1,4,5-trisphosphate antagonist heparin and the Ca2+ entry blocker SKF&96365 were without effect. This pharmacological approach was confirmed by a molecular knock-down approach. Jurkat T cell clones with largely reduced expression of ryanodine receptors did not respond to microinjections of NAADP. Taken together, our data suggest that the Ca2+ release channel sensitive to NAADP in T-lymphocytes is the ryanodine receptor.

Production and bioavailability of autochthonous dissolved organic carbon: effects of mesozooplankton

A phytoplankton bloom and decay sequence was created in 2 laboratory containers and mesozooplankton was added to one container before the peak of algal biomass. Each day for 22 d, the net production of autochthonous dissolved organic carbon (DOC) was measured and on 5 occasions the degradation kinetics and the total pool of biodegradable DOC (BDOC) were assayed in experi- ments lasting 230 d. Net accumulation of new DOC was 235 and 280 µM in the containers with and without zooplankton, respectively. The best description of microbial DOC degradation was a 2-pool model and 1st order exponential decay. Without mesozooplankton present, the degradation experi- ments showed accumulation of a large pool of labile BDOC characterised by decay coefficients >0.2 d -1 . The least labile pools in the 2 containers had similar coefficients (average 0.02 d -1 ). The amount of newly produced recalcitrant DOC (RDOC) accounted for about 12% of new DOC. The differences observed with respect to degradation kinetics and net DOC production are explained by food web interactions and nutrient limitation. The presence of mesozooplankton resulted in high bacterial production keeping labile BDOC at low concentrations. In the container without meso- zooplankton, the bacterial uptake capacity was reduced, probably by a combination of protist grazing and nutrient limitation. Consequently, about 75 µM BDOC with a half-life of less than 3 d accumu- lated during the experiment. Mineralisation of the accumulated dissolved organic matter (DOM) during microbial degradation in a nutrient replete environment was measured as the decrease in DOC and net mineralisation/immobilisation of inorganic N and P. The mineralisation of DOC was accompanied by low mineralisation of N and P and even immobilisation of phosphate during degra- dation of DOM produced in the container with mesozooplankton present. Bacterial production of DON and DOP is believed to result in a recalcitrant DOM pool enriched in N and P, and the activity of mesozooplankton seems to enhance this scenario.

Equilibrium of phosphointermediates of sodium and potassium ion transport adenosine triphosphatase: action of sodium ion and Hofmeister effect.

Sodium and potassium ion transport adenosine triphosphatase accepts and donates a phosphate group in the course of its reaction sequence. The phosphorylated enzyme has two principal reactive states, E1P and E2P. E1P is formed reversibly from ATP in the presence of Na+ and is precursor to E2P, which equilibrates with P(i) in the presence of K+. We studied equilibrium between these states at 4 degrees C and the effect of Na+ on it. To optimize the reaction system we used a Hofmeister effect, replacing the usual anion, chloride, with a chaotropic anion, usually nitrate. We phosphorylated enzyme from canine kidney with [32P]ATP. We estimated interconversion rate constants for the reaction E1P <--> E2P and their ratio. To estimate rate constants we terminated phosphorylation and observed decay kinetics. We observed E1P or E2P selectively by adding K+ or ADP respectively. K+ dephosphorylates E2P leaving E1P as observable species; ADP dephosphorylates E1P leaving E2P as observable species. We fitted a 2-pool model comprising two reactive species or a twin 2-pool model, comprising a pair of independent 2-pool models, to the data and obtained interconversion and hydrolysis rate constants for each state. Replacing Na+ with Tris+ or lysine+ did not change the ratio of interconversion rate constants between E1P and E2P. Thus Na+ binds about equally strongly to E1P and E2P. This conclusion is consistent with a model of Pedemonte (1988. J. Theor. Biol. 134:165-182.). We found that Na+ affected another equilibrium, that of transphosphorylation between ATP x dephosphoenzyme and ADP x E1P. We used the reactions and model of Pickart and Jencks (1982. J. Biol. Chem. 257:5319-5322.) to generate and fit data. Decreasing the concentration of Na+ 10-fold shifted the equilibrium constant 10-fold favoring ADP x E1P over ATP x dephosphoenzyme. Thus Na+ can dissociate from E1P x Na3. Furthermore, we found two characteristics of Hofmeister effects on this enzyme.

