Relative to day 0, samples from days 3 and 7 showed gradually increasing levels of both essential and nonessential amino acids (Fig. 3A) and urea cycle metabolites (Fig. 3B). Specifically, from day 0 to day 7, plasma concentrations for both citrulline and ornithine increased (p = 0.04 and 0.05, respectively).
We then determined metabolite pathways which differed over time among subjects who started PN. Overall, after false discovery rate correction, 5 of 102 identified metabolites showed statistically significant variation over the first week after exposure to PN (Table 2).
Generally, the initiation of PN was also associated with increased concentrations of plasma amino acids. However, subjects also showed decreased concentrations of urea cycle metabolites and essential fatty acids over time (Fig. 2C and D).
Relative to day 0, samples from days three and seven showed a gradual rise in both essential and non-essential amino acids (Fig. 4A), decreased urea cycle metabolites (Fig. 4B), and decreasing essential fatty acids (Fig. 4C). Specifically, from day 0 to day 7, subjects who started PN demonstrated rising plasma concentrations for taurine and phenylalanine (p = 0.04 and 0.05, respectively), decreasing levels of urate (p = 0.03), and decreasing omega-6 and omega-3 fatty acids (p = 0.05 and 0.03, respectively).
In this study of critically ill surgical patients, we applied broad-based metabolomics in a novel way to determine the metabolic response to artificial nutritional support. The institution of EN was associated with a number of metabolic changes including amino-acid repletion, urea cycle upregulation, antioxidant restoration, and increased RNA synthesis, which, taken together, reflect anabolism. Subjects receiving parenteral nutrition had fewer changes in metabolic pathways. Although there was evidence of amino acid repletion, this could simply be measurement of the amino acids that were infused as part of the parenteral formula. The reductions in circulating fatty acids perhaps reflect intermittent administration of lipids; of note, omega-6 fatty acids are absent in parenteral lipids. These observations suggest that parenteral nutrients do not promote urea cycling, antioxidant metabolism, or RNA transcription to a similar degree that enteral nutritional support does. This supports the notion that parenteral nutrients are not used as effectively as enteral nutrients.2
Initiation of EN was associated with restoration of antioxidant equilibrium over time. This was seen as progressively increasing levels of vitamin and antioxidant substrates (betaine and biotin), and diminishing levels of antioxidant catabolites (4-pyridoxic acid). Biotin is known as vitamin B7 and is a cofactor in carboxylase-based reactions in protein lysis.19 Betaine is an antioxidant and methyl donor which is thought to protect against osmotic stress, modulate inflammation, and regulate lipid metabolism.20 Finally, 4-pyridoxic acid is a catabolite of vitamin B6 involved in nicotinamide metabolism.19 Restoration of these antioxidants with EN may be particularly relevant for trauma patients, who generally have ongoing depletion of antioxidant stores over the first week of injury.11
Of note, prior studies have found similar associations with EN and antioxidant repletion. For example, Windsor and colleagues21 studied 34 patients with acute pancreatitis who started on EN or PN, and found that the group on EN had lower markers of acute inflammation and an increased total antioxidant capacity. Other systemic reviews have also confirmed that EN appears to restore antioxidant balance more effectively than parenteral nutrition.22
Plasma amino acid concentrations and urea cycle products increased after starting enteral nutrition and approached levels measured in healthy volunteers. This suggests that EN is associated with restoration of circulating amino acids, coupled with processing of excess amino acids to their end-products in the urea cycle.19 This interpretation is consistent with prior literature showing that gut absorption of amino acids is tightly regulated to maintain a steady-state in the plasma, and any excess enteral amino acids are catabolized directly to urea cycle end-products in the liver.23,24 The increases in urea cycle products likely indicate that enteral amino acids are effectively used as a source for energy.
