An evaluation of four methods for measuring cholesterol absorption by the intestine in man

Critical comparisons have been made in 12 patients of four methods for measuring cholesterol absorption from the intestine. Methods I-III depend on the use of labeled cholesterol (intravenously or continuous labeling orally) in conjunction with sterol balance measurements; Method IV can be carried out with only a single test dose containing labeled cholesterol plus labeled beta-sitosterol. In the latter technique absorption is calculated as the loss of cholesterol relative to beta-sitosterol during intestinal transit. Method III (isotopic steady-state method) proved to be undependable because of uncertainties in determining the existence of an isotopic steady state. However, Method IV gave good agreement with Methods I and II, and it appears to have certain practical as well as theoretical advantages. Although Method IV requires collections of stools for up to 8 days, it is nevertheless the most rapid and the simplest of all the methods for estimating absorption. It can also be used in certain situations, such as in fur-licking animals, when Methods I and II are inadequate. Therefore, this method would seem to be a valuable addition to other isotopic techniques for estimating cholesterol absorption in man.     

Sources of error in the isotopic cholesterol balance method in African green monkeys consuming a cholesterol-free diet

Six African green monkeys were labeled intravenously with [1,2-(3)H]cholesterol while consuming a cholesterol-free liquid formula diet. The plasma cholesterol specific activity was compared with the specific activity of the biliary cholesterol and bile acids and with the fecal neutral steroids in order to determine whether the traditional isotopic balance method was valid for the calculation of endogenous cholesterol excretion. The specific activity of biliary cholesterol and bile acids averaged 10-15% lower than plasma cholesterol specific activity. Fecal cholesterol and coprostanone specific activities were similar to that of the biliary cholesterol, but the specific activity of fecal coprostanol was approximately 25% lower. This suggests that biliary cholesterol and bile acids were derived from a pool of hepatic cholesterol that did not completely equilibrate with the whole body exchangeable cholesterol pool. In addition, there was further reduction in the specific activity of coprostanol, the major fecal neutral steroid, presumably by cholesterol synthesized in the lower intestine and preferentially converted to coprostanol. As a result, the traditional isotopic balance procedure underestimated endogenous neutral steroid excretion by 46% and bile acid excretion by 31% in African green monkeys fed the cholesterol-free diet. Within 7 days after the addition of 1 mg cholesterol/kcal to the diet, the specific activities of plasma and biliary cholesterol and biliary bile acids were identical and there was no difference in the specific activities of the individual fecal neutral steroids. Thus, the traditional isotopic balance procedure (DPM fecal neutral steroids + bile acids/specific activity [DPM/mg] plasma cholesterol) can be used for calculation of endogenous cholesterol excretion in cholesterol-fed animals during the nonsteady state when plasma cholesterol concentrations are rapidly increasing, as well as after a new steady state has been achieved.-Henderson, G. R., and R. W. St. Clair. Sources of error in the isotopic cholesterol balance method in African green monkeys consuming a cholesterol-free diet.     

Direct measurement of apolipoprotein B synthesis in human very low density lipoprotein using stable isotopes and mass spectrometry

Stable isotope methodology has been adapted to the study of lipoprotein turnover in human subjects. Using endogenous [15N]glycine labeling and gas-liquid chromatographic-mass spectrometric analysis, synthesis of apolipoprotein B in very low density lipoprotein (VLDL) was measured directly in five normal and two hyperlipidemic subjects. An isotopic precursor steady state was achieved during the studies by utilizing a priming dose and constant infusion containing [15N]glycine. Measurement of the plateau in 15N enrichment in the urinary hippurate produced during each study was used to estimate the 15N enrichment of the hepatic glycine precursor pool. The range of values for the fractional synthetic rate of VLDL apoB in the normal subjects obtained by this method was 5.9 to 11.5 day-1, with a mean of 9.2 +/- 2.4 (SD). This value agrees with the results of previous investigations which have utilized other methods. The method was also tested in two hypertriglyceridemic subjects and gave fractional synthetic rates of VLDL apoB that were significantly lower than in normals (1.5 and 2.8 day-1). This stable isotope method allows calculation of the fractional synthetic rate of VLDL apoB by maintaining an isotopic steady state throughout the study. It makes possible repeated studies in the same individual since no risk of exposure to radioisotopes is involved.     

Cholesterol catabolism in the rabbit in fasted and fed states.