Accuracy of urea removal estimated by kinetic models

The most accurate method for assessing the dialysis dose delivered during high efficiency/flux hemodialysis has not been established. Most current indices of dialysis dose are based on blood-side urea measurements, and thus estimate urea removal. Unfortunately, these methods may lead to inappropriately short dialysis during high flux or high efficiency dialysis, perhaps because of inaccuracies in estimating the amount of urea removal. It is unknown whether these clearance-based approaches can accurately predict either absolute or fractional net urea removal, the latter being equivalent to the solute removal index (SRI). Therefore, we compared the urea removal calculated by five blood-side kinetic methods: (1) urea reduction ration, (2) 1-pool, (3) 2-pool models, and the (4) Smye and (5) Daugirdas formulae. These were compared with the gold standard measurement by direct dialysate quantification. Eight stable patients receiving high-flux hemodialysis were studied over four sessions each. BUN was measured at 0, 45 minutes, 90 minutes, end dialysis, one hour after dialysis (equilibrium value), and 48 hours later. Total body water was determined from the dialysate urea removal; the urea generation rate was calculated using one hour post-dialysis and 48-hour BUN values. Both the total body water and urea generation rate were provided to the 1- and 2-pool models to optimize accuracy. The urea reduction ratio overestimated SRI. The 1-pool model overestimated both absolute urea removal and SRI in 28 of 32 sessions. The 2-pool model slightly underestimated both absolute urea removal and SRI. In contrast, the Smye and Daugirdas formulas accurately estimated SRI.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-stimulated GLUT4 glucose transporter recycling. A problem in membrane protein subcellular trafficking through multiple pools

The subcellular trafficking of GLUT4 in isolated rat adipose cells and 3T3-L1 adipocytes exhibits many of the properties observed in regulated secretory processes and neurosecretion. GLUT4 is sorted and sequestered from endosomes into a specialized secretory compartment in the basal state and the initial stimulation of its exocytosis by insulin is more rapid than its recycling through the endosomes and secretory compartment during the steady-state response to insulin. We present a mathematical analysis which shows that this behavior is inconsistent with a simple 2-pool model with one plasma membrane and one intracellular compartment, but that a 3-pool model, with two intracellular compartments, can simulate these properties. We extend this model to include the presence of occluded pools in the plasma membrane. Our analysis compares the behavior expected when these occluded pools are precursors in stimulation and/or clathrin-associated-like intermediates in endocytosis. The presence of a precursor occluded pool can account for a lag between the appearance of GLUT4 in the membrane and before the full stimulation of glucose transport activity. The analysis also shows that since the pool size of the occluded GLUT4 is relatively small, the formation of endocytic occluded intermediates such as GLUT4 in clathrin-coated pits is likely to be slow compared with the rate of endocytosis of the coated vesicles.

Estimation of treatment dose in high-efficiency haemodialysis.

Blood urea concentration is artificially low immediately following high-efficiency dialysis of duration T and will rise ('rebound') due to the continued diffusion of urea from the intracellular to the extracellular space. This leads to an overestimate of the efficiency of the dialysis given by KT/V (where V is the total distribution volume of urea and K is the urea clearance of the dialyser) if the true equilibrium blood concentration of urea is not used in the calculation of KT/V by the single-pool urea kinetic model (UKM). The measurement of the equilibrium urea concentration entails an additional blood sample 60 min after dialysis, but an estimate may be calculated using a blood sample taken 80 min following the start of dialysis together with an approximate formula derived from the 2-pool model. In 14 patients, an average error in the calculation of KT/V of 35% (range 19-75%) by the single-pool UKM was reduced to 13% (range 1-55%, but 8 measurements to < 7%) using the approximate technique. It is concluded that the approximate technique significantly improves the accuracy of dose estimation in high-efficiency dialysis without inconveniencing the patient.

