The effect of amino acid infusion on leg protein turnover assessed by L-[15N]phenylalanine and L-[1-13C]leucine exchange

A stable isotope technique depending on the use of [15N]phenylalanine and [1-13C]leucine to assess exchange was utilized to measure the components of protein turnover of the human leg and the effects of amino acid infusion. Eight healthy subjects (28.5 +/- 2.5 years) were studied when post-absorptive in the basal state and again during infusion of a mixed amino acid solution (55 g l-1, 1.52 ml kg-1 h-1). During the basal period leucine oxidation by the leg was 4.4 +/- 2.0 nmol 100 g-1 min-1 and this increased threefold during amino acid infusion (13.6 +/- 3.1 nmol 100 g-1 min-1, mean +/- SEM, P = 0.003). Amino acid infusion abolished the net negative balance between incorporation of leucine into, and release from, protein (basal, -31.8 +/- 5.8; during infusion, +3.1 +/- 7.1 nmol 100 g-1 P = 0.001). Phenylalanine exchange showed a similar pattern (basal, -13.7 +/- 1.8; during infusion, -0.8 +/- 3.0 nmol 100 g-1 min-1, P = 0.003). Basal entry of leucine into leg protein (i.e. protein synthesis) was 70.0 +/- 10.8 nmol 100 g-1 min-1 and this increased during amino acid infusion to 87.3 +/- 14.1 nmol 100 g-1 min-1 (P = 0.11). Phenylalanine entry to protein also increased with amino acid infusion (29.1 +/- 4.5 vs. 38.3 +/- 5.8 nmol 100 g-1 min-1, P = 0.09). Release from protein of leucine (101.8 +/- 9.1 vs. 84.2 +/- 9.1 nmol 100 g-1 min-1, P = 0.21) and of phenylalanine (42.8 +/- 4.2 vs. 39.1 +/- 4.2 nmol 100 g-1 min-1, P = 0.50) was unchanged by amino acid infusion. The results suggest that, in the post-absorptive state in man, infusion of mixed amino acids, without additional energy substrates; reverses negative amino acid balance by a mechanism which includes stimulation of muscle protein synthesis but which does not alter protein breakdown. Interpretation of the results obtained concurrently on whole-body protein turnover suggests that the increase in muscle protein synthesis contributes substantially to the whole-body increase, but the fall in whole-body breakdown with exogenous amino acids is independent of changes in muscle.     

Amino acid and protein metabolism in hospitalized patients as measured by L-[U-14C]tyrosine and L-[1-14C]leucine

Plasma amino acid kinetics were determined in hospitalized patients receiving one of three intravenous solutions: isotonic amino acids, isotonic sodium chloride, or total parenteral nutrition. Whole body amino acid appearance, oxidation and incorporation into protein were estimated with two different isotopically labelled amino acids: L-[1-14C]leucine and L-[U-14C]tyrosine. A positive correlation was obtained between whole body amino acid appearance, oxidation and incorporation into protein with the two isotopically labelled amino acids. Derivation of whole body protein kinetics with L-[U-14C]tyrosine consistently gave higher values than those obtained from L-[1-14C]leucine, presumably due in part to the contribution of phenylalanine hydroxylation to plasma tyrosine appearance. However, the percentages of amino acid appearance oxidized and used for protein synthesis were similar. It can be concluded that estimates of whole body protein kinetics are qualitatively similar when measured with L-[U-14C]tyrosine and L-[1-14C]leucine in hospitalized patients receiving either isotonic sodium chloride solutions or balanced amino acid intakes.     

Direct determination of leucine metabolism and protein breakdown in humans using L-[1-13C, 15N]-leucine and the forearm model

Abstract. We have used the forearm model to study protein metabolism in six normal healthy subjects in the fed state using L-[1 –¹³C, ¹⁵N]-leucine as the substrate tracer.
Deep venous and arterialized venous blood samples from the forearm were collected at 10-min intervals 2±5 h into a primed-continuous infusion of the dilabelled tracer. Arterialized venous blood was obtained using a 'hot-box' technique and forearm blood flow was measured by mercury strain-gauge plethysmography.
The concentration and isotope enrichment of leucine and its metabolites, α-ketoisocaproic acid and CO₂, in deep venous and arterialized venous blood were measured by gas chromatography-mass spectrometry and isotope ratio-mass spectrometry.
The rates of leucine deamination and reamination were 388 ± 24 (mean ± SEM) and 330 ± 23 nmol (100 ml)^-1 min^-1 respectively, whilst protein synthesis and breakdown rates were 127 ± 11 and 87 ± 10 nmol (100 ml)^-1 min^-1 respectively across the forearm in the fed state. We have demonstrated that the use of doubly labelled leucine as tracer and application of the mathematical model developed in this study, permits the comprehensive quantification of leucine kinetics including protein breakdown.     