Urinary and fecal endogenous steroid excretion of fed or fasted New Zealand white rabbits was determined by the isotopic steady state method after subcutaneous implantation of radioactive cholesterol. While plasma cholesterol was increasing during a 9-day fast, fecal steroid excretion decreased to 10% of the excretion rates in the fed state. Refeeding the fasted rabbits led to a decrease in plasma cholesterol and an increase in fecal endogenous steroid excretion. Urinary steroid excretion, which represented 18% of total endogenous steroid excretion for fed animals, decreased during fasting and increased during refeeding, but these changes were relatively small. The small intestine, cecum, and colon of fed or fasted rabbits had similar endogenous steroid was acidic steroid. During attempts to alter the circulating bile acid concentration by supplying deoxycholate (200 mg/day) to fed rabbits or cholestyramine (2 g/day) to fasted rabbits, plasma cholesterol concentration did not change to the same extent as during fasting or refeeding, respectively. The decreased cholesterol catabolism and the hypercholesterolemia that are seen in the fasting rabbit may result from decreased clearance of plasma cholesterol.     

Formation of cholic acid via 3 alpha, 7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid in the dog.

The proposed cholic precursor, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-[3H]cholestan-26-oic acid, and [14C]cholesterol were infused intravenously at a constant rate into two dogs for 25 days. If the specific activities of trihydroxy[3H]cholestanoic acid and [3H]cholic acid will be equal after an isotopic steady-state is achieved. The specific activities of [14C]deoxycholic acid (formed from [14C]cholic acid) isolated in the stool of these two dogs were equal the last four days of the infusion indicating that labeled deoxycholic acid (and presumably labeled cholic acid) was in an isotopic steady-state. However, the specific activities of trihydroxy[3H]cholestanoic acid were 3.3 and 5.7 times greater than the specific activities of [3H]cholic acid, respectively. These data suggest that either an alternate route of cholic acid synthesis exists exclusive of trihydroxycholestanoic acid or that an isotopic steady state of trihydroxycholestanoic acid cannot be reached during an infusion of labeled trihydroxycholestanoic acid.     

Validation of a dual-isotope plasma ratio method for measurement of cholesterol absorption in rats

Several methods for measuring cholesterol absorption in the rat have been compared. After administration of an oral dose of labeled cholesterol ((14)C or (3)H) and an intravenous dose of colloidal labeled cholesterol ((3)H or (14)C) the ratio of the two labels in plasma or whole blood 48 hr or more after dosing compared closely to the ratio of areas under the respective specific activity-time curves. The area ratio method is independent of a time lag between the appearance of oral and intravenous label in the bloodstream. Both measures of cholesterol absorption agree fairly well with a method based on measuring the unabsorbed dietary cholesterol in a pooled fecal sample. The plasma isotope ratio method gave more reproducible results than the fecal collection method when the measurement was repeated in the same animals 5 days after the first measurement. Cholesterol absorption was overestimated by the use of Tween 20-solubilized labeled cholesterol for the intravenous dose. The plasma disappearance curves of injected labeled colloidal cholesterol and cholesterol-labeled chylomicrons infused intravenously over a 3.5-hr period in the same animal coincided within experimental error from the first day until 75 days after injection. The plasma isotope ratio method for cholesterol absorption gave the same results in rats practicing coprophagy as in those in which this practice was prevented. The addition of sulfaguanidine to the diet lowered cholesterol absorption as measured by the plasma isotope ratio to the same degree as that measured by the fecal collection method.     

Accumulation of cholesterol in Acholeplasma laidlawii membranes in the steady-state phase of culture growth

In early logarithmic phase of growth of the A. laidlawii cells the lipid composition of plasma membrane is changed: the total lipid, glycolipid and phospholipid contents are decreased, while that of cholesterol changes only insignificantly. In late logarithmic and steady state phases the cholesterol level in the membrane is increased in parallel with the decrease of the phospholipid content. Throughout the growth period a quantitative redistribution of membrane phospholipids in fatty acids and an increase of the molar content of saturated fatty acids are observed. Accumulation of cholesterol in the steady state phase is accompanied by an increase in the membrane viscosity which results in inhibition of membrane processes in the cell.     