Empirical Assessment of Model Validity

The metabolism of amino acids is far more complicated than a 1- to 2-pool model. Yet, these simple models have been extensively used with many different isotopically labeled tracers to study protein metabolism. A tracer of leucine and measurement of leucine kinetics has been a favorite choice for following protein metabolism. However, administering a leucine tracer and following it in blood will not adequately reflect the complex multi-pool nature of the leucine system. Using the tracer enrichment of the ketoacid metabolite of leucine, alpha-ketoisocaproate (KIC), to reflect intracellular events of leucine was an important improvement. Whether this approach is adequate to follow accurately leucine metabolism in vivo or not has not been tested. From data obtained using simultaneous administration of leucine and KIC tracers, we developed a 10-pool model of the in vivo leucine-KIC and bicarbonate kinetic system. Data from this model were compared with conventional measurements of leucine kinetics. The results from the 10-pool model agreed best with the simplified approach using a leucine tracer and measurement of KIC enrichment.

Leucine Metabolism in Man: Lessons from Modeling

The metabolism of amino acids is far more complicated than a 1- to 2-pool model, yet, such simple models have been extensively used with many different isotopically labeled tracers to study protein metabolism. A tracer of leucine and measurement of leucine kinetics has been a favorite choice for following protein metabolism. However, administering a leucine tracer and following it in blood will not adequately reflect the complex, multi-pool nature of the leucine system. Using the tracer enrichment of the ketoacid metabolite of leucine, alpha-ketoisocaproate (KIC), to reflect intracellular events of leucine was an important improvement. Whether this approach is adequate to follow accurately leucine metabolism in vivo or not has not been tested. From data obtained using simultaneous administration of leucine and KIC tracers, we developed a 10-pool model of the in vivo leucine-KIC and bicarbonate kinetic system. Data from this model were compared with conventional measurements of leucine kinetics. The results from the 10-pool model agreed best with the simplified approach using a leucine tracer and measurement of KIC enrichment.

Measurements of Plasma Colloid Osmotic Pressure, Total Protein and Sodium Concentration during Haemodialysis: Can Single-Pool Sodium Modelling Explain the Results?

Considering the plasma colloid osmotic pressure (COP) as a possible parameter for the monitoring of dialysis treatment compatibility, a characteristic time course was found. The COP and the total protein concentration very often do not increase significantly during the first treatment hour in spite of ultrafiltration. An increase in the plasma sodium concentration, which was higher than expected, was found to be the reason for a plasma dilution effect. This can be explained by a transcapillary sodium transfer coefficient which is not infinitely high as assumed in single-pool sodium modelling. From a 2-pool model considering the plasma volume as a separate pool and including capillary filtration time courses for plasma sodium, total protein concentration and COP could be calculated, which was very similar to the measured curves.

Computerprogramm zur interaktiven Auswertung von15N-tracerkinetischen Stoffwechselexperimenten

The theoretical concept of the computer program is based on the compartment theory for the 3-pool model using single pulse administration of the tracer. The code estimates the model parameters by means of the non-linear method of least squares fit under steady state conditions. Furthermore the parameters of the protein metabolism are calculated. The program works interactively and allows reading and modifying the experimental 15N tracer data via terminal and controlling the program.

Urea utilization in growing lambs. 7. NPN- and pure protein-N-utilization at various ages

The utilization quota of NPN and pure feed protein for body protein synthesis was calculated on the basis of N balance experiments with 15N labelled urea with the help of model concepts of a 3-pool model and its mathematical usage. In lambs weighing 13 kg, the efficiency of amino acid and nucleic acid synthesis in the non-amino acid N pool was 64%. This results in a total utilization quota for NPN and pure protein in the ration of 40% and 60%, resp. Lambs weighing 27 kg showed an efficiency in amino acid and nucleic acid synthesis in the non-AA N pool of 77% and in the AA N pool of 60%. The total utilization quota of NPN was 47% and that of pure protein 56%. The pure protein in the ration was thus about twice as well utilized for total protein synthesis and for protein synthesis for crude protein retention as the NPN compounds in the ration.

