Metabolic Effects of HAY's Diet

Abstract

The aim of the study was, to evaluate the metabolic effect of HAY's diet on protein turnover, fat oxidation, respiratory quotient, body fat and weight loss. Twelve healthy adults received an individually regular diet and thereafter a corresponding isocaloric and isonitrogenous 10-day HAY-diet. Protein turnover and ¹³C-fat oxidation were investigated after administration of [¹⁵N]glycine and an [U-¹³C]algae lipid mixture. The ¹⁵N and ¹³C enrichment in urine and breath were measured by isotope ratio mass spectrometry. The respiratory quotient was measured by indirect calorimetry. Body fat, total body water and lean body mass were estimated by bio-electric impedance analysis. HAY's diet led to a significantly higher ¹³C-fat oxidation (15.4 vs. 22.0% P < 0.01), corresponding to a lower respiratory quotient (0.88 vs. 0.81; P < 0.01), whereas the protein turnover remained constant in both diets (3.06 vs. 3.05 g/kg/day). HAY's diet did not reduce total body water, lean body mass, body fat and body weight (72.2 vs. 71.4 kg).     

Body Water,Lean Body and Fat Mass of Healthy Children as Measured by Deuterium Oxide Dilution

Abstract

Body composition of 165 healthy children was measured using the well-established method of deuterium oxide (²H₂O) dilution. After distribution of an oral load of 2.0 ml ²H₂O/kg body weight body water was estimated from the ²H₂O concentration in urine. Lean body mass was then calculated from body water using previously published age dependent ratios of the water content of the lean body mass. Fat mass was calculated as the difference of body weight and lean body mass. A good correlation was found between body water and body weight. Linear regression revealed TBW = 0.589 BW + 0.728 (r = 0.99). Body water, lean body mass and fat mass were found to change with age. The fat content of the body increases during the first six months of life. It then decreases until four to five years then rising again until 15 years of age.     

Whole-body protein turnover in nephrotic subjects: a comparison between different degrees of proteinuria

In contrast to the increased hepatic albumin synthesis in subjects with proteinuria, little is known about the corresponding whole-body protein turnover rates (WPTR). The WPTR and the reutilisation rates (R) were investigated in 20 patients divided in three groups of different degrees of proteinuria (groups I-III: < 1, 1-3, > 3 g/m2/day, respectively). [15N]glycine was administered as a single oral pulse. Urine samples were taken over 2 days. After removing urinary proteins by trichloracetic acid, 15N-enrichment in the supernatant was measured by isotope ratio mass spectrometry. A three-compartment model was used to calculate WPTR and R, which showed a statistically significant difference between groups I and III (2.64 vs. 4.63 g/kg/day, and 70.4% vs. 80.8%, respectively, P < 0.01), whereas the net protein gain remained unchanged (0.13 vs. 0.22 g/kg/day). The higher the protein loss the higher the WPTR and the corresponding R. The severe protein loss provokes increased WPTR and R as well.     

Whole-body protein turnover in nephrotic subjects: A comparison between different degrees of proteinuria

In contrast to the increased hepatic albumin synthesis in subjects with proteinuria, little is known about the corresponding whole-body protein turnover rates (WPTR). The WPTR and the reutilisation rates (R) were investigated in 20 patients divided in three groups of different degrees of proteinuria (groups I–III: < 1, 1–3, > 3 g/m²/day, respectively). [¹⁵N]glycine was administered as a single oral pulse. Urine samples were taken over 2 days. After removing urinary proteins by trichloracetic acid, ¹⁵N-enrichment in the supernatant was measured by isotope ratio mass spectrometry. A three-compartment model was used to calculate WPTR and R, which showed a statistically significant difference between groups I and III (2.64 vs. 4.63 g/kg/day, and 70.4% vs. 80.8%, respectively, P < 0.01), whereas the net protein gain remained unchanged (0.13 vs. 0.22 g/kg/day). The higher the protein loss the higher the WPTR and the corresponding R. The severe protein loss provokes increased WPTR and R as well.     