Placental transfer of cholesterol-4-14C into rabbit and guinea pig fetus

A tracer dose of cholesterol-4-(14)C was given daily in the diet of six pregnant guinea pigs to establish an isotopic steady state. At the time of parturition, maternal and fetal blood and fetal tissues were collected and analyzed for cholesterol content and cholesterol specific activity. A comparison of these specific activities in neonatal and maternal serum indicated that about 22% of the fetal serum cholesterol was transferred from maternal blood. In the newborn, tissues generally had the same cholesterol specific activity as serum. Brain tissue was an exception in having a specific activity only 8.4% of that of serum. Dietary cholesterol did not increase serum cholesterol levels in the newborn but did increase the percentage of fetal cholesterol derived from the maternal circulation. The rapid transfer of cholesterol-4-(14)C across the placenta was indicated by the appearance of this isotope in the newborn 2 days after its administration to pregnant rabbits. A considerable amount of the cholesterol content of newborn guinea pigs and rabbits originated from the maternal blood.     

Measurements of cholesterol turnover, synthesis, and absorption in man, carried out by isotope kinetic and sterol balance methods

We have estimated the daily synthesis of cholesterol in man by measuring the excretion of cholesterol and its conversion products during periods of controlled sterol intake (sterol balance method), using isotopic or chromatographic procedures (or a combination of the two). Estimates of daily synthesis by this method are based on the premise that the subject is in the metabolic steady state; i.e., the synthesis of cholesterol is equal to the balance (or difference) between the intake of cholesterol and the excretion of cholesterol and its products. To test this premise, we carried out sterol balances in 11 patients; simultaneously, after administration of isotopic cholesterol, turnover was calculated according to previously described models (one-pool, two-pool, or isotopic steady state models for the distribution of radioactive cholesterol within various pools of the body). With calculations based on the one-pool model, turnover rates were considerably higher than estimates based on all other models, and reasons are given for considering these to be overestimates. Good agreement was obtained between results calculated from the two-pool model and those based on sterol balance data; neither method is theoretically preferable to the other. However, with the sterol balance method supplemented by isotopic techniques, valid measurements of cholesterol absorption can be obtained; this in turn permits the essential distinction to be made between daily synthesis and daily turnover of cholesterol when the diet contains cholesterol. In addition, the use of chromatographic isolation procedures provides an accurate measurement of the balance of -sitosterol. This in turn permits valid corrections to be made for losses (which may be large) of neutral steroids during intestinal transit; this is a unique advantage of the chromatographic method.     

Steady state analysis and experimental validation of a model for hepatic high-density lipoprotein transport

Transport of high-density lipoprotein (HDL) in the hepatocyte plays a fundamental role in reverse cholesterol transport and regulation of plasma HDL levels. On the basis of a recently developed kinetic model, the steady state distribution of HDL was analyzed. Fractional fluorescence of labeled HDL in the basolateral membrane, sorting endosomes (SE), the subapical compartment/ apical recycling compartment, the biliary canaliculus and in late endosomes and lysosomes (LE/LYS) including expected standard deviation is predicted. Improved parameter estimation was obtained by including kinetic data of apical endocytosis of fluorescent markers for LE/LYS, asialoorosomucoid and Rhodamine-dextran, in the regression. Predicted values using the refined kinetic parameters are in good agreement with experimental values of compartmental steady state fluorescence of Alexa488-HDL in polarized hepatic HepG2 cells. From calculated steady state fluxes, it is suggested that export of HDL from basolateral SE is the key step for determining the transport of HDL through the hepatocyte. The analysis provides testable predictions for high-throughput fluorescence microscopy screening experiments on potential inhibitors of hepatic HDL processing. By quantitative fluorescence imaging and model analysis, it is shown that the phosphoinositide kinase inhibitor wortmannin prevents apical transport of fluorescent HDL from basolateral SE. The results support that endosomes of polarized hepatic cells have different sorting functions and that apical endocytosis is an integrative trafficking step in hepatocytes.     

Biosynthesis of cholesterol in rabbit reticulocytes and its exchange with plasma

When rabbit reticulocytes were incubated in normal blood plasma containing mevalonic acid-2-(14)C, radioactivity was incorporated into cholesterol, cholesteryl esters, and squalene in the cells. The squalene reached a steady level of radioactivity much more rapidly than did cholesterol. Rabbit reticulocytes which were labeled as a result of previous incubation with mevalonic acid-2-(14)C were incubated with normal autologous blood plasma. The specific activity of the cholesterol in the plasma rapidly became higher than that of the cells. This suggests that there is compartmentation of cholesterol in the reticulocyte and that a pool involved in exchange with plasma cholesterol has a specific activity which is much higher than the average for the whole cell.     