Metabolism of lactose-[13C]ureide and lactose-[15N,15N]ureide in normal adults consuming a diet marginally adequate in protein.

Oral lactose-ureide is resistant to human digestive enzymes, but is fermented by the colonic microflora. Nine normal adults consuming a diet which provided 36 g of protein/day were given oral doses of lactose-[(13)C]ureide and lactose-[(15)N,(15)N]ureide. The appearance on breath of (13)CO(2) derived from lactose-[(13)C]ureide was followed for 48 h. The fate of (15)N derived from lactose-[(15)N, (15)N]ureide was determined by measuring the recovery of (15)N in stools and urine in various forms. About 80% of the label given as lactose-[(13)C]ureide was recovered on the breath, and about 80% of label given as lactose-[(15)N,(15)N]ureide was not recovered in stool, indicating that 80% of the dose was completely fermented. At least 5% of the labelled urea was absorbed and excreted as the intact molecule. Of the (15)N derived from lactose-[(15)N, (15)N]ureide and available for further metabolic interaction, 67% was retained and 33% was excreted in urine. The time taken for [(15)N,(15)N]urea to appear in urine was similar for all subjects, but the appearance of either (13)CO(2) on the breath or [(15)N, (14)N]urea in urine varied. It is concluded that the hydrolysis of the sugar-urea bond may reflect oro-caecal transit time, but that other factors related to colonic bacterial metabolism determine the duration and extent of hydrolysis of urea by urease enzymes. Lactose-ureide can be used to probe the metabolic activity of the colonic microflora in normal individuals.     

Measurement of the rate of protein synthesis in muscle of postabsorptive young men by injection of a 'flooding dose' of [1-13C]leucine

1. The 'flooding dose' technique for measuring the rate of protein synthesis in tissues in vivo involves the injection of a large amount of unlabelled amino acid together with the tracer to minimize differences in isotopic enrichment of the free amino acid in plasma and tissue compartments. This approach has been investigated in human muscle by taking biopsies from postabsorptive male volunteers given [1-13C]leucine. 2. Intravenous injection of 4 g of unlabelled leucine resulted in a rapid rise in free leucine concentration of seven- to eleven-fold in plasma and five-fold in muscle. Values were still elevated by two-fold after 2 h. 3. Five minutes after injection of [1-13C]leucine (0.05 g/kg) the isotopic enrichment of plasma leucine was 82% that of the injected material, falling to 44% at 120 min. The enrichment of free leucine in sequential muscle biopsies was close to that in plasma and almost identical to that for plasma alpha-ketoisocaproate. 4. The rate of protein synthesis was determined from the increase in leucine enrichment in protein of muscle biopsies taken before and 90 min after injection of [1-13C]leucine (0.05 g/kg; 19 or 39 atom% excess) and the average plasma alpha-ketoisocaproate enrichment over this period (taken to represent muscle free leucine). The mean rate of muscle protein synthesis in 10 subjects was 1.95 (SEM 0.12) %/day. Rates of protein synthesis calculated from plasma leucine as precursor enrichment were only 5% lower than those calculated from plasma alpha-ketoisocaproate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase in anterior tibialis muscle protein synthesis in healthy man during mixed amino acid infusion: studies of incorporation of [1-13C]leucine

1. Anterior tibial muscle protein synthesis in seven healthy postabsorptive men was determined from increases in muscle protein bound leucine enrichment during a primed continuous infusion of L-[1-13C]leucine. Biopsies were taken 30 min after the beginning of leucine infusion (when plasma 13C enrichment was steady), 240 min later during continued fasting and again after 240 min of infusion of a mixed amino acid solution which increased plasma total amino acid concentrations by 37%. The mean enrichment of 13C in plasma alpha-ketoisocaproate was used as an index of the enrichment of the precursor pool for leucine metabolism. 2. Anterior tibial muscle mixed protein synthetic rate during fasting was 0.055 (SD 0.008)%/h and this increased by an average of 35% during infusion of mixed amino acid to 0.074 (SD 0.021)%/h (P less than 0.05). 3. Whole-body protein breakdown (expressed as the rate of endogenous leucine appearance in plasma) was 121 (SD 8) mumol h-1 kg-1 during fasting and decreased (P less than 0.01) by an average of 12% during amino acid infusion. Leucine oxidation was 18 (SD 3) mumol h-1 kg-1 during fasting and increased (P less than 0.001) by 89% during amino acid infusion. Whole-body protein synthesis (non-oxidative leucine disappearance) was 104 (SD 6) mumol h-1 kg-1 during fasting and rose by 13% (P less than 0.001) during mixed amino acid infusion. 4. 13C enrichment of muscle free leucine was only 61 (SD 19)% of that in plasma alpha-ketoisocaproate and this increased to 74 (SD 16)% (P less than 0.02) during mixed amino acid infusion. 5. The results suggest that increased availability of amino acids reverses whole-body protein balance from negative to positive and a major component of this is the increase in muscle protein synthesis.     

