A simple and cost-effective protocol for high-yield expression of deuterated and selectively isoleucine/leucine/valine methyl protonated proteins in Escherichia coli grown in shaker flasks.

A simple and cost-effective protocol is presented for expression of perdeuterated, Ile/Leu/Val 1H/13C methyl protonated proteins from 100 ml cultures in M9 ++ /D2O medium induced at high (OD600 ~ 10) cell density in shaker flasks. This protocol, which is an extension of our previous protocols for expression of 2H/15N/13C and 1H/13C labeled proteins, yields comparable quantities of protein from 100 ml cell culture to those obtained using a conventional 1 L culture with M9/D2O medium, while using three-fold less α-ketoisovaleric (1,2,3,4-13C4; 3,4',4',4'-d4) and α-ketobutyric (13C4; 3,3-d2) acid precursors.

Application of Bayesian analysis to the doubly labelled water method for total energy expenditure in humans.

RATIONALE: The doubly labelled water (DLW) method is the reference method for the estimation of free-living total energy expenditure (TEE). In this method, where both 2 H and 18 O are employed, different approaches have been adopted to deal with the non-conformity observed regarding the distribution space for the labels being non-coincident with total body water. However, the method adopted can have a significant effect on the estimated TEE. METHODS: We proposed a Bayesian reasoning approach to modify an assumed prior distribution for the space ratio using experimental data to derive the TEE. A Bayesian hierarchical approach was also investigated. The dataset was obtained from 59 adults (37 women) who underwent a DLW experiment during which the 2 H and 18 O enrichments were measured using isotope ratio mass spectrometry (IRMS). RESULTS: TEE was estimated at 9925 (9106-11236) [median and interquartile range], 9646 (9167-10540), and 9,638 (9220-10340) kJ·day-1 for women and at 13961 (12851-15347), 13353 (12651-15088) and 13211 (12653-14238) kJ·day-1 for men, using normalized non-Bayesian, independent Bayesian and hierarchical Bayesian approaches, respectively. A comparison of hierarchical Bayesian with normalized non-Bayesian methods indicated a marked difference in behaviour between genders. The median difference was -287 kJ·day-1 for women, and -750 kJ·day-1 for men. In men there is an appreciable compression of the TEE distribution obtained from the hierarchical model compared with the normalized non-Bayesian methods (range of TEE 11234-15431 kJ·day-1 vs 10786-18221 kJ·day-1 ). An analogous, yet smaller, compression is seen in women (7081-12287 kJ·day-1 vs 6989-13775 kJ·day-1 ). CONCLUSIONS: The Bayesian analysis is an appealing method to estimate TEE during DLW experiments. The principal advantages over those obtained using the classical least-squares method is the generation of potentially more useful estimates of TEE, and improved handling of outliers and missing data scenarios, particularly if a hierarchical model is used.

Historical and contemporary stable isotope tracer approaches to studying mammalian protein metabolism.

Over a century ago, Frederick Soddy provided the first evidence for the existence of isotopes; elements that occupy the same position in the periodic table are essentially chemically identical but differ in mass due to a different number of neutrons within the atomic nucleus. Allied to the discovery of isotopes was the development of some of the first forms of mass spectrometers, driven forward by the Nobel laureates JJ Thomson and FW Aston, enabling the accurate separation, identification, and quantification of the relative abundance of these isotopes. As a result, within a few years, the number of known isotopes both stable and radioactive had greatly increased and there are now over 300 stable or radioisotopes presently known. Unknown at the time, however, was the potential utility of these isotopes within biological disciplines, it was soon discovered that these stable isotopes, particularly those of carbon (13 C), nitrogen (15 N), oxygen (18 O), and hydrogen (2 H) could be chemically introduced into organic compounds, such as fatty acids, amino acids, and sugars, and used to "trace" the metabolic fate of these compounds within biological systems. From this important breakthrough, the age of the isotope tracer was born. Over the following 80 yrs, stable isotopes would become a vital tool in not only the biological sciences, but also areas as diverse as forensics, geology, and art. This progress has been almost exclusively driven through the development of new and innovative mass spectrometry equipment from IRMS to GC-MS to LC-MS, which has allowed for the accurate quantitation of isotopic abundance within samples of complex matrices. This historical review details the development of stable isotope tracers as metabolic tools, with particular reference to their use in monitoring protein metabolism, highlighting the unique array of tools that are now available for the investigation of protein metabolism in vivo at a whole body down to a single protein level. Importantly, it will detail how this development has been closely aligned to the technological development within the area of mass spectrometry. Without the dedicated development provided by these mass spectrometrists over the past century, the use of stable isotope tracers within the field of protein metabolism would not be as widely applied as it is today, this relationship will no doubt continue to flourish in the future and stable isotope tracers will maintain their importance as a tool within the biological sciences for many years to come. © 2016 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Mass Spec Rev.