Whole body protein metabolism estimated by (15)n tracer experiments and body composition of mice selected for different growth parameters

Abstract Whole body protein synthesis was investigated in growing male mice which were long-time selected for high carcass protein amount (DU-6P, protein line) or for high body weight (DU-6, growth line) and in the unselected randomly bred control (DU-Ks). Six mice/line were housed singly in metabolic cages for the estimation of N balance, whole body protein synthesis (end-product method, single dose of (15)N-labelled amino-acid mixture), and N distribution in the body. Another six mice/line were used for the determination of the body composition. All mice had free access to a commercial stock diet (crude protein 268 g, gross energy 19 MJ/kg dry matter) and to water. Body weight of both selection lines was about twice that of control mice at the same age. Selection for high body weight resulted in higher body fat content. Scaled to the corresponding body protein pools, the protein synthesis rates of selected mice were significantly higher than in controls, but were not significantly different between both selection lines in contrast to the protein deposition rates. The higher protein accretion in the protein line in comparison to the growth line seems to be due to a combination of a lower protein breakdown and an increased protein synthesis rate.     

Effect of alcohol consumption on whole-body protein turnover in healthy adults

The aim of the study was to investigate the whole-body protein turnover, either before or after continuous, moderate ethanol-induced oxidative stress by red wine consumption over a relatively short period in healthy volunteers. Ten healthy adults received an individual regular diet over 20 days. After 10 days, the subjects consumed 0.4 ml ethanol kg(-1) day(-1) as red wine together with dinner over a 10-day period. After 8 and 18 days, respectively, a (15)N-labelled yeast protein was administered in a dosage of 4.2 mg kg(-1) body weight. Urine and faeces were collected over 48 h, respectively. The (15)N-enrichment was measured by isotope ratio mass spectrometry, whereas the protein flux rates were calculated by a three-compartment model. The whole-body protein turnover without/with red wine consumption amounted to 3.73±0.6 and 3.49±0.6 g kg(-1) day(-1) (not significant), respectively. Moderate alcohol consumption does not induce significant short-term changes in the whole-body protein turnover of healthy adults.     

The measurement of muscle protein synthesis in broilers with a flooding dose technique: use of 15N-labelled phenylalanine, GC-MS and GC-C-IRMS

An experiment was carried out to measure fractional muscle protein synthesis rates (k(s)) in broilers with injection of a flooding dose of phenylalanine (1 ml/100 g body weight of 150 mM phenylalanine; 38 atom percent excess (APE) [15N]phenylalanine). K(s) was calculated from the [15N] enrichment in phenylalanine of tissue-free and protein-bound phenylalanine using both gas chromatography mass spectrometry (GC-MS) and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) for measurements after a 10 min isotope incorporation period. The tertiary-butyldimethylsilyl (t-BDMS) derivatives of phenylalanine were used for gas chromatographic separation in both systems. GC-MS and GC-C-IRMS were calibrated for a range of 7 to 37 [15N]APE and 0 to 0.62 [15N]APE, respectively, and for sample sizes of 0.45 to 4.5 nmol phenylalanine and 7 to 40 nmol phenylalanine, respectively. Reproducibility of standards as a measure of precision varied from 0.06 to 0.29 [15N]APE and from 0.0004 to 0.0018 [15N]APE in GC-MS and GC-C-IRMS, respectively. K(s) was measured in the m. pectoralis major of broilers fed rye based diets (56%) which were provided either unsupplemented (-) or supplemented (+) with an enzyme preparation containing xylanase. K(s) in breast muscles was significantly increased from 21.8%/d to 23.9%/d due to enzyme supplementation. It can be concluded from the study that the measurement of protein synthesis in broilers with the flooding dose technique can be carried out by using [15N]phenylalanine, GC-MS and GC-C-IRMS.     