Quantification of hepatic carbohydrate metabolism in conscious mice using serial blood and urine spots

In vivo studies of hepatic carbohydrate metabolism in (genetically modified) conscious mice are hampered by limitations of blood and urine sample sizes. We developed and validated methods to quantify stable isotope dilution and incorporation in small blood and urine samples spotted onto filter paper. Blood glucose and urinary paracetamol-glucuronic acid were extracted from filter paper spots reproducibly and with high yield. Fractional isotopomer distributions of glucose and paracetamol-glucuronic acid when extracted from filter paper spots were almost identical to those isolated from the original body fluids. Rates of infusion of labeled compounds could be adjusted without perturbing hepatic glucose metabolism. This approach was used in mice to find the optimal metabolic condition for the study of hepatic carbohydrate metabolism. In fed mice, no isotopic steady state was observed during a 6-h label-infusion experiment. In 9-h-fasted mice, isotopic steady state was reached after 3 h of label infusion and important parameters in hepatic glucose metabolism could be calculated. The rate of de novo glucose-6-phosphate synthesis was 143 +/- 17 micromol kg(-1) min(-1) and partitioning to plasma glucose was 79.0 +/- 5.2%. In 24-h-fasted mice, abrupt changes were noticed in whole body and in hepatic glucose metabolism at the end of the experiment.     

Isotopic non-stationary 13C gluconate tracer method for accurate determination of the pentose phosphate pathway split-ratio in Penicillium chrysogenum

Current (13)C labeling experiments for metabolic flux analysis (MFA) are mostly limited by either the requirement of isotopic steady state or the extremely high computational effort due to the size and complexity of large metabolic networks. The presented novel approach circumvents these limitations by applying the isotopic non-stationary approach to a local metabolic network. The procedure is demonstrated in a study of the pentose phosphate pathway (PPP) split-ratio of Penicillium chrysogenum in a penicillin-G producing chemostat-culture grown aerobically at a dilution rate of 0.06h(-1) on glucose, using a tracer amount of uniformly labeled [U-(13)C(6)] gluconate. The rate of labeling inflow can be controlled by using different cell densities and/or different fractions of the labeled tracer in the feed. Due to the simplicity of the local metabolic network structure around the 6-phosphogluconate (6pg) node, only three metabolites need to be measured for the pool size and isotopomer distribution. Furthermore, the mathematical modeling of isotopomer distributions for the flux estimation has been reduced from large scale differential equations to algebraic equations. Under the studied cultivation condition, the estimated split-ratio (41.2+/-0.6%) using the novel approach, shows statistically no difference with the split-ratio obtained from the originally proposed isotopic stationary gluconate tracing method.     

Analysis of the distribution of cholesterol in the intact cell

We have used the enzyme cholesterol oxidase, which catalyzes the oxidation of cholesterol to cholest-4-en-3-one, to examine the distribution of cholesterol in cultured fibroblasts, Chinese hamster ovary cells, and isolated rat liver hepatocytes. While the plasma membrane normally was not attacked by cholesterol oxidase, we found that treating cells with low ionic strength buffer and glutaraldehyde rendered their cholesterol highly susceptible to oxidation. Most of the cholesterol was oxidized in all three cell types: 94% in fibroblasts, 92% in Chinese hamster ovary cells, and 80% in hepatocytes. Given that the enzyme had access only to the outer surface of the cells and cholesterol can move rapidly across the fixed plasma membrane, these values are taken to reflect the fraction of cellular cholesterol present in the plasma membrane. Additional experiments confirmed this interpretation. Fibroblasts were labeled with [3H]cholesterol by brief exposure to exogenous radiolabel and incubated with [14C]mevalonic acid to label cholesterol biosynthetically. Cholesterol oxidase attacked at least 97% of the exogenous label but as little as 10% of the biosynthetically labeled cholesterol. These data suggest that the cholesterol oxidase did not reach the intracellular pool and that cholesterol in the plasma membrane is not in rapid equilibrium with internal membranes. A study of the transfer of cholesterol to plasma from cells labeled biosynthetically with [3H]cholesterol and exogenously with [14C]cholesterol confirmed the different subcellular distribution of the two labels. These studies demonstrate that an unexpectedly high proportion of cell cholesterol is associated with plasma membranes and that this cholesterol pool can be rapidly and selectively labeled and oxidized. These features make cholesterol a useful specific marker for the plasma membrane.     