Use of deuterium labeling by high-temperature solid-state hydrogen-exchange reaction for mass spectrometric analysis of bradykinin biotransformation.

RATIONALE: Studies of molecular biodegradation by mass spectrometry often require synthetic compounds labeled with stable isotopes as internal standards. However, labeling is very expensive especially when a large number of compounds are needed for analysis of biotransformation. Here we describe an approach for qualitative and quantitative analysis using bradykinin (BK) and its in vitro degradation metabolites as an example. Its novelty lies in the use of deuterated peptides which are obtained by a high-temperature solid-state exchange (HSCIE) reaction. METHODS: Deuterated and native BK were analyzed by positive electrospray ionization high-resolution mass spectrometry (ESI-HRMS) using an Orbitrap Fusion mass spectrometer. High-energy collision-induced dissociation (HCD) experiments were performed on [M+H](+) and [M+2H](2+) ions in targeted-MS(2) mode with adjusted normalized HCD value. RESULTS: After the HSCIE reaction, each amino acid residue of the deuterated peptide contained deuterium atoms and the average degree of substitution was 5.5 atoms per the peptide molecule. The deuterated peptide demonstrated the same chromatographic mobility as the unlabeled counterpart, and lack of racemization during substitution with deuterium. Deuterium-labeled and unlabeled BKs were incubated with human plasma and their corresponding fragments BK(1-5) and BK(1-7), well known as the major metabolites, were detected. CONCLUSIONS: Quantitative assays demonstrated applicability of the heavy peptide for both sequencing and quantification of generated fragments. Applicability of the HSCIE deuterated peptide for analysis of routes of its degradation has been shown in in vitro experiments. Copyright © 2016 John Wiley & Sons, Ltd.

Maximal sustained levels of energy expenditure in humans during exercise.

UNLABELLED: Migrating birds have been able to sustain an energy expenditure (EE) that is five times their basal metabolic rate. Although humans can readily reach these levels, it is not yet clear what levels can be sustained for several days. PURPOSE: The study's purposes were 1) to determine the upper limits of human EE and whether or not those levels can be sustained without inducing catabolism of body tissues and 2) to determine whether initial body weight is related to the levels that can be sustained. METHODS: We compiled data on documented EE as measured by doubly labeled water during high levels of physical activity (minimum of five consecutive days). We calculated the physical activity level (PAL) of each individual studied (PAL = total EE / basal metabolic rate) from the published data. Correlations were run to examine the relationship between initial body weight and body weight lost with both total EE and PAL. RESULTS: The uppermost limit of EE was a peak PAL of 6.94 that was sustained for 10 consecutive days of a 95-d race. Only two studies reported PALs above 5.0; however, significant decreases in body mass were found in each study (0.45-1.39 kg·wk(-1) of weight loss). To test whether initial weight affects the ability to sustain high PALs, we found a significant positive correlation between TEE and initial body weight (r = 0.46, P < 0.05) but no correlation between PAL and body weight (r = 0.27, not statistically significant). CONCLUSIONS: Some elite humans are able to sustain PALs above 5.0 for a minimum of 10 d. Although significant decreases in body weight occur at this level, catabolism of body tissue may be preventable in situations with proper energy intake. Further, initial body weight does not seem to affect the sustainability of PALs.

An investigation of a novel three-dimensional activity monitor to predict free-living energy expenditure.

The aim of this study was to assess the capability of the 3dNX accelerometer to predict energy expenditure in two separate, free-living cohorts. Twenty-three adolescents and 14 young adults took a single dose of doubly labelled water and wore a 3dNX activity monitor during waking hours for a 10-day period while carrying out their normal routines. Multiple linear regression with backward elimination was used to establish the strength of the associations between various indices of energy expenditure, physical activity counts, and anthropometric variables. 3dNX output accounted for 27% and 35% of the variance in the total energy expenditure of the adolescent and young adult cohort, respectively. The explained variance increased to 78%, with a standard error of estimate of 7%, when 3dNX output was combined with body composition variables. The 3dNX accelerometer can be used to predict free-living daily energy expenditure with a standard error of estimate of 1.65 MJ in adolescents and 1.52 MJ in young adults. The inclusion of anthropometric variables reduces the error to approximately 1 MJ. Although it remains to cross-validate these models in other populations, early indications suggest that the 3dNX provides a useful method of predicting energy expenditure in free-living individuals.

