Alexandra C. Small

A novel oral tracer procedure for measurement of habitual myofibrillar protein synthesis

RATIONALE Conventionally, myofibrillar protein synthesis is measured over time periods of hours. In clinical studies, interventions occur over weeks. Functional measures over such periods may be more representative. We aimed to develop a novel method to determine myofibrillar protein fractional synthetic rate (FSR) to estimate habitual rates, while avoiding intravenous tracer infusions. METHODS Four healthy males were given 100 g water enriched to 70 Atom % with (2)H2O as a single oral bolus. Vastus-lateralis needle biopsies were performed and plasma samples collected, 3-13 days post-dose. (2)H enrichment in body water was measured in plasma using continuous flow isotope ratio mass spectrometry (IRMS). Myofibrillar protein was isolated from muscle biopsies and acid hydrolysed. (2)H enrichment of protein-bound and plasma-free alanine was measured by gas chromatography (GC)/pyrolysis/IRMS. Myofibrillar protein FSR was calculated (% day(-1)). RESULTS The tracer bolus raised the initial enrichment of body water to 1514 ppm (2)H excess. Water elimination followed a simple exponential. The average elimination half-time was 8.3 days. Plasma alanine, labelled during de novo synthesis, followed the same elimination kinetics as water. The weighted average myofibrillar protein FSR from the four subjects was 1.38 % day(-1) (range, 1.0-1.9 % day(-1) ). CONCLUSIONS Myofibrillar protein FSR was measured in free-living healthy individuals over 3-13 days. Using a single oral (2)H2O bolus, endogenous labelling of alanine occurred in a predictable manner giving estimates of synthesis comparable with published values. Furthermore, the protocol does not compromise the ability to measure other important metabolic processes such as total energy expenditure.

Influence of the type of indigestible carbohydrate on plasma and urine short-chain fatty acid profiles in healthy human volunteers

Background/Objectives:Health effects of whole grain foods are becoming more evident. In this study, we analysed the short-chain fatty acid profiles in urine and serum derived from the colonic fermentation process of 13C-barley meals, prepared from barley grown under 13CO2 atmosphere.Subjects/Methods:In a crossover study, five volunteers ingested intact barley kernels (high content of non-starch polysaccharides (NSP) and resistant starch (RS)) and barley porridge (high content of NSP only). Using a newly developed stable isotope technology, we monitored 14 and 24 h postprandially 13C-acetate, 13C-propionate and 13C-butyrate in plasma and urine, respectively. The oro-cecal transit time (OCTT) of the meals was measured with the hydrogen breath test.Results:The OCTT was 6 h and did not differ between the two test meals. An increase of 13C-acetate was observed already early after ingestion of the meals (<6 h) and was attributed to early fermentation of the test meal. A rise in plasma 13C-propionate in the fermentation phase could only be detected after the porridge and not after the kernel meal. An increase in 13C-butyrate was only found in the fermentation phase and was higher after the barley kernels. Urine 13C-short-chain fatty acids data were consistent with these observations.Conclusions:The difference in the profiles of 13C-acetate, 13C-propionate and 13C-butyrate indicates that NSP combined with RS results in an altered fermentation profile than dietary fibre alone.

Habitual Myofibrillar Protein Synthesis Is Normal in Patients with Upper GI Cancer Cachexia

Purpose: Skeletal muscle wasting and weight loss are characteristic features of cancer cachexia and contribute to impaired function, increased morbidity, and poor tolerance of chemotherapy. This study used a novel technique to measure habitual myofibrillar protein synthesis in patients with cancer compared with healthy controls. Experimental design: An oral heavy water (87.5 g deuterium oxide) tracer was administered as a single dose. Serum samples were taken over the subsequent week followed by a quadriceps muscle biopsy. Deuterium enrichment was measured in body water, serum alanine, and alanine in the myofibrillar component of muscle using gas chromatography–pyrolysis–isotope ratio mass spectrometry and the protein synthesis rate calculated from the rate of tracer incorporation. Net change in muscle mass over the preceding 3 months was calculated from serial CT scans and allowed estimation of protein breakdown. Results: Seven healthy volunteers, 6 weight-stable, and 7 weight-losing (≥5% weight loss) patients undergoing surgery for upper gastrointestinal cancer were recruited. Serial CT scans were available in 10 patients, who lost skeletal muscle mass preoperatively at a rate of 5.6%/100 days. Myofibrillar protein fractional synthetic rate was 0.058%, 0.061%, and 0.073%/hour in controls, weight-stable, and weight-losing patients, respectively. Weight-losing patients had higher synthetic rates than controls (P = 0.03). Conclusion: Contrary to previous studies, there was no evidence of suppression of myofibrillar protein synthesis in patients with cancer cachexia. Our finding implies a small increase in muscle breakdown may account for muscle wasting. Clin Cancer Res; 21(7); 1734–40. ©2014 AACR.