Measurement of whole-body protein turnover in insulin-dependent (type 1) diabetic patients during insulin withdrawal and infusion: comparison of [13C]leucine and [2H5]phenylalanine methodologies

1. The aims of this study were twofold: (i) to investigate the ability of a recently described [2H5]phenylalanine method for quantifying whole-body protein turnover during acute physiological perturbation; (ii) to determine specifically whether the previously observed increase in protein synthesis on insulin withdrawal in insulin-dependent (type 1) diabetic patients seen when employing the [13C]leucine technique could be corroborated by using [2H5]phenylalanine. 2. Whole-body protein turnover was measured by both the [2H5]phenylalanine and [13C]leucine primed continuous infusion methods applied simultaneously to six type I post-absorptive diabetic patients during insulin withdrawal and infusion. 3. Values were determined by the [13C]leucine method by measuring either [13C]leucine (primary pool) or alpha-[13C] ketoisocaproic acid (reciprocal pool) enrichment in plasma. 4. Values of whole-body protein breakdown during insulin withdrawal derived from the [2H5]phenylalanine and primary and reciprocal pool [13C]leucine models respectively were 3.54 +/- 0.43, 3.85 +/- 0.41 and 4.62 +/- 0.44 g day-1 kg-1 (means +/- SD). Insulin infusion resulted in a significant reduction (P less than 0.02) to 3.07 +/- 0.34, 3.05 +/- 0.26 and 3.82 +/- 0.4 g day-1 kg-1, respectively. Synthesis values fell significantly but by a smaller amount than breakdown, resulting in increased (P less than 0.05) net protein deposition, regardless of the model used. 5. These data demonstrate that the [2H5]phenylalanine and [13C]leucine methods generate similar results both in absolute and relative terms in response to short-term insulin infusion. 6. The confirmation of increased whole-body protein synthesis during insulin withdrawal by two independent methods supports the validity of this observation.     

A new application of pre-normalized principal component analysis for improvement of image quality and clinical diagnosis in human brain PET studies--clinical brain studies using [11C]-GR205171, [11C]-L-deuterium-deprenyl, [11C]-5-Hydroxy-L-Tryptophan, [11C]-L-DOPA and Pittsburgh Compound-B

Principal component analysis (PCA) is one of the most applied multivariate image analysis tool on dynamic Positron Emission Tomography (PET). Independent of used reconstruction methodologies, PET images contain correlation in-between pixels, correlations in-between frame and errors caused by the reconstruction algorithm including different corrections, which can affect the performance of the PCA. In this study, we have investigated a new approach of application of PCA on pre-normalized, dynamic human PET images. A range of different tracers have been used for this purpose to explore the performance of the new method as a way to improve detection and visualization of significant changes in tracer kinetics and to enhance the discrimination between pathological and healthy regions in the brain. We compare the new results with the results obtained using other methods. Images generated using the new approach contain more detailed anatomical information with higher quality, precision and visualization, compared with images generated using other methods.     

老年患者疼痛评估的应用进展

前言：疼痛已被护理人员作为除体温、脉搏、呼吸和血压以外的“第五个生命体征”来评估与处理[1],北美将疼痛的控制作为一项基本医疗服务。从伦理及人道主义的角度而言,“缓解疼痛是基本人权(pain relief is a basic human right)”。随着全球人口老龄化程度的日趋加重,关注老年人的身心健康,控制治疗疼痛,提高生活质量又显得十分重要。慢性疼痛在老年人中很普遍,严重影响了老年人的生活质量[2],认知功能异常可能使老年人疼痛的评估变得复杂[3]而如何对老年人进行正确疼痛评估尤显重要,现将其综述如下。     

Influence of Multidrug Resistance-Associated Proteins on the Excretion of the ABCC1 Imaging Probe 6-Bromo-7-[11C]Methylpurine in Mice

Molecular Imaging and Biology - Multidrug resistance-associated proteins (MRPs) mediate the hepatobiliary and renal excretion of many drugs and drug conjugates. The positron emission tomography...     