Effect of late-onset sepsis on energy expenditure in extremely premature infants.

The purpose of this study was to compare total energy expenditure (TEE) in extremely premature infants during and after an episode of sepsis. We hypothesized that TEE in the sepsis group (SEP) would be higher during the septic period and higher than an age-matched control group (CTL). We further hypothesized that the TEE of the SEP group during the recovery period would be similar to that of the CTL group. The doubly labeled water method was used to determine TEE in both groups. Infant characteristics were as follows: SEP group, n = 10, gestation = 26 +/- 1 wk, birth weight = 854 +/- 218 g; CTL group, n = 10, gestation = 26 +/- 1 wk, birth weight = 880 +/- 158 g. TEE of the SEP group during the septic period was significantly greater than during the recovery period (96 +/- 25 kcal/kg/d versus 55 +/- 17 kcal/kg/d) and significantly greater than the CTL group during the first study period (96 +/- 25 kcal/kg/d versus 67 +/- 12 kcal/kg/d). TEE in the SEP group during the recovery period was similar to the CTL group. These increases in TEE may contribute to impaired growth and need to be considered when providing nutritional support for extremely premature infants.

Assessment of postprandial glucose metabolism: conventional dual- vs. triple-tracer method.

The dual-tracer method has been used conventionally for assessment of postprandial fluxes, i.e., appearance in plasma of ingested glucose (R(a meal)), endogenous glucose production (EGP), and disposal (R(d)). To quantify the magnitude of errors affecting the calculations and their dependence on model assumptions, this method was assessed and compared with the triple-tracer method, which provides model-independent estimates. For this purpose, the dual-tracer protocol was performed twice in eight normal subjects, with [1-(13)C]glucose to trace ingested glucose and [6,6-(2)H(2)]glucose constantly infused. A third tracer, [6-(3)H]glucose, was infused at variable rates to render the calculation of R(a meal) and EGP virtually model independent. The dual-tracer method analyzed with a one-compartment model performed poorly, since R(a meal) peak was significantly lower and delayed compared with triple-tracer reference, resulting in a significantly lower estimation of the amount of absorbed glucose (9,036 +/- 558 vs. 11,316 +/- 823 micromol/kg, P = 0.0117). EGP showed a paradoxical pattern, with an initial overshoot followed by a rapid decay to negative values, resulting in a significant underestimation of EGP suppression (57 +/- 3 vs. 65 +/- 4%, P = 0.0117). A two-compartment model performed better but did not overcome the limitations of the dual-tracer approach, since the amount of absorbed glucose was still significantly underestimated (10,231 +/- 661 vs. 12,169 +/- 838 micromol/kg, P = 0.0117) and EGP still showed a paradoxical behavior. R(d), estimated from R(a meal) and EGP, was significantly underestimated with the dual-tracer method, irrespective of adopted model. We conclude that three suitably infused tracers are required for accurate assessment of postprandial R(a meal), EGP, and R(d).

Assessment of the amount of body water in the Red Knot (Calidris canutus): an evaluation of the principle of isotope dilution with 2H, (17)O, and (18)O as measured with laser spectrometry and isotope ratio mass spectrometry.

We have used the isotope dilution technique to study changes in the body composition of a migratory shorebird species (Red Knot, Calidris canutus) through an assessment of the amount of body water in it. Birds were quantitatively injected with a dose of water with elevated concentrations of 2H, (17)O, and (18)O. Thereafter, blood samples were taken and distilled. The resulting water samples were analysed using an isotope ratio mass spectrometry (for 2H and (18)O only) and a stable isotope ratio infrared laser spectrometry (2H, (17)O, and (18)O) to yield estimates of the amount of body water in the birds, which in turn could be correlated to the amount of body fat. Here, we validate laser spectrometry against mass spectrometry and show that all three isotopes may be used for body water determinations. This opens the way to the extension of the doubly labelled water method, used for the determination of energy expenditure, to a triply labelled water method, incorporating an evaporative water loss correction on a subject-by-subject basis or, alternatively, the reduction of the analytical errors by statistically combining the (17)O and (18)O measurements.

Total energy expenditure estimated using a foot-contact pedometer.

