Naturally occurring stable carbon isotope ratios (13C/12C) in body proteins, fats and carbohydrates reflect the isotope ratios of the diet, however, the 13C isotopes are not equally distributed in these tissue types. As energy is stored during the conversion of dietary glucose to lipids, enzymatic processes discriminate against 13C, resulting in stored lipids being about 3.5 parts per mil lighter than carbohydrates. Energy metabolism is fueled by a mixture of carbohydrate, protein and lipid as they are converted to CO2 and expired. Changes in the ratio of macronutrient oxidation, either due to exercise or starvation, influence the 13CO2/12CO2 delta value of expired breath (BDV). Previous work has shown that the BDV is correlated to respiratory quotient (RQ) during exercise. Therefore, the BDV may be useful for simple and non‐invasive tracking changes in macronutrient oxidation during exercise. Tracking macronutrient metabolism via the BDV in performance trained athletes during conditioning exercise is a previously unexplored application of this concept. By measuring and understanding shifts in macronutrient metabolism during exercise, athletes and trainers can re‐program nutrition and exercise regimens to optimize energy availability, recovery, injury prevention, and weight management.We hypothesized that the BDV correlates to workload only during energy balance and can serve as a biomarker for metabolic energy need in performance‐trained athletes. We recruited subjects from the University of Wisconsin football program, and categorized them into 3 groups based on body type (i.e. ectomorphic, mesomorphic and endomorphic body types). Breath samples were taken immediately before, 30 and 60 minutes into, immediately after, and 20 after conditioning training.Breath samples were analyzed using two breath 13CO2/12CO2 spectrometers, the Picarro G2101i (Picarro, Santa Clara, CA, USA) and the LIR100 (MIRICO LTD, Harwell Oxford, Didicot, UK). The change in the BDV from the pre‐workout baseline was calculated for each timepoint and reported as delta over baseline (DOB). Breath samples were compared against the reported relative perceived exertion (RPE) and total workload measured by the Catapult monitoring system (Catapult Sports, Chicago, IL).Both the G2101i and the LIR100 breath spectrometers yielded similar results. During one per day conditioning trainings there was a linear increase in the BDV during the workout period that persisted up to 20 minutes after exercise in each of the body type groups, indicating an increased reliance on glucose metabolism for energy relative to baseline. The increase in the BDV was correlated to the intensity of the workout, and the total work load of the individual players. During the second practice on a two a day schedule, the BDV decreased linearly during the workout period, particularly in the endomorphic and mesomorphic body type groups. According to our hypothesis, a decrease in the BDV is likely due to an increased reliance on lipid oxidation to perform work when muscle glycogen is depleted due to negative energy balance.These data suggest that athletes and trainers can use the BDV during performance workout trainings to understand and optimize the metabolic fuel sources being used to perform work, and may provide immediate feedback on rest, recovery programs and nutritional strategies during intense training.Support or Funding InformationResearch supported in part by NIGMS SBIR Grant 1R44GM110844‐ 01, and the Wisconsin Alumni Research Foundation. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the funding agencies. Data presented here were part of a non‐provisional patent application P140010US01 entitled “Methods of Determining Energy Balance Using Breath Carbon Isotope Ratios” filed in January, 2014. Daniel E. Bütz has an ownership interest in Isomark, LLC, which has licensed the technology discussed in this publication. Damien Weidmann is also Chief Scientific Officer of MIRICO Ltd.

Introduction: Increasing the usage of hearts from marginal donors and from donors after circulatory death (DCD) has the potential to expand the donor pool. Such hearts may be resuscitated when subjected to ex vivo perfusion. In addition, this may provide an opportunity for viability testing prior to transplant. We describe a novel ex vivo perfusion system designed to reanimate porcine DCD hearts. The same system was subsequently tested using a human heart from a marginal brainstem-dead donor. Method: In the first phase of the study 23 porcine hearts were procured following circulatory death. All hearts were subjected to a period of primary warm ischemia followed by 120 minutes of hypothermic preservation. The period of hypothermic preservation was initially static cold storage (SCS); then oxygenated machine perfusion and finally a combination of static cold storage and oxygen persufflation via the coronary sinus. Ex vivo perfusion of the hearts was performed with a normothermic, oxygenated blood-based solution in our Langendorff system. In the second phase of the study the same system was used to perfuse a human heart from a marginal brainstem-dead donor. This donor had not met criteria for consideration of heart donation. Results: 15 of the 23 (65.2%) DCD porcine hearts reanimated following reperfusion on the ex vivo system. Reanimation was achieved with 63.6% (7/11) in the SCS group; 33.3% (2/6) in the machine perfusion group and 100% (6/6) in the persufflation group. The human heart was placed in the system after a cold ischemic period of 7 hours 4 minutes and perfused with a mid-thermic temperature solution for a further 2 hours 40 minutes to allow for correction of hyperkalemia before warming. The heart started to work after a further hour and then maintained until the experiment was terminated after a further two hours. Conclusion: The ex vivo perfusion system described can potentially resuscitate marginal hearts including those from DCD. The system devised in this study could also be used as a platform to functionally assess marginal human hearts. This mode of viability testing would be an essential step to determine suitability for transplant.