In contrast, subjects on PN showed plasma amino acid concentrations which were often higher than those in healthy volunteers, and urea cycle products that were not clearly increased. These data are consistent with the fact that PN is not subject to the same hepatic “first-pass” effect as EN,23 and therefore subjects on PN do not have tightly regulated plasma amino acid concentrations, or shunting of excess amino-acids directly to the urea cycle. Therefore, it appears that parenteral infusion can increase amino acid concentrations, but these amino acids are not efficiently metabolized. Our data are corroborated by a prior study of 49 trauma patients and 43 healthy volunteers, where PN initiation was associated with higher plasma levels of amino acids.25
As expected, subjects on PN showed downtrending levels of essential omega fatty acids, which are not supplemented in our PN formulas. In addition, PN subjects showed increasing levels of carnitine, which is an amino acid involved in fatty acid transport from plasma to both skeletal and smooth muscle.19,26 A gradual increase in carnitine can be expected in subjects who receive regular intravenous lipid infusions, where continuous transport of lipid out of the intravascular space must occur.
Several limitations are relevant to the interpretation of this study and are related to the current capabilities of mass spectrometry–based metabolomics. First, all metabolite changes in this study should be interpreted with caution. Metabolites are often involved in multiple pathways, and change in a metabolite's concentration could represent a change in utilization, or a change in production, or both. Therefore, individual metabolite changes should be interpreted in concert with other metabolites in the pathways of interest.29 Second, given the large quantity of data and multiple analyses in this study, there are more opportunities for random highly biased results (false positives). We have partly accounted for this fact using a false discovery rate correction in all our analyses, but our findings still need to be replicated in independent larger studies before any definitive conclusions are drawn. Third, observed effects may be due differences between the subjects in the two cohorts. For example, patients receiving parenteral nutrition were more likely to have an ileus, gastrointestinal tract dysfunction, and perhaps an altered microbiome; these may have influenced the metabolic response to nutrients, irrespective of the route of administration. However, we accounted for most other major confounding factors by excluding those subjects with cancer, chronic organ dysfunction, pregnancy, obesity, active infection, or a recent major operation. Fourth, it is possible that observed effects are due to differences in timing of nutrition initiation, which could lead to bias. However, we attempted to account for such individual variation by adjusting our analyses for clustering of serial observations within subjects,30 thereby accounting for potential bias due to individual factors like nutrition timing.
The metabolic response to enteral nutrition includes a cascade of events related to amino acid metabolism, urea cycling, RNA synthesis, and antioxidant repletion. Parenteral nutrition appears to increase plasma amino acid concentrations, without concomitant increase in their metabolism. Also, fatty acid concentrations dropped markedly. This suggests that parenteral nutrients are used less effectively than enteral nutrients. Biomarkers reported in this study may eventually become clinically useful in guiding nutrition therapy for critically ill patients.
B.A.P., M.S., D.D., H.G., S.L.N., D.R., and G.E.O. designed this study. B.A.P., B.W., and G.E.O. conducted the literature search. All authors contributed to data collection. B.A.P., M.S., D.D., H.G., S.L.N., D.R., and G.E.O. performed data analysis and interpretation. B.A.P. and G.E.O. wrote the article, which all authors critically revised.
This article was prepared with financial support from the National Institute of Health grant 2T32 GM007037 (GEO and BP). We wish to thank Lauren Jacobson, MS, Peter Louras, MS and Laura Hennessey, RN for their contributions to sample collection for this study.
The authors declare no conflicts of interest.
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Dr. Panna A. Codner (Milwaukee, Wisconsin): The manuscript is extremely well-written and actually a fascinating read with just a huge amount of data. And I’m sure the authors had a hard time to compress it into such a short presentation.
Indeed, there are innumerable questions that can come from this preliminary study and it really fits very well within the precision medicine model that is of interest to so many people.
The gist of the manuscript, although not necessarily the gist of the presentation, really was around the molecular evidence that the body metabolizes nutrients given parenterally very different than it does with nutrients given enterally.
This is something we have known intrinsically but maybe this is the beginning of figuring out why, how, and if it can be modified.
I really tried to keep my questions simple as points of clarification because the authors, I’m sure, are working on many of the potential follow-up questions for the future. But I really have five questions.