BACKGROUND: Total energy expenditure (TEE) assessment using pedometers provide an easy and less expensive method than doubly labeled water (DLW). This study assessed TEE by a new pedometry method (TEEpedo) compared to the doubly labeled water method (TEEdlw). MATERIAL/METHODS: Shipboard sailors (7 men, age: 23.0+/-3.9 yrs; ht: 180.2+/-6.5 cm; wt: 83.8+/-11.8 kg, and 10 women, age: 24.7+/-4.4 yrs; ht: 165.2+/-8.0 cm; wt: 63.5+/-14.0 kg) (Mean +/-SD) were studied for 8 days. The energy cost of activity was estimated using (a) total body weight, (b) foot-ground contact times [Tc] during running, walking, and non-exercise activity [NEAT], and (c) the known proportion of time spent in each activity category. Resting metabolic rate (RMR) was estimated from lean body mass. RESULTS: TEEPEDO was calculated as: TEEpedo (MJ) = (1440 x [%Run Time x ((0.0761 x [Total Body Weight/TcRun]) - 7.598) +%Walk Time x ((0.056 x [Total Body Weight/TcWalk]) - 2.938) + (%NEAT Time x 0.1 x [RMR/Minute])] + RMR)/239. This method, explained 79% of the variance of TEEpedo with a 95% confidence interval of +/-0.81 MJ/day, relative to TEEdlw (12.55+/-3.3MJ/day). Mean TEEpedo (12.65+/-3.1 MJ/day) did not differ from mean TEEdlw (p=0.95). CONCLUSIONS: At TEEs >14 MJ/day, the TEEpedo method underestimated actual TEE, possibly due to unaccounted for upper body exercise. At more moderate TEEs of 9 to 14 MJ/day, the Tc pedometry method provided accurate estimates of TEE.

Validation of three alternative methods to measure total energy expenditure against the doubly labeled water method for older Japanese men.

In a previous study using young Japanese men as subjects, Ebine et al. found that accelerometer (AC) represents a promising technique for measuring free-living total energy expenditure (TEE) when compared to activity records (AR) and heart rate monitoring (HR). Thus, the present study was designed to validate the use of an AC and to determine whether or not the previous findings regarding the three alternative field methods (AC, AR, and HR) could be extended to older Japanese men (n = 24; mean +/- SD age 48 +/- 10 y, body mass index 23.1 +/- 2.7 kg/m2 and body fat 18.7 +/- 4.8%). TEE values obtained over a 3 d period by AR, HR, and AC (3dAC), and AC over a 14 d period (14dAC) were simultaneously validated against TEE measured by the doubly labeled water (DLW) method applied within a 14 d period. TEE values obtained by AR, HR, 3dAC, and 14dAC ranged from 1,750 to 3,447 kcal/d, 1,691 to 5,286 kcal/d, 1,716 to 2,765 kcal/d, and 1,700 to 2,855 kcal/d, respectively. Expenditures obtained by HR were similar to those obtained using the DLW method, with a mean difference of 57 +/- 603 kcal/d (2%), but those obtained using AR, 3dAC, and 14dAC differed substantially from the DLW method, with mean differences of -335 +/- 289 kcal/d (12%), -542 +/- 249kcal/d (-19%), and -566 +/- 223kcal/d (-20%), respectively. AR, HR, 3dAC, and 14dAC were significantly correlated with the DLW method, with r values of 0.76 (p < 0.0001), 0.67 (p < 0.001), 0.78 (p < 0.0001), and 0.83 (p < 0.0001), respectively. Intra-individual variation indicated by the coefficient of variation (CV) was significantly higher for HR (15 +/- 11%, p < 0.001) than for AR (7 +/- 4%), 3dAC (7 +/- 5%), and 14dAC (8 +/- 31%). The same findings were obtained using Bland and Altman plots at the population level. Interestingly, 3dAC and 14dAC were significantly correlated with r = 0.97 (p < 0.0001), with a lower mean difference of 24 kcal/d. These results suggest that, same as the previous study, AC is superior to HR in estimating TEE, and seems to be satisfactory for estimation at both group and individual levels, particularly for large-scale studies of older individuals when compared to the DLW method. However, some modifications of the AC method may be needed to compensate for the underestimation of TEE.

Energy intake by multiple pass 24 h recall and total energy expenditure: a comparison in a representative sample of 3-4-year-olds.

The accuracy of the multiple pass 24 h recall for assessment of habitual energy intake in pre-school children is unclear. The primary aim of this study was to assess its accuracy by comparison with measurement of total energy expenditure by doubly-labelled water in a representative sample of forty-one 3-4-year-olds. The recall method was well tolerated by subjects and was administered quickly and easily. However, it produced estimates of energy intake which significantly exceeded measures of total energy expenditure from doubly-labelled water, mean paired difference 660 kJ/d (P<0.01). Agreement between the two methods was poor at the individual level: limits of agreement 660+/-3018 kJ/d. Error and imprecision in both methods contribute to individual differences, but the present study suggests that the multiple pass 24 h recall does not provide accurate estimates of dietary energy intake in individual children.