Compartmental Approach for Evaluation of Plasma Kinetics and 13Co2-Exhalation after Oral Loading with L-[1-13C]Leucine

Abstract

A seven compartment model was applied for evaluation of oral L-[1-¹³C]leucine loading tests (38 μmol/kg body wt.) in healthy volunteers. The model comprises transport and absorption in stomach and gut into a central L-leucine-compartment which is connected to a protein compartment and to the compartment of the corresponding 2-oxo acid. CO₂ release from the latter occurs in a fast and a slow compartment into the central CO₂ compartment for exhalation. Using the fmins routine of MATLAB for parameter estimation, a good agreement was obtained between calculated and actually measured kinetics of ¹³C-labelled metabolites and a mean in vivo L-leucine oxidation of 0.365 ± 0.071 μmol/kg per min (n = 5) was computed. Plausibility of the model was checked by predicting in vivo leucine oxidation rates from primed continuous infusion tests (priming: L-[1-¹³C]leucine, 5 μmol/kg; NaH¹³CO₂, 1.2 μmol/kg; infusion: L-[1-¹³C]leucine, 5 μmol/kg per h). In 5 tested volunteers, the experimental L-leucine oxidation rate amounted to 0.358 ± 0.105 μmol/kg per min versus predicted 0.324±0.099 μmol/kg per min. Possible causes for some observed intraindividual variations are discussed.     

Compartmental approach for evaluation of plasma kinetics and (13)co(2)-exhalation after oral loading with L-[1-(13)c]leucine

Abstract A seven compartment model was applied for evaluation of oral L-[1-(13)C]leucine loading tests (38 μmol/kg body wt.) in healthy volunteers. The model comprises transport and absorption in stomach and gut into a central L-leucine-compartment which is connected to a protein compartment and to the compartment of the corresponding 2-oxo acid. CO(2) release from the latter occurs in a fast and a slow compartment into the central CO(2) compartment for exhalation. Using the fmins routine of MATLAB for parameter estimation, a good agreement was obtained between calculated and actually measured kinetics of (13)C-labelled metabolites and a mean in vivo L-leucine oxidation of 0.365 ± 0.071 μmol/kg per min (n = 5) was computed. Plausibility of the model was checked by predicting in vivo leucine oxidation rates from primed continuous infusion tests (priming: L-[1-(13)C]leucine, 5 μmol/kg; NaH(13)CO(2), 1.2 μmol/kg; infusion: L-[1-(13)C]leucine, 5 μmol/kg per h). In 5 tested volunteers, the experimental L-leucine oxidation rate amounted to 0.358 ± 0.105 μmol/kg per min versus predicted 0.324±0.099 μmol/kg per min. Possible causes for some observed intraindividual variations are discussed.     

Whole Body Protein Metabolism Estimated by 15N Tracer Experiments and Body Composition of Mice Selected for Different Growth Parameters

Abstract

Whole body protein synthesis was investigated in growing male mice which were long-time selected for high carcass protein amount (DU-6P, protein line) or for high body weight (DU-6, growth line) and in the unselected randomly bred control (DU-Ks). Six mice/line were housed singly in metabolic cages for the estimation of N balance, whole body protein synthesis (end-product method, single dose of ¹⁵N-labelled amino-acid mixture), and N distribution in the body. Another six mice/line were used for the determination of the body composition. All mice had free access to a commercial stock diet (crude protein 268 g, gross energy 19 MJ/kg dry matter) and to water. Body weight of both selection lines was about twice that of control mice at the same age. Selection for high body weight resulted in higher body fat content. Scaled to the corresponding body protein pools, the protein synthesis rates of selected mice were significantly higher than in controls, but were not significantly different between both selection lines in contrast to the protein deposition rates. The higher protein accretion in the protein line in comparison to the growth line seems to be due to a combination of a lower protein breakdown and an increased protein synthesis rate.     

Studies on Body Composition in Irradiated Mice

A total of 40 male and 40 female mice of equal body weight were used in the present study. Whole body gamma (⁶⁰Co) irradiation was carried out at 400, 800 and 1200 rad. Life span and several parameters of body composition were measured. Cumulative mortality precentage was lower in irradiated females in comparison to males. Ionizing radiation induced reduction (P < 0.01) in body weight, total body water with its two compartments (extra-and intracellular), fat-free body with its component (body cell mass and extracellular tissue), fat-free dry solids with its constituent (body protein and body ash), body potassium and body fat. While all of the above mentioned body composition parameters decreased (P ≤ 0.01) following whole body irradiation, the rations between the body water to these parameters increased (P ≤ 0.01). Sex difference was noticed either for the parameters or for the ratios.     