The effect of grinding and pelleting hay on digestibility, fermentation rate, digesta passage and rumen and faecal particle size in cows

High-quality grass hay was either chopped (CH) or ground and pelleted (PH) and given to two rumen-fistulated cows at about the maintenance level. Digestibility in vivo was measured as well as neutral detergent fibre (NDF) rate and extent of fermentation in sacco. Solids and liquid marker excretions in faeces were measured after a pulse dose in the rumen of Cr-mordanted NDF and LiCoEDTA. A 3-pool model with lag was fitted to the data and retention times were calculated for individual pools and for the total digestive tract. Core samples were taken from the rumen three times daily for particle size analysis by wet sieving. Faecal and feed mean particle size (MPS) was determined. Pelleting lowered dry matter and NDF digestibility from 0.73 to 0.67 and from 0.79 to 0.68, respectively. Fermentation rate decreased from an average of 0.094 to 0.051 h −1 and the unavailable NDF fraction was increased from 0.23 to 0.37 by pelleting. Digesta liquid retention was unaffected by pelleting, but total solids retention decreased from 73 to 54 h. The effect of pelleting on retention in different pools varied between the animals. Rumen MPS was largest, and faecal MPS smallest, when feeding on CH. Hay PH gave the lowest MPS after evening feeding and the highest before morning feeding. The opposite was observed for CH. The decrease in fermentation rate, increase in unavailable fibre and decrease in rumen digesta retention are believed to be the main factors responsible for decreased digestibility in vivo.

Methodologic aspects of the determination of N-exchange parameters from 15N-tracer studies in swine on the basis of models of N-metabolism. 3. Calculation of protein synthesis and decomposition rate on the basis of lysine flux, ascertained from the amount of 15N in the urine

The final product method introduced here is based on the use of 15N L-Lysine as tracer amino acid for the determination of the protein synthesis and decomposition quotas of the total body with the flux of lysine. For this purpose it is necessary to complete the N flow and N compartments of the 3-pool model by the values of lysine content. The ascertained values of protein synthesis--8.4 g/d/kg--and protein decomposition--6.9 g/d/kg--agree very well with those determined with 15N glycine as tracer amino acid.

Investigations on the Turnover of Plant Proteins. II. The Protein Turnover in Epicotyl Segments of Pisum sativum L. Influenced by 2’ -Methyl-4’ -chlorophenoxy Acetic Acid (MCPA)

Summary Based on a kinetic analysis of amino acid and protein turnover by means of compartment models the synthesis and degradation of the soluble proteins in etiolated epicotyl segments of Pisum sativum 1. as well as their dependence on the herbicice 2'-methyl-4'-chlorophenoxyacetic acid (MOPA) were quantitatively determined. The segments were incubated for 10 h in a medium containing l4O-leucine and subsequently placed in an inactive medium; the radioactivity in the soluble proteins and in the amino acid fraction was experimentally followed up for a total of 24 h. A 3-pool model in which the total measurable amino acid pool was divided into a direct precursor pool for protein synthesis and into a degradation pool was best suited to interpret the data. The turnover rate for the soluble proteins of untreated epicotyl segments was determined to be 0.058 h-1 ; at an MOPAconcentration of 10-4 M this value was nearly doubled. An increased proteolytic activity in the epicotyl segments ran parallel to the change of the turnover rate in dependence on MOP A-concentration. The heterogeneity of the soluble protein with respect to the turnover rate was investigated by means of 3H/140 double labelling for individual protein fractions separated by gel electrophoresis. The results obtained in this way are comparable with those of the investigation of the total pool turnover.

Investigations on the Turnover of Plant Proteins. I. The Determination of Protein Turnover as a Problem of Tracer-Kinetic Modelling

The turnover of proteins is investigated in most cases by means of tracer kinetic experiments which are to be interpreted on the basis of mathematical models. Here models are formulated for use in the evaluation of experiments with 14C-leucine on the protein turnover of epicotyl segments of Pisum sativum. The given data imply that incorporation of radioactivity by protein synthesis and elimination by protein degradation must be taken into account simultaneously. At first a 2-pool model was formulated mathematically which equates the precursor pool for protein synthesis with the total measured pool of free amino acids. This hypothesis proved to be incompatible with the time course of the experiment~,l data. In an extension of the model the pool of amino acids was divided into the precursor pool and a pool fed by protein degradation. This version gives a good fit to the data. The limitations of the model are discussed and the kinetic approach applied here is compared with similar possibilities and methods proposed in the literature.

Arginine vasopressin metabolism in dogs. II. Modeling and system analysis.