Synthesis, characterization, and monkey positron emission tomography (PET) studies of [18F]Y1-973, a PET tracer for the neuropeptide Y Y1 receptor

Neuropeptide Y receptor subtype 1 (NPY Y1) has been implicated in appetite regulation, and antagonists of NPY Y1 are being explored as potential therapeutics for obesity. An NPY Y1 PET tracer is useful for determining the level of target engagement by NPY Y1 antagonists in preclinical and clinical studies. Here we report the synthesis and evaluation of [(18)F]Y1-973, a novel PET tracer for NPY Y1. [(18)F]Y1-973 was radiolabeled by reaction of a primary chloride with [(18)F]KF/K2.2.2 followed by deprotection with HCl. [(18)F]Y1-973 was produced with high radiochemical purity (>98%) and high specific activity (>1000 Ci/mmol). PET studies in rhesus monkey brain showed that the distribution of [(18)F]Y1-973 was consistent with the known NPY Y1 distribution; uptake was highest in the striatum and cortical regions and lowest in the pons, cerebellum nuclei, and brain stem. Blockade of [(18)F]Y1-973 uptake with NPY Y1 antagonist Y1-718 revealed a specific signal that was dose-dependently reduced in all regions of grey matter to a similarly low level of tracer uptake, indicative of an NPY Y1 specific signal. In vitro autoradiographic studies with [(18)F]Y1-973 in rhesus monkey and human brain tissue slices revealed an uptake distribution consistent with the in vivo PET studies. Highest binding density was observed in the dentate gyrus, caudate-putamen, and cortical regions; moderate binding density in the hypothalamus and thalamus; and lowest binding density in the globus pallidus and cerebellum. In vitro saturation binding studies in rhesus monkey and human caudate-putamen homogenates confirmed a similarly high B(max)/K(d) ratio for [(18)F]Y1-973, suggesting the tracer may provide a specific signal in human brain of similar magnitude to that observed in rhesus monkey. [(18)F]Y1-973 is a suitable PET tracer for imaging NPY Y1 in rhesus monkey with potential for translation to human PET studies.     

Measurement of the global lumped constant for 2-deoxy-2-[18F]fluoro-D-glucose in normal human brain using [15O]water and 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography imaging. A method with validation based on multiple methodologies.

PURPOSE: This study aims to determine a lumped constant (LC) value that can be applied to the 2-deoxy-2[18F]fluoro-D-glucose positron emission tomography (FDG-PET) study to yield a physiological value of cerebral metabolic rate of glucose (CMR(glc)) in normal brain. PROCEDURES: We developed a more robust method for determining the global FDG LC. Dynamic FDG and H(2)(15)O PET studied were acquired in 18 normal subjects. Arterial-venous difference of blood glucose level was measured. RESULTS: A global LC of 0.65 +/- 0.15 was obtained if a 3-microparameter FDG model (k*(4)=0)was assumed. Assumption of a 4-microparameter FDG model (k*(4) not equal 0) in analyzing the FDG data resulted in a higher LC value of 0.81 +/- 0.18. CONCLUSION: The value of LC used for quantitating CMR(glc) should match the assumption inherent to the method of data analysis. The LC results in this study agree well with recent findings in the literature.     

Mapping of the norepinephrine transporter in the human brain using PET with (S,S)-[18F]FMeNER-D2

We have evaluated the detailed mapping of the norepinephrine transporter (NET) in the human brain with (S,S)-[(18)F]FMeNER-D(2) using a template method and the generation of functional ROIs based on the PET information. Brain PET measurements were performed from 90 to 210 min after the injection of (S,S)-[(18)F]FMeNER-D(2) in 20 healthy male subjects. Binding potential (BP(ND)) was calculated as late time ratio of the target region to the reference region (caudate) minus one. BP(ND) template images were generated from mean parametric images obtained in a group of 10 subjects using SPM2. On the BP(ND)/MRI template images, functional ROIs based on several different BP(ND) thresholds for the thalamus and brainstem were generated automatically using PMOD 2.8 software in addition to anatomical ROIs. PET/MRI data of another group of 10 subjects were used to evaluate the validity of the template method and the functional ROIs. NET BP(ND) template images demonstrated higher binding in the medial thalamus whereas the anterior and the pulvinar divisions had lower binding. In the brainstem, high binding was detected around the cerebral aqueduct of the midbrain and within the dorsal pons, in a volume comprising locus coeruleus. Functional ROIs with higher BP(ND) thresholds naturally yielded higher BP(ND) and lower coefficients of variance than did anatomical ROIs. This study indicated that (S,S)-[(18)F]FMeNER-D(2) combined with a template method provides detailed information on the distribution of NET in vivo and that functional ROIs on the template would be useful in further clinical studies.     