Background: This study reports on the development of a novel method for achieving ex vivo reanimation of hearts from a porcine donation after circulatory death (DCD) model without the use of donor pretreatment.Methods: Porcine hearts (n = 23) were procured 10-29 min after confirmation of asystole. All hearts underwent initial flush with AQIX RS-I solution (London, UK). A 2-h preservation period followed: group 1 hearts (n1-n11) were preserved using static cold storage, group 2 hearts (n12-n17) were preserved using oxygenated, hypothermic machine perfusion (MP), and group 3 hearts (n18-n23) were subjected to retrograde oxygen persufflation. Reperfusion was performed on a Langendorff modification of a Model 33 Functional Circulation circuit. In hearts n16-n23, a dialysis circuit was incorporated into the circuit to facilitate removal of metabolites. The experimental protocol was allowed to follow an evolutionary course, with the aim of achieving greater success with reanimation.Results: In group 1 (static cold storage), 7 of the 11 hearts (63.6%) achieved reanimation on the ex vivo circuit. Two of the six hearts (33.3%) in group 2 (MP) were successfully reanimated. All the six hearts (100%) in group 3 (persufflation) were successfully reanimated. The period of sustained reanimation increased when dialysis was incorporated into the circuit with a maximum of 300 min.Conclusions: Porcine DCD hearts after 29 min of warm ischemia can be reanimated using the method described. A mechanism of reoxygenation (oxygenated MP or coronary sinus oxygen persufflation) during preservation appears mandatory for hearts from DCDs. Persufflation was associated with a higher probability of successful reanimation. Dialysis in the warm phase was useful in removing metabolites that could interfere with reanimation.

Purpose. Kidneys from DCD donors represent a significant pool, but preservation problems exist. The study objective was to test the importance of machine type for hypothermic preservation of DCD kidneys. Methods. Adult Beagle dog kidneys underwent 45 minutes of warm in situ ischemia followed by hypothermic perfusion for 24 hours (Belzer-MPS Solution) on either an ORS LifePort or a Waters RM3 using standard perfusion protocols. Kidneys were then autotransplanted, and renal function was assessed over 7 days following contralateral nephrectomy. Results. Renal vascular resistance was not different between the two pumps. After 24 hours, the oxygen partial pressure and oxygen delivery in the LifePort perfusate were significantly lower than those in the RM3 but not low enough to change lactate production. TheLifePort ran significantly colder than RM3 (2° versus 5°C). The arterial pressure waveform of the RM3 was qualitatively different from the waveform of the LifePort. Preservation injury after transplantation was not different between the devices. When the LifePort was changed to nonpulsatile flow, kidneys displayed significantly greater preservation injury compared to RM3. Conclusions. Both LifePort and RM3 can be used for hypothermic machine perfusion preservation of DCD kidneys with equal outcomes as long as the duty cycle remains pulsatile.

Introduction: Utilisation of hearts from Donors after Circulatory Death (DCD) could increase the donor pool and stem the decline in heart transplantation. Persufflation is a method of introducing oxygen into the organ during the cold preservation phase. This may offset the effects of warm ischaemia inflicted on the DCD heart, enabling a greater chance of functional recovery. Methods: Seventeen cross-Yorkshire Landrace pigs (divided into three groups) were euthanased humanely by Schedule-1 (intravenous administration of phenobarbitone) or exsanguination. The non-beating hearts were procured after being subjected to 10 - 29 minutes of warm ischaemia. All hearts underwent initial antegrade flush with AQIX® RS-I solution (a novel non-phosphate pH buffered preservation solution). Group I and II hearts were subjected to static cold storage (SCS). The Group III hearts were subjected to retrograde oxygen persufflation via the coronary sinus, at a pressure of 12mmHg, whilst the heart was immersed in cold AQIX® RS-I solution. 10-12 small holes were made in the myocardium, using a 21G needle, to create an outlet for excess oxygen. Reperfusion was performed on a Langendorff modification of Model 30 Functional Circulation circuit, using a mixture of heparinised, leukocyte-depleted blood and AQIX® RS-I solution. Drugs (adrenaline, calcium gluconate, dopamine) and DC cardioversion were used to initiate left ventricular activity. Results:[Figure 1Discussion: DCD hearts subjected to preservation using coronary sinus oxygen persufflation can be successfully reanimated. This study compared persufflation with a retrospective control group (SCS). However, persufflation has demonstrated promise as a potential method for introducing oxygen into the heart during the cold preservation phase, with subsequent ability to achieve sustained reanimation.

