During critical illness, hypermetabolism and decreased nutritional intake lead to malnutrition within days, which can complicate recovery. As an adjunct to care, specialized nutritional support, delivered as enteral or parenteral feedings, provides macronutrients and micronutrients until oral dietary intake can resume. By helping in part to offset the hypermetabolism of critical illness, early nutritional delivery is known to improve overall outcome and has become an essential component of modern critical care. Whereas parenteral (intravenous) nutrition (PN) held considerable promise during its development, comparisons between parenteral and enteral feedings generally demonstrate fewer infectious complications and better clinical outcomes with enteral nutritional (EN) delivery. Therefore, when no barriers to gut feeding exist, enteral feedings have emerged as the standard of care (1–3).
Preclinical studies of different nutrition modalities suggest that EN helps to sustain both innate and adaptive immune function more effectively than PN (4–7). Compared with enterally fed animals, those that received PN demonstrated increased gut bacterial translocation (8) and had increased mortality when challenged with bacterial lipopolysaccharide (endotoxin) or with intra-abdominal infection (9). Dysregulation of both innate and adaptive immune functions is believed to influence these adverse outcomes.
Most clinical analyses of the influence of feeding modality on immune function document increased levels of proinflammatory cytokines in postoperative patients who had received preoperative PN versus those fed enterally (10, 11). Similarly, studies in healthy volunteer subjects suggest that the route of nutrition influences innate immune responsiveness. We have previously shown that a 7-day period of continuous PN in healthy subjects resulted in an enhanced systemic inflammatory and acute-phase response after subsequent in vivo challenge with endotoxin compared with those receiving intermittent enteral feedings (12). Interestingly, however, a later study by Santos et al (13) in 1994 failed to reveal differences in systemic inflammatory mediators after endotoxin challenge between normal subjects fed orally compared with those who received 7 days of PN.
Proposed mechanisms underlying the relationship between innate immunity and route of nutritional delivery, enteral versus parenteral, include the alterations in the balance of gut-mediated autonomic nervous system outflows. In particular, efferent vagus nerve activity appears to highly influence peripheral immune cell populations. This “cholinergic anti-inflammatory pathway” (14–16) involves the nutrient-mediated release of cholecystokinin (CCK), which acts both centrally and locally via vagal afferent pathways to stimulate α-7-nicotinic acetylcholine receptors on immune cells, especially macrophages, by way of vagal efferents.
Although the influence of specialized nutrition support upon this pathway has not, to the best of our knowledge, been examined in humans subjected to differing routes of feeding, preclinical studies suggest that intestinal luminal nutrients influence autonomic activity, in part, by modulating vagal afferent signals and the balance of efferent autonomic signals. Intestinal gavage with long-chain lipids enhances afferent vagus nerve activity via stimulation of intestinal CCK receptors (17, 18). This nutrient-specific stimulation of parasympathetic activity is also associated with improved survival in a model of acute hemorrhagic stress (19).
These observations of parasympathetic stimulation by enteral feedings led us to conduct a preliminary study with the hypothesis that differing routes of nutrient delivery, enteral versus parenteral, might have a systemic impact on autonomic activity and on the pattern of gene activation within peripheral immune cell populations. To determine if an autonomic impact was detectible in healthy subjects, we compared measures of heart rate variability (HRV) to determine if continuous EN or PN differentially influenced outputs, with orally fed subjects as controls. Heart rate variability analysis is a noninvasive technique of studying patterns between successive QRS complexes using continuous electrocardiography (ECG) over various time and frequency scales. It is used to detect physiologic complexity and can reflect homeostatic feedback between organ systems such as the central nervous system and the heart. Specifically, vagal and parasympathetic tone and sympathovagal balance can be evaluated with this technique (20). To assess the impact of differential feeding on patterns of peripheral blood monocyte (PBM) gene expression, microarray analysis was used to compare continuous EN and PN groups.
MATERIALS AND METHODS
Ten healthy subjects between the ages of 18 and 36 years were recruited by public advertisement for participation. All subjects provided written, informed consent under guidelines approved by the institutional review board of the UMDNJ–Robert Wood Johnson Medical School. Inclusion criteria were as follows: adults aged 18 to 40 years with “normal general health” as demonstrated by medical history and physical examination and by laboratory testing. Women of child-bearing potential were screened for pregnancy risk and were included if using reliable contraception.
Subjects underwent an initial screening visit that included comprehensive history and physical and laboratory testing to establish suitability for inclusion; eligible subjects returned within 3 weeks for admission to the Clinical Research Center (CRC). Throughout admission, all subjects were attended to by nursing staff on a 24-h basis. Subjects were precluded from any physical activity other than walking about the study unit.
Subjects were randomized to two feeding groups: (i) a continuous feeding PN group (n = 7) and (ii) a continuous nasogastric EN group (Nutren; Nestle Nutrition, Minnetonka, Minn) (n = 3). The feeding regimens were designed to provide a nonprotein calories intake of 22 to 24 kcal/kg per day over each 24-h study day. This rate of substrate administration was chosen to approximate the resting energy expenditure of unstressed normal subjects and to eliminate any confounding influence of significant undernutrition or overnutrition. An additional cohort (n = 5) of healthy subjects, recruited, screened, and monitored in the same fashion, was admitted to the CRC and allowed an oral diet ad libitum and served as the control group (PO) for HRV analysis.
All subjects fasted overnight beginning at 11 PM on the day of CRC admission. Subjects in the PN group had a peripherally inserted central catheter placed by the interventional radiology department between 7 and 10 AM the morning following admission (day 1). The EN feeding subjects had placement of an eight French Dobbhoff silastic tube (Kendall, Mansfield, Mass) during the same time frame. Proper placement of all feeding devices was confirmed by radiograph. The PO group required no feeding device placement.