Preferential ATP-binding cassette transporter A1-mediated cholesterol efflux from late endosomes/lysosomes

Recently, ATP-binding cassette transporter A1 (ABCA1), the defective molecule in Tangier disease, has been shown to stimulate phospholipid and cholesterol efflux to apolipoprotein A-I (apoA-I); however, little is known concerning the cellular cholesterol pools that act as the source of cholesterol for ABCA1-mediated efflux. We observed a higher level of isotopic and mass cholesterol efflux from mouse peritoneal macrophages labeled with [(3)H]cholesterol/acetyl low density lipoprotein (where cholesterol accumulates in late endosomes and lysosomes) compared with cells labeled with [(3)H]cholesterol with 10% fetal bovine serum, suggesting that late endosomes/lysosomes act as a preferential source of cholesterol for ABCA1-mediated efflux. Consistent with this idea, macrophages from Niemann-Pick C1 mice that have an inability to exit cholesterol from late endosomes/lysosomes showed a profound defect in cholesterol efflux to apoA-I. In contrast, phospholipid efflux to apoA-I was normal in Niemann-Pick C1 macrophages, as was cholesterol efflux following plasma membrane cholesterol labeling. These results suggest that cholesterol deposited in late endosomes/lysosomes preferentially acts as a source of cholesterol for ABCA1-mediated cholesterol efflux.     

Transport of GABA at the Blood-CSF Interface

Abstract: The entry of GABA into cerebrospinal fluid (CSF) was studied in dogs anesthetized with pentobarbital and relaxed with suxamethonium. GABA was administered intravenously as a priming dose and subsequent maintenance infusion to compensate for the rapid elimination of the amino acid. Steady state concentrations of GABA in CSF were reached between 10 and 60 min after injection, the rate of entry tending to decrease with increasing plasma levels. During steady state conditions CSF concentrations showed great interin-dividual differences and varied between 0.03 and 5.1% of those in plasma. Probenecid and sodium valproate considerably enhanced the CSF/plasma concentration ratio of GABA. When GABA was directly injected into the liquor space, probenecid slowed down the elimination of GABA from CSF. The results suggest a transport of GABA into and out of CSF, the outward transport being inhibited by probenecid and sodium valproate.     

Evidence for different isotopic enrichments of acetyl-CoA used for cholesterol synthesis in the liver and intestine: a study in the rat by mass fragmentography after intravenous infusion of [13C]acetate

Wistar rats were killed 4 h after an intravenous infusion of [1,2-13C]- and [1-14C]acetic acid sodium salt (39 mg, 12.5 microCi/ml, constant rate: 1.2 ml/h). At this time, labeled free cholesterol movements between the organs are still weak and cholesterol labeling in each tissue mainly originates from the in situ incorporation of the exogenous substrate. In male rats, the specific radioactivity of free cholesterol was found to be higher in the intestine (mucosa and wall) than in the liver and plasma. In female and in cholestyramine-fed male rats, cholesterol 14C labeling was close to that of male rats in the intestine, and was markedly higher in the liver. The same variations of 13C excess, calculated by mass fragmentography, indicated that there was no isotopic effect between 13C and 14C precursors. The advantage of this method consisted in obtaining the proportions of labeled molecules according to their molecular weight (M + 1-M + 11) for each sample. Then the distribution of 13C atoms in newly synthesized cholesterol was assessed in each sterogenesis site. In the intestine, about 3/4 of the 13C atoms were found in molecules of weight of at least M + 4 (after incorporation of at least two labeled acetate units). This proportion was only 1/3 in hepatic and plasma free cholesterol. These distinct 13C-labeling patterns clearly indicate that local variations occurred in the isotopic enrichment of acetyl-CoA used for cholesterol formation. Whatever the experimental conditions of this study, cholesterol was synthesized from an acetyl-CoA more 13C enriched in the intestine than in the liver. Such variations probably result from the different dilutions of exogenous acetyl-CoA by the endogenous pool in the liver and intestine. Consequently, the 14C or 13C incorporations measured in the liver and intestinal sterols do not account for absolute rates of cholesterol production by these organs. This study also indicated that after a few hours of infusion, free cholesterol labeling in the plasma originated mainly from cholesterol newly formed in the liver, even when acetate incorporation into cholesterol was higher in the intestine than in the liver.     