Unusual patterns in 15N blood values after a diet switch in red knot shorebirds

When a diet switch results in a change in dietary isotopic values, isotope ratios of the consumer's tissues will change until a new equilibrium is reached. This change is generally best described by an exponential decay curve. Indeed, after a diet switch in captive red knot shorebirds (Calidris canutus islandica), the depletion of ¹³C in both blood cells and plasma followed an exponential decay curve. Surprisingly, the diet switch with a dietary ¹⁵N/¹⁴N ratio (δ¹⁵N) change from 11.4 to 8.8 ‰ had little effect on δ¹⁵N in the same tissues. The diet-plasma and diet-cellular discrimination factors of ¹⁵N with the initial diet were very low (0.5 and 0.2 ‰, respectively). δ¹⁵N in blood cells and plasma decreased linearly with increasing body mass, explaining about 40 % of the variation in δ¹⁵N. δ¹⁵N in plasma also decreased with increasing body-mass change (r ²=.07). This suggests that the unusual variation in δ¹⁵N with time after the diet switch was due to interferences with simultaneous changes in body-protein turnover.     

The first in vivo observation of (13)C-(15)N coupling in mammalian brain.

[5-(13)C,(15)N]Glutamine, with (1)J((13)C-(15)N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by (13)C MRS at 4.7 T. The brain [5-(13)C]glutamine peak consisted of the doublet from [5-(13)C,(15)N]glutamine and the center [5-(13)C,(14)N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-(13)C,(15)N]glutamine was monitored in vivo with a time resolution of 20-35 min. This [5-(13)C,(15)N]glutamine was formed by glial uptake of released neurotransmitter [5-(13)C]glutamate and its reaction with (15)NH(3) catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively (13)C-enriched by intravenous [2,5-(13)C]glucose infusion to (13)C-label whole-brain glutamate C5, followed by [(12)C]glucose infusion to chase (13)C from the small and rapidly turning-over glial glutamate pool, leaving (13)C mainly in the neurotransmitter [5-(13)C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-(13)C,(15)N]glutamine arises from a coupling between (13)C of neuronal origin and (15)N of glial origin. Measurement of the rate of brain [5-(13)C,(15)N]glutamine formation provides a novel noninvasive method of studying the kinetics of neurotransmitter uptake into glia in vivo, a process that is crucial for protecting the brain from glutamate excitotoxicity.     

Effect of alcohol consumption on the liver detoxication capacity as measured by [13C]methacetin- and [methyl-13C]methionine-breath tests

The aim of this study was to investigate the hepatic microsomal and mitochondrial functions by using the 13CO2-breath test in healthy subjects either before or after the consumption of red wine. Fourteen adults received [13C]methacetin and [methyl-13C]methionine together with a standardised dinner. Expired air samples were taken over 6 h. After a wash-out period, the subjects consumed 0.4 ml ethanol/kg/day together with dinner over a 10-day period. Thereafter, 13C-tracer administration was repeated under identical conditions. The 13CO2-enrichments were measured by isotope ratio mass spectrometry. The mean cumulative percentage 13C-dose recovery (CPDR) after administration of [13C]methacetin and [methyl-13C]methionine either without or with red wine consumption amounted to 38.2+/-6.3 vs. 36.3+/-6.7% (p=0.363) and 9.5+/-3.3 vs. 8.8+/-2.5% (p=0.47), respectively. Moderate alcohol consumption does not induce significant short-term changes of the microsomal and the mitochondrial functions of the human liver in healthy subjects.     