System modeling and analysis methods were applied to interpret data regarding arginine vasopressin (AVP) metabolism in dogs. Based on this analysis a new nonlinear 3-pool model of AVP distribution and disposal was proposed and quantified; the model pools included the plasma, a receptor pool, and an extravascular nonreceptor pool. The receptor pool mediated a portion of the rapid flux of hormone between the plasma and the extravascular pool. Mathematical analysis indicated that the plasma AVP impulse response (bolus) data would be insufficient to uniquely estimate all the model constants, but additional plasma impulse data using 125I-labeled AVP, which does not bind to physiologic hormone receptors, would allow unique model quantification. Other required measurements were the urinary excretion of intact hormone, and plasma AVP degradation. The model was successfully fitted to the data from 10 dogs. The results suggest that, in the normal dogs studied, plasma contained 25% of the total AVP, 19% was bound to receptors, and the remaining 56% was in the extravascular pool. Eighty percent of the flux of AVP from the vascular compartment was mediated by the receptor pool; 98% of AVP degradation occurred in the extravascular pool; and urine excretion and plasma degradation made up the remainder.

Relationships between plasma lipoprotein cholesterol concentrations and the pool size and metabolism of cholesterol in man

The plasma concentrations of unesterified and esterified cholesterol within very low density (VLDL), low density (LDL) and high density (HDL) lipoproteins have been examined in relation to the metabolism and pool size of cholesterol in normal and hyperlipidaemic subjects. Cholesterol metabolism was assessed as faecal endogenous neutral and acidic steroid excretion, a 2-pool model of cholesterol turnover, and in vitro plasma cholesterol esterifying activity. VLDL total cholesterol (TC) concentration was positively correlated with cholesterol turnover, endogenous neutral steroid excretion, bile acid excretion and the absolute rate of plasma cholesterol esterification. The correlations with cholesterol turnover and neutral steroid excretion, but not that with bile acid excretion, remained significant when these were corrected for their relationships to body weight. LDL-TC was negatively correlated with the fractional rate of plasma cholesterol esterification and, in subjects with primary type IIa hyperlipoproteinaemia, also with the rate constant for cholesterol elimination from the rapidly exchanging cholesterol pool. No correlation was found between LDL-TC concentration and bile acid excretion. HDL-TC concentration was negatively correlated with both the rapidly and slowly exchanging pools of tissue cholesterol, after correction for their relationships to body weight and adiposity. In contrast, cholesterol pool sizes were not correlated with the concentration of VLDL or LDL-TC; nor was there any relationship to plasma cholesterol esterifying activity. No correlation was found between the relative proportions of unesterified and esterified cholesterol within any lipoprotein fraction and either the pool size or metabolism of cholesterol. These findings accord with previous reports of enhanced cholesterol metabolism in subjects with elevated VLDL concentrations and of impaired plasma LDL and cholesterol clearance in patients with primary type IIa hyperlipoproteinaemia. The demonstration that HDL-TC concentration is negatively correlated with body cholesterol pool size supports in vitro evidence for a role of HDL in tissue cholesterol clearance.

Origin of Plasma Fatty Acids in Lactating Cows Fed High Grain or High Fat Diets

Palmitate-1-14C was given to lactating cows in 20 trials involving four dietary treatments and two different routes of tracer administration. The specific activitytime curve of 14-C activity in milk fat was resolved by curve analysis into two components, a rapid-turnover component attributed to exogenous (dietary) fatty acid and a slower turnover component attributed to adipose tissue. Administration of palmitate-1-14C abomasally or orally gave greater (P<0.01) estimates of exogenous fatty acid than did intravenous dosing. A high grain-restricted roughage diet reduced (P<0.01) the exogenous estimate, presumably due to increased uptake of dietary fatty acid by adipose tissue. A low-fat diet also lowered the estimate of exogenous fatty acid. A high-fat diet reduced the turnover time of the adipose tissue pool by 30% whereas the high grain restricted roughage diet increased the turnover tirne by 26%. Estimates of the effects of dietary treatments on rate constants of fatty acid iransfer in a 2-pool model are presented.Low fat and high grain restricted roughage diets reduced plasma free fatty acids (P<0.01 and 0.001). The high grain restricted rouglhage diet increased plasma glucose and serum heparin-precipitable lipoprotein esters (P<0.01 and 0.001).