Contribution of exogenous substrates to acetyl coenzyme A: measurement by 13C NMR under non-steady-state conditions

A method is presented for the rapid determination of substrate selection in a manner that is not restricted to conditions of metabolic and isotopic steady state. Competition between several substrates can be assessed directly and continuously in a single experiment, allowing the effect of interventions to be studied. It is shown that a single proton-decoupled 13C NMR spectrum of glutamate provides a direct measure of the contribution of exogenous 13C-labeled substrates to acetyl-CoA without measurement of oxygen consumption and that steady-state conditions need not apply. Two sets of experiments were performed: one in which a metabolic steady state but a non-steady-state 13C distribution was achieved and another in which both metabolism and labeling were not at steady state. In the first group, isolated rat hearts were supplied with [1,2-13C]acetate, [3-13C]lactate, and unlabeled glucose. 13C NMR spectra of extracts from hearts perfused under identical conditions for 5 or 30 min were compared. In spite of significant differences in the spectra, the measured contributions of acetate, lactate, and unlabeled sources to acetyl-CoA were the same. In the second set of experiments, the same group of labeled substrates was used in a regional ischemia model in isolated rabbit hearts to show regional differences in substrate utilization under both metabolic and isotopic non steady state. This sensitive probe of substrate selection was also demonstrated in intact hearts where excellent time resolution (3 min) of substrate selection was feasible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of dietary nutrients on ileal endogenous losses of threonine,cysteine, methionine,lysine, leucine and protein in broiler chicks

An isotope dose technique was utilized (i) to determine endogenous amino acid (AA) and protein losses and (ii) to propose adjusted values for AA requirements. The endogenous flow rate was calculated from the pool of enrichment in plasma AA, assuming similitude to enrichment of endogenous AA. In experiment 1, chicks were orally administered D4-lysine at 2% of estimated lysine intake from 16 to 24 days to find the isotopic steady state of the atom percent excess (APE) of lysine for plasma and jejunal and ileal digesta. The APE of D4-lysine in plasma, jejunal digesta and ileal digesta reached the isotopic steady state at 5.5, 3.4 and 2.0 days, respectively, by using the broken-line model. It was assumed that the isotopic steady state at 5 days identified for D4-lysine is also representative for the ¹⁵N-labeled AA. In experiment 2, chicks were fed diets from 1 to 21 days with increasing levels of fat (6%, 8%, 12%, 13% extract ether), protein (26%, 28.5%, 31% CP) or fiber (14%, 16%, 18% NDF) by adding poultry fat, soybean meal, blended animal protein or barley. Chicks were orally administered ¹⁵N-threonine, ¹⁵N-cysteine, ¹⁵N-methionine, ¹⁵N-lysine and ¹⁵N-leucine at 2% of estimated daily intake for 5 days from 17 to 21 days of age. Dietary nutrients influenced endogenous losses (EL), where dietary fat stimulated EL of lysine (P=0.06), leucine and protein (P=0.07); dietary protein enhanced EL of leucine and protein; and finally the dietary fiber increased EL of leucine. Dietary nutrients also affected apparent ileal digestibility (AID). Dietary fat increased AID of cysteine but decreased AID of lysine. Dietary protein reduced AID of protein, threonine, lysine and leucine, and similarly dietary fiber decreased AID of protein, threonine, methionine, lysine and leucine. In contrast, dietary fat or protein did not affect real ileal digestibility (RID) of protein and AA except threonine and leucine. The dietary fiber reduced the RID of protein, threonine and leucine. This indicate that variations of some endogenous AA and protein losses due to dietary nutrients almost eliminates the effects of RID, and thus the EL coming from the body should be utilized to adjust the AA requirement instead of changing the true digestible nutrients of ingredients. The present data suggest that 5 days’ feeding labeled AA was enough to reach the isotopic steady state and AA requirements should be adjusted when additional dietary protein, fat or fiber is fed.     

D2 18O (deuterium oxide) method for CO2 output in small mammals and economic feasibility in man.

A test of the validity of the isotopic steady state relationships of the doubly labeled water (H2O) method has been carried out with D2 18O in small mammals (three chipmunks and one mouse). CO2 outputs calculated just from 1) the rate of water intake and 2) the ratios of the isotopic concentrations in the body water to the intake water agreed satisfactorily with observed values. Moreover, reconstructed energy and material balances agreed reasonably with similar balances reconstructed for an immediately succeeding period on the same animals studied by the previously validated decay procedure. We conclude from an error analysis that by expressing the isotopic specific activities as abundances in excess of the body water of a subject on a given regimen, the decay procedure is economically feasible in the human with available accuracy of isotopic analyses and the present cost of H2 18O. The method therefore appears to be a useful tool ready for application to the field of human energy metabolism.