Effects of risedronate in a rat model of osteopenia due to orchidectomy and disuse: densitometric, histomorphometric and microtomographic studies

Dual energy X-ray absorptiometry (DXA), histomorphometry and X-ray microtomography (microCT) were used to assess effects of risedronate and testosterone in a combined rat model of orchidectomy (ORX) and local paralysis induced by botulinum neurotoxin (BTX). Four groups of mature rats were studied for 1 month: SHAM operated; ORX and right hindlimb immobilization (BTX); ORX+BTX+risedronate or testosterone. Changes in bone and body composition were measured by DXA (BMC, lean and fat mass), histomorphometry (BV/TV(2D), Tb.Th and microarchitectural parameters) and microCT (BV/TV(3D), SMI and cortical parameters). ORX and BTX had additive effects on bone loss since differences were maximized on the immobilized bone. The decrease in BMC on the tibial metaphysis reached -33.6% vs. -11.3% in the non-immobilized limb. BV/TV and Tb.N decreased and Tb.Sp increased in both hindlimbs whereas Tb.Th was significantly lower only in the immobilized limb. Decrease of tibial cortical area and thickness was greater in the immobilized limb. Risedronate prevented BMC, BV/TV and architecture loss but not reduction in Tb.Th. Cortical bone was preserved only in the non-immobilized limb. Testosterone was unable to prevent trabecular and cortical bone loss, but it prevents loss of whole body lean mass. In conclusion, ORX and BTX resulted in additive effects on bone loss. Risedronate had protective effects on trabecular bone loss but was less effective on cortical bone.     

The Measurement of Muscle Protein Synthesis in Broilers with a Flooding Dose Technique: Use of 15N-Labelled Phenylalanine,GC-MS and GC-C-IRMS

Abstract

An experiment was carried out to measure fractional muscle protein synthesis rates (k _s ) in broilers with injection of a flooding dose of phenylalanine (1 ml/100 g body weight of 150 mM phenylalanine; 38 atom percent excess (APE) [¹⁵N]phenylalanine). K _s was calculated from the [¹⁵N] enrichment in phenylalanine of tissue-free and protein-bound phenylalanine using both gas chromatography mass spectrometry (GC-MS) and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) for measurements after a 10 min isotope incorporation period.

The tertiary-butyldimethylsilyl (t-BDMS) derivatives of phenylalanine were used for gas chromatographic separation in both systems. GC-MS and GC-C-IRMS were calibrated for a range of 7 to 37 [¹⁵N]APE and 0 to 0.62 [¹⁵N]APE, respectively, and for sample sizes of 0.45 to 4.5 nmol phenylalanine and 7 to 40 nmol phenylalanine, respectively. Reproducibility of standards as a measure of precision varied from 0.06 to 0.29 [¹⁵N]APE and from 0.0004 to 0.0018 [¹⁵N]APE in GC-MS and GC-C-IRMS, respectively.

K _s was measured in the m. pectoralis major of broilers fed rye based diets (56%) which were provided either unsupplemented (-) or supplemented (+) with an enzyme preparation containing xylanase. K _s in breast muscles was significantly increased from 21.8%/d to 23.9%/d due to enzyme supplementation.

It can be concluded from the study that the measurement of protein synthesis in broilers with the flooding dose technique can be carried out by using [¹⁵N]phenylalanine, GC-MS and GC-C-IRMS.     

The effect of resistant starches on fat oxidation in healthy adults as measured by a 13CO2-breath test

A resistant starch (RS) mixture (MIX) consisting of fibre of potatoes (FP) and wrinkled pea starch (WPS), and high amylose maize starch (HAMS) were supplemented in adults to evaluate their effects on fat oxidation by means of a ¹³CO₂-breath test. Sixteen subjects received a regular diet either without or with the supplementation of MIX and HAMS in randomised order. After administration of a [U-¹³C]algal lipid mixture, exhaled air was collected over 14?h in 0.5- and 1-h intervals. The ¹³C abundances were measured by nondispersive infrared spectroscopy. In comparison to the dry run (DR), supplementation with MIX and with HAMS increased the cumulative percentage dose recovery: (DR: 16.7?%, MIX: 16.9?%, HAMS: 18.0?%), but without statistical significance. The colonic degradation of MIX and HAMS to short-chain fatty acids tends to lower the formation of carbohydrate-derived acetyl-CoA and contributes to a postprandial lipid oxidation increase by using fat-derived acetyl-CoA as a compensatory fuel source.     