Assessment of the role of an omega loop of cholesterol oxidase: a truncated loop mutant has altered substrate specificity.

The function of an active site loop (70-90) of cholesterol oxidase has been ascertained by deleting five contiguous residues (79-83) from the tip of the loop. From the crystal structure of the wild-type enzyme, it appears that this truncation will not significantly perturb the structure of the rest of the enzyme. The UV/vis and CD spectra of the mutant confirm that the enzyme is properly folded with FAD bound. The mutant enzyme still transfers 2H from the 4beta-carbon of the intermediate, cholest-5-en-3-one, to the 6beta-carbon of the product, cholest-4-en-3-one, during isomerization. The kcat/Km of the mutant is increased 6-fold with dehydroepiandrosterone as substrate. Thus, the enzyme is still catalytically active after deletion of the five loop-tip residues. With micellar cholesterol, the kcat/Km of the mutant is decreased 170-fold relative to wild type. This suggests that the tip of the loop is necessary for packing with the "tail" of cholesterol and is responsible for substrate specificity at C17. Increased release of intermediate cholest-5-en-3-one in the mutant-catalyzed reaction is not observed. Truncation of the loop, therefore, does not affect the grip of the enzyme on the intermediate. With lipid vesicle substrates (egg phosphatidylcholine/cholesterol, 1:1), the initial velocity of the mutant is reduced 3000-fold. The binding affinity for the vesicles, however, is only reduced 2-fold. Consequently, the loop is not the primary determinant of binding affinity for vesicles. It is concluded that the loop is important for movement of cholesterol from the lipid bilayer. The tip residues form a hydrophobic pathway between lipid membrane and active site to facilitate movement of substrate and product in to and out of the active site.

Interactions between 2H and 18O natural abundance variations and DLW measurements of energy expenditure.

Appropriate corrections for 2H and 18O natural abundance are necessary in energy expenditure measurements with the doubly labeled water (DLW) method. The contribution of natural abundance variations to errors in the method is generally ignored if an appropriate dose level is given. Calculation of the appropriate dose level assumes that 2H and 18O natural abundance values are covariant and have a fixed slope. This study has investigated the between- and within-subject natural abundance variations, the latter over periods of time similar to those used in DLW experiment. Although 2H and 18O natural abundance values are covariant between subjects, the slope of their relationship is different from that of the Meteoric Water Line. Within subjects there is no such demonstrable covariance. It is concluded that: 1) the determinants of 2H and 18O natural abundance are different within and between subjects; 2) the concept of an optimal ratio of doses is not valid, and a safer strategy is probably to use a ratio > 10; 3) natural abundance variations may contribute significantly to the magnitude of error in the DLW measurements of energy expenditure.

Total energy expenditure in burned children using the doubly labeled water technique.

Total energy expenditure (TEE) was measured in 15 burned children with the doubly labeled water technique. Application of the technique in burned children required evaluation of potential errors resulting from nutritional intake altering background enrichments during studies and from the high rate of water turnover relative to CO2 production. Five studies were discarded because of these potential problems. TEE was 1.33 +/- 0.27 times predicted basal energy expenditure (BEE), and in studies where resting energy expenditure (REE) was simultaneously measured, TEE was 1.18 +/- 0.17 times REE, which in turn was 1.16 +/- 0.10 times predicted BEE. TEE was significantly correlated with measured REE (r2 = 0.92) but not with predicted BEE. These studies substantiate the advantage of measuring REE to predict TEE in severely burned patients as opposed to relying on standardized equations. Therefore we recommend that optimal nutritional support will be achieved in convalescent burned children by multiplying REE by an activity factor of 1.2.

Validation of doubly labeled water for measuring energy expenditure during parenteral nutrition.

The doubly labeled water method was compared with intake-balance for measuring energy expenditure in five patients receiving total parenteral nutrition (TPN). Because parenteral solutions were isotopically different from local water, patients had to be placed on TPN at least 10 days before the metabolic period. Approximately 0.1 g 2H2O and 0.25 g H2(18)O per kg total body water were given orally. We collected saliva before, 3 h, and 4 h after the dose for measurement of total body water and urine before, 1 day, and 14 days after the dose for measurement of isotope eliminations. On day 14, total body weight was remeasured and change in body energy stores was calculated, assuming constant hydration. Intake was assessed from weights of TPN fluids plus dietary record for any oral intake. Energy expenditure from doubly labeled water (+/- SD) averaged 3 +/- 6% greater than intake-balance. Doubly labeled water method is a noninvasive, nonrestrictive method for measuring energy expenditure in patients receiving TPN.