Patient-focused endpoints in advanced cancer: Criterion-based validation of accelerometer-based activity monitoring

BACKGROUND & AIMS Objective assessment of daily physical activity (PA) by body-worn accelerometers offers potential as a novel endpoint in the clinical management of advanced cancer patients. This study aimed to assess criterion-based validity of an accelerometer-based activity monitoring system (AM-system), ActivPAL™, using two different methods. METHODS Advanced cancer in patients and outpatients (Karnofsky Performance Status (KPS) 40-100). ActivPAL™ measurements were validated against (i) observations and (ii) energy expenditure (EE) measured by 2-week doubly-labelled water (DLW) protocol. RESULTS Absolute errors for mean time spent in different body positions (<0.1%) and number of transfers (0%) were low. Step count error was significantly higher in patients with KPS 40-60 (non-self caring) compared to KPS 70-100 (self-caring) (33 vs. 24%, p = 0.006). Post-hoc mathematical analysis demonstrated that absolute errors for the mean energy expenditure of activity (EEA) (1.4%) and mean total EE (0.4%) were low, but agreement was also low. CONCLUSIONS AM-systems provide valid estimates of body positions and transfers, but not step count, especially in non-self caring patients. ActivPAL™ can derive estimates of EE but there is considerable variability in results, which is consistent, in part, with the inaccuracy in step count. Further studies are required to assess the validity of different endpoints derived from AM-systems in advanced cancer patients.

A curve fitting approach to estimate the extent of fermentation of indigestible carbohydrates

Background  Information about the extent of carbohydrate digestion and fermentation is critical to our ability to explore the metabolic effects of carbohydrate fermentation in vivo. We used cooked 13C‐labelled barley kernels, which are rich in indigestible carbohydrates, to develop a method which makes it possible to distinguish between and to assess carbohydrate digestion and fermentation.

In Vivo Assessment of Acetic, Propionic and Butyric Acid Production Following Colonic Fermentation of Non-Digestible Carbohydrates From Two 13C-Labelled Barley Meals

Extrinsic afferent neurons play an important role in the regulation of gut function. These neurons exhibit extensive plasticity in phenotype and cell number in response to a variety of conditions. We have previously reported that TNBS colitis increased the mRNA and protein levels of calcitonin gene-related peptide (CGRP) in rat dorsal root ganglia (DRG). The promoter of CGRP contains a binding site for cAMP response element binding (CREB) protein. The activation of CREB was regulated by its phosphorylation on Ser133, for which Ca2+-regulated pathways may have a role. Thus the AIMS of this study are to examine 1) if phospho (p)-CREB was regulated by colitis in DRG; 2) if p-CREB was co-localized with CGRP; and 3) the association of p-CREB with Ca2+/calmodulin-dependent protein kinase (CaMK) II in the DRG during colitis. METHODS: Colonic inflammation was induced in rats by intracolonic instillation of TNBS (1.5 mL/kg of 60 mg/mL solution in 50 % EtOH). Control animals received 50 % EtOH. Rats were sacrificed by intracardiac perfusion with 4% paraformaldehyde on day 7 or 21 following the induction of colitis. The lumbar L1-L2 DRG sections (20 μm thickness) underwent single or double immunostaining for p-CREB, CaMKII, or/and CGRP. RESULTS: In all animals examined, p-CREB immunoreactivity was expressed in the nucleus of small diameter DRG neurons (10-20 μm). Following TNBS treatment, the number of p-CREB immunoreactive neurons in L1-L2 DRG was significantly increased (p<0.05) at day 7 (29 cells per 105 μm2 DRG section) and day 21 (22 cells per 105 μm2 DRG section) when compared to control (11 cells per 105 μm2 DRG section). The number of CGRP immunoreactive neurons was also increased by 2-fold (control: 7 cells; TNBS 7 days: 13 cells; TNBS 21 days: 16 cells per 105 μm2 DRG section, p<0.05). Colocalization studies showed that the number of DRG neurons co-expressing CGRP and pCREB was increased by 2-2.5 fold in the lumbar L1-L2 DRG at 7 and 21 days following colitis induction. The neurons that contained CGRP also expressed p-CREB, however, only 50 % of p-CREB immunoreactive cells expressed CGRP. Double immunostaining also showed co-localization of p-CREB with CaMKII in the DRG during colitis. CONCLUSIONS: TNBS colitis increased the expression level of CGRP in lumbar L1-L2 DRG at day 7 and day 21, and also increased the phosphorylation level of CREB in these DRG neurons. The colocalization of p-CREB with CGRP and with CaMKII in the DRG suggested a correlation of CaMKII/p-CREB activation and the CGRP expression in DRG during colitis.