Investigation of Enterohepatic Bile Acid Circulation with [15N]Taurocholate

Abstract

[¹⁵N]Taurocholate has been prepared and used as a simple urine test substrate as a substitute for ¹⁴C- and ¹³C breath test substrates. [¹⁵N]Taurocholate was administered instead of [¹⁵N] glycocholate assuming that taurine is mainly used in the cholate cycle. Differences were found between healthy and diseased subjects, although deconjugated [¹⁵N]taurine was recovered in urine much less than expected. To get additional information on the actual excess of free taurine, [¹⁵N]taurine was administered orally in parallel investigations. Apparently, large fluctuations in the whole body taurine metabolism are responsible for the unexpected results.     

The Influence of Feeding Frequency on Nitrogen Turnover and Whole Body Protein Synthesis in Adult Rats (Estimation using [14C]- and [15N]- Labelled Leucine)

Abstract

Male Wistar rats (17 wks. old, body weight ～400 g), fitted with an intra gastric cannula and with a catheter in the vena jugularis were divided into 3 groups and given a marginal ration of the feeding solution Nutrison Standard (1g protein and 350 kJ ME per day). Group 1 had ad lib. access to the drinking bottle, the groups 2 and 3 were pair fed by gastric infusion, splitted up into 2 greater meals for group 2 respectively into 6 smaller meals for group 3. After adaptation all animals get an i.p. injection of doubly labelled tracer solution (200μl) containing 2.5mg L-[¹⁵N]leucine (72 atom% ¹⁵N) combined with either [1-¹⁴C]- or [U-¹⁴C] leucine (37 kBq).

The course of ¹⁴CO₂ expiration was estimated by breath test over 4h in intervals of 15 min and the course of urinary ¹⁵N excretion over 24h in intervals of 45 resp. 90 min. An infusion of saline (0.9% 5ml/h) into the vena jugularis was used to provoke sustained urine production of the animals during the experiment.

From the parameters of the excretion curves of breath ¹⁴CO₂ resp. urine ¹⁵N (cumulative end value) and from the N balance the portions of leucine-C and leucine-N used for protein synthesis, transamination decarboxylation and total oxidation as well as the kinetic parameters for whole body protein metabolism were computed.

The following conclusions were drawn:

6 x feeding regime produces a small but measurable amino acid economy effect in comparison to 2 x feeding regime.

Protein gain for 2 x feeding group was significant smaller than for 6 x feeding group, though protein synthesis rate was higher, but was overcompensated by a greater increase of protein breakdown rate for the 2 x feeding group. Energy storage in form of fat and glycogen built from decarboxylation was unaffected by feeding frequency. The amount of leucine oxidized for heat production was 4% higher for the 6 x feeding group. Transamination rate for leucine was estimated to 8–15%. Absolute values for protein flux, protein synthesis and protein breakdown may be overestimated or underestimated because the metabolism of [¹⁵N] leucine does not exactly agree with that of total N; but the proportions of them and therefore also the conclusions will be true. Better results for absolute values will be obtained using a mixture of ¹⁵N labelled AA, ¹⁵N labelled protein or hydrolysate of ¹⁵N labelled protein (yeast) as the tracer source.     

The Effect of Diet Quality on δ(13)C and δ(15)N in the Tissues of Locusts, Locusta migratoria L

Abstract Locust nymphs were raised from hatching to adult locusts on either seedling wheat (C(3)) or maize (C(4)), to determine whether relative enrichments/depletions of (15)N and (13)C within body tissues are influenced by diet. The maize contained less hexose sugars and protein per gram than wheat. The isotopic spacing between the food and the whole insect was found to differ between the two diets. The lower quality maize diet showed an overall +5.1‰ enrichment in δ(15)N compared to + 2.8‰ for wheat, possibly due to increased fractionation due to protein recycling. The maize diet resulted in increased depletion in lipid and trehalose and depletion in chitin relative to diet. The results for both δ(15)N and δ(13)C suggest that substrate recycling was occurring on the low quality maize diet. Therefore diet quality determines the enrichment/depletion in δ(15)N and δ(13)C within organisms.