Comparison of [11C]-(R)-PK 11195 and [11C]PBR28, two radioligands for translocator protein (18 kDa) in human and monkey: Implications for positron emission tomographic imaging of this inflammation biomarker

Ten percent of humans lack specific binding of [¹¹C]PBR28 to 18 kDa translocator protein (TSPO), a biomarker for inflammation. “Non-binders” have not been reported using another TSPO radioligand, [¹¹C]-(R)-PK 11195, despite its use for more than two decades. This study asked two questions: (1) What is the cause of non-binding to PBR28? and (2) Why has this phenomenon not been reported using [¹¹C]-(R)-PK 11195? Methods: Five binders and five non-binders received whole-body imaging with both [¹¹C]-(R)-PK 11195 and [¹¹C]PBR28. In vitro binding was performed using leukocyte membranes from binders and non-binders and the tritiated versions of the ligand. Rhesus monkeys were imaged with [¹¹C]-(R)-PK 11195 at baseline and after blockade of TSPOs. Results: Using [¹¹C]PBR28, uptake in all five organs with high densities of TSPO (lung, heart, brain, kidney, and spleen) was 50% to 75% lower in non-binders than in binders. In contrast, [¹¹C]-(R)-PK 11195 distinguished binders and non-binders in only heart and lung. For the in vitro assay, [³H]PBR28 had more than 10-fold lower affinity to TSPO in non-binders than in binders. The in vivo specific binding of [¹¹C]-(R)-PK 11195 in monkey brain was ～80-fold lower than that reported for [¹¹C]PBR28. Conclusions: Based on binding of [³H]PK 11195 to leukocyte membranes, both binders and non-binders express TSPO. Non-binding to PBR28 is caused by its low affinity for TSPO in non-binders. Non-binding may be differentially expressed in organs of the body. The relatively low in vivo specific binding of [¹¹C]-(R)-PK 11195 may have obscured its detection of non-binding in peripheral organs.     

Evaluation of enzymatic and magnetic properties of γ-glutamyl-[1-13C]glycine and its deuteration toward longer retention of the hyperpolarized state

Dynamic nuclear polarization (DNP) is an emerging cutting-edge method of acquiring metabolic and physiological information in vivo. We recently developed γ-glutamyl-[1-¹³C]glycine (γ-Glu-[1-¹³C]Gly) as a DNP nuclear magnetic resonance (NMR) molecular probe to detect γ-glutamyl transpeptidase (GGT) activity in vivo. However, the detailed enzymatic and magnetic properties of this probe remain unknown. Here, we evaluate a γ-Glu–Gly scaffold and develop a deuterated probe, γ-Glu-[1-¹³C]Gly-d₂, that can realize a longer lifetime of the hyperpolarized signal. We initially evaluated the GGT-mediated enzymatic conversion of γ-Glu–Gly and the magnetic properties of ¹³C-enriched γ-Glu–Gly (γ-Glu-[1-¹³C]Gly and γ-[5-¹³C]Glu–Gly) to support the validity of γ-Glu-[1-¹³C]Gly as a DNP NMR molecular probe for GGT. We then examined the spin-lattice relaxation time (T₁) of γ-Glu-[1-¹³C]Gly and γ-Glu-[1-¹³C]Gly-d₂ under various conditions (D₂O, PBS, and serum) and confirmed that the T₁ of γ-Glu-[1-¹³C]Gly and γ-Glu-[1-¹³C]Gly-d₂ was maintained for 30 s (9.4 T) and 41 s (9.4 T), respectively, even in serum. Relaxation analysis of γ-Glu-[1-¹³C]Gly revealed a significant contribution of the dipole–dipole interaction and the chemical shift anisotropy relaxation pathway (71% of the total relaxation rate at 9.4 T), indicating the potential of deuteration and the use of a lower magnetic field for realizing a longer T₁. In fact, by using γ-Glu-[1-¹³C]Gly-d₂ as a DNP probe, we achieved longer retention of the hyperpolarized signal at 1.4 T.

By examining enzymatic and magnetic properties, γ-Glu-[1-¹³C]Gly-d₂ was developed as a long-lived DNP molecular probe for detecting γ-glutamyl transpeptidase.