First, most of your exclusion criteria which, in the manuscript, include a history of cancer, endocrine disorders, pregnancy, chronic organ dysfunction, obesity and active infection, make sense in this preliminary type of study.
You also included only immediate postoperative patients, but according to the manuscript only abdominal orthopedic ones. Is there something intrinsically in these groups that would make them different from, say, a thoracic or a neurosurgical procedure?
Second, your study had three groups: healthy volunteers—all, presumably, on enteral nutrition—critically-ill patients on enteral nutrition, and those on parenteral nutrition.
You constructed two models: one for those on enteral and one for those on parenteral and really focused on each group’s before and after comparisons rather than comparing between groups.
It’s unclear from the manuscript where the healthy controls fit in here. Were they included in the enteral nutrition group? And would that skew the results?
Third, there was a difference in time between the insult and initiation of nutrition and, presumably, study enrollment in the two groups. Do you think that played a role in the differences between the subjects’ response to initiation of nutrition? That difference, I believe, was close to a week.
Fourth, along those same lines, all ten of the enteral patients were victims of trauma; whereas, only half were trauma victims in the parenteral group. Why add in the potential confounder of a more varied study group?
Finally, ileus, itself, I think, is an organ dysfunction. The parenteral group consisted almost exclusively of patients with intolerance of enteral feeds. Do you think that some of the lack of anabolic response seen with the parenteral nutrition was not necessarily the fault of the route but related to something within the GI tract which wasn’t happening in patients with GI tract dysfunction?
Again, I think this is a phenomenal piece of work and the manuscript clearly reflects the authors’ enthusiasm and expertise on the subject. This type of individual metabolic information may be invaluable to future clinicians in mitigating some of the catabolic consequences and I look forward to the authors’ continued work.
Dr. Carrie Sims (Philadelphia, Pennsylvania): A quick question, actually two. Was there any crossover in your design? Where all the patients who were initially in the enteral group kept on enteral nutrition for seven days or did some people in the parenteral group crossover to enteral?
And when I looked at your data for Day 0, those numbers actually looked different, whether you were assigned to the parenteral or enteral of nutrition group so maybe those people are inherently different, aside from the fact they were given different sources of nutrition.
The second thing is did you look at fatty acid metabolism? There is some interest in whether or not when you switch from glucose metabolism to predominantly fatty acid metabolism it’s a marker for a change in acute inflammation to chronic inflammation so I would be interested in knowing if you found any differences. Thank you so much.
Dr. Grant E. O’Keefe (Seattle, Washington): Thank you. I did have a lot of trouble getting everything down into this talk, as you could probably tell.
We enrolled non-trauma patients primarily out of necessity. We could have waited longer but I think that in the interest of getting the study done we wanted to get some more patients who were treated with parenteral nutrition.
In regard to the question of the healthy controls, they weren’t part of the two artificial nutrition groups and I hope we’ve clarified that now. They were just used as a reference for the metabolomic measurements.
And you are correct. The time of starting either enteral or parenteral nutrition probably addresses some of the other questions as well but these are quite different patients.
I think that it’s probably not fair to compare the patients in the enteral nutrition group directly to the patients in the parenteral nutrition group. There are probably many factors that are related to when we started their nutritional support that determine their metabolomic status.
On average, enteral nutrition started on about Day 3 whereas the median start day was Day 8 for the parenteral group.
I think an ileus is probably one of the most underappreciated components of organ failure and perhaps that is, at least in part, related to some of the differences in the metabolic responses is that the parenteral nutrition patients see and not just their inability or differences in handling the nutrients.
You asked whether any patients crossed over from one group to the other. No, none of the patients crossed over. They were all independent. That was deliberate.
The fatty acid metabolism is fascinating. There are so much data here I had to cut some of it out but I think the notion that there is more fatty acid metabolism later on in a critically-ill patient’s course was borne out in our data.
What we see is that some of the fatty acids are much lower in the parenteral nutrition patients. And that’s probably not simply due to the mode of nutritional support. We observed not just the omega-3s and the omega-6s, but all the fatty acids are lower in the parenteral nutrition patients.