Pancreas procurement for islet isolation and transplantation is limited by concerns for the detrimental effects of postmortem ischemia. Hypothermic machine perfusion (HMP) preservation technology has had a major impact in circumventing ischemic injury in clinical kidney transplantation and is applied here to the preservation and procurement of viable islets after hypothermic perfusion preservation of porcine pancreata because pigs are now considered the donor species of choice for xenogeneic islet transplantation. Pancreases were surgically removed from young (<6 months) domestic Yorkshire pigs (25–32 kg), either before or after 30 min of warm ischemia time (WIT), and cannulated for perfusion. Each pancreas was assigned to one of six preservation treatment groups: fresh controls—processed immediately (cold ischemia <1 h) (G1, n = 7); static cold storage—flushed with cold UW-Viaspan and stored in UW-Viaspan at 2–4°C for 24 h with no prior WIT (G2, n = 9); HMP perfused on a LifePort® machine at 4–6°C and low pressure (10 mmHg) for 24 h with either KPS1 solution (G3, n = 7) or Unisol-UHK (G4, n = 7). Additional treatment groups to evaluate the effects of prior warm ischemia examined islet isolation after 30 min WIT in situ without (G5, n = 6) or with subsequent 24-h HMP with KPS1 (G6, n = 7). The pancreas was intraductally distended with Liberase PI enzyme and normothermically digested. The isolated islets were purified by a continuous density-gradient centrifugation. Perfusion-induced glandular edema was G3 = 138 ± 19%, G4 = 160 ± 16%, and G6 = 127 ± 22%. Islet yield (IEQ/g of pancreas) varied between the groups: G1 = 1,425 ± 610, G2 = 1,002 ± 262, G3 = 2,242 ± 449 ( p < 0.05 vs. G2), G4 = 1,901 ± 420 ( p < 0.05 vs. G2), G5 = 1,756 ± 329, and G6 = 1,396 ± 243. Islet stimulation indices were equivalent between the groups and similar to controls (G1). Insulin content (ng/IE) was different between the treatment groups with the highest insulin content in islets harvested from HMP pancreata. Dithizone staining for islets consistently showed more uniform digestion of the perfused organs, with greater separation of the tissue, less entrapped islets, and higher islet yield and purity. The salutary effects of HMP for 24 h were also manifest after 30-min prior warm ischemia. We conclude that 24 h of HMP is well tolerated, leading to moderate edema but no loss of function of the harvested islets. The edema appears to aid in enzymatic digestion, producing a greater yield and purity of islets compared with pancreas subjected to 24 h of static cold storage.

Purpose of review This review considers the potential of machine perfusion to preserve livers for clinical transplantation, including steatotic or ischaemically damaged grafts and aims to go over the most significant achievements in liver machine perfusion over the last year. To reach acceptance in liver preservation, machine perfusion will need to improve outcomes compared with simple cold storage (SCS), provide objective measures of graft viability, and resuscitate less-than-ideal grafts before transplantation. Recent findings Current machine perfusion protocols comprise both hypothermic (HMP) and normothermic (NMP) approaches. HMP increases energy stores compared to SCS, and NMP shows additional resuscitative potential. Dutkowski transplanted ischaemically damaged pig livers after HMP following SCS, which avoided graft failure observed after SCS alone. Guarrera performed 20 clinical transplants after 4–7 h HMP. Friend has performed porcine transplantations after NMP of 4–20 h and univocally demonstrated the significant resuscitative effects on ischaemically damaged grafts otherwise destined to fail. Whereas NMP promises resuscitative effects, it demands challenging, near-physiologic conditions. Subnormothermic perfusion is being tested as a promising medium in between. Summary Despite recent substantial improvements, liver preservation by machine perfusion remains limited and in contrast to the global revival of kidney machine perfusion. However, liver machine perfusion may be close to returning to clinical practice if it has not already done so. History shows that superiority alone does not guarantee immediate clinical use. Further clear-cut benefits of machine perfusion such as viability assessment will have to be accompanied by usability and human factors, and innovative and improved perfusion solutions applied in novel perfusion protocols.