Whole blood was collected in EDTA and heparin tubes from patients on day 1 before initiation of the PN or EN feedings. Blood was again collected after 72 h of continuous feeding (day 4) while the assigned feeding regimen was being administered.
Monocyte isolation from peripheral blood
Blood was collected in CPT tubes (BD Biosciences, Franklin Lakes, NJ), and 400 μL of RosetteSep (Stem Cell Technologies, Vancouver, British Columbia, Canada) was immediately added as previously described (21). After 20 min at room temperature, tubes were centrifuged at 1,200g for 25 min at room temperature. The interfacial layer was collected; the top of the CPT tube was washed and added to the interfacial layer. The monocytes were recovered by centrifugation. Residual red blood cells were lysed with EL buffer (Qiagen, Valencia, Calif) following centrifugation, and the monocyte pellet was subsequently lysed in TRIzol (Invitrogen, Carlsbad, Calif) and immediately frozen at −70°C.
Assessment of monocyte purity was performed on cells triple-stained with CD66b–fluoroscein isothiocyanate (Beckman-Coulter, Miami, Fla), CD2-phycoerythrin (Becton-Dickinson Biosciences, San Jose, Calif) and CD33-peridinin-chlorophyll-protein complex, and cyanine dye 5.5 PerCP-Cy5.5 (BD Biosciences) for 30 min at 4°C. After washing 1× with phosphate-buffered saline containing 0.5% bovine serum albumin, cells were analyzed with flow cytometry FACSCalibur (Becton-Dickinson Biosciences). Data were collected and analyzed using the CELLQuest software. Monocyte purity was 82% ± 1.7% (mean ± SEM) for all samples.
Preparation of RNA, cDNA, and labeled cRNA
Cell lysates in TRIzol were thawed and treated with chloroform. RNA was isolated from the aqueous phase and precipitated with isopropyl alcohol. Following alcohol wash, the RNA pellet was dried and dissolved in DEPC water. The quality and quantity of the isolated RNA were evaluated using the 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Calif).
First-strand cDNA synthesis was performed using reverse transcription (SuperScript II; Invitrogen) with 5 µg of total RNA, T7-oligo (dt)24 primer, DTT, and dNTP mix. Second-strand cDNA synthesis was then carried out with DNA polymerase I, DNA ligase, and dNTP mix, followed by additional reaction with T4 DNA polymerase (Invitrogen). Double-stranded cDNA was purified using the Focus GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, Calif).
Biotinylated cRNA was synthesized from the double-stranded cDNA using GeneChip expression 3′-amplification reagents for IVT labeling (Affymetrix) using MEGAscript T7 polymerase (Life Technologies, Grand Island, NY) in the presence of four natural ribonucleotides and one biotin-conjugated analog. The generated biotinylated cRNA was purified using the Focus GeneChip Sample Cleanup Module (Affymetrix).
Steps outlined in this section were performed by the microarray core facility at this institution. Following fragmentation of the biotinylated cRNA, 15 μg was placed in hybridization cocktail, heated to 95°C, centrifuged, and then hybridized to the Focus GeneChip microarray (Affymetrix) for 16 h at 45°C. Chips were then washed, stained with streptavidin phycoerythrin, and scanned on the Agilent Gene Array Scanner (Agilent Technologies, Santa Clara, Calif).
Analysis of microarray data
Focus chip data CEL files were imported, grouped, and analyzed using GeneSpring software (Agilent Technologies). Primary analysis was carried out by log 2 transformation followed by transformation to the median and RMA (quantile) normalization. Advanced significance analysis was performed on normalized-transformed data using paired or unpaired Student t tests as appropriate. Unless otherwise indicated below, we further defined significant probes as those with a P < 0.05 and fold change of 1.5 or greater from the prefeeding baseline (day 1) or in comparison to the alternative feeding regimen on day 4. Data were also exported for analysis by Ingenuity Pathway Analysis (Ingenuity, Palo Alto, Calif) as previously described (22). In addition, monocyte gene expression for each group was subjected to analysis by filtering for a consensus group of 266 innate immunity genes shown to be responsive to in vivo endotoxin stimulation (22). The microarray data have been submitted to Gene Expression Omnibus (accession number GSE21534).
Assessment of HRV
A supine recording of electrocardiographic output was obtained before initiation of feedings on day 1 and at 12-h intervals (9 AM and 9 PM) throughout the study period in all subjects. Each recording interval consisted of two consecutive 5-min epochs. During these determinations, heart rate was monitored using a continuous ECG technique with three standard limb leads and CardioPro 2.0 software with one Infiniti and one Procomp Plus recorder (Thought Technology, Ltd, Montreal, Quebec, Canada). Heart rate variability parameters as well as interbeat intervals were collected using ECG data at a rate of 256 samples per second as previously described (23, 24).
Analysis of HRV data
In a continuous ECG record, each QRS complex was detected, and the “normal-to-normal” intervals (all intervals between adjacent QRS complexes resulting from sinus node depolarization) were tabulated. For each epoch, noise artifact and irregular heartbeats were manually edited by visual inspection and interpolation before calculation of interbeat intervals using CardioPro software. We analyzed each epoch as previously described (23, 24). The power spectral density then was calculated using a fast Fourier transformation algorithm. All signals were exported in standard ASCII format to Excel (Microsoft Corp, Redmond, Wash) and SAS 9.0 (SAS Institute, Inc., Cary, NC) for analysis and graphics as previously described (23, 24).
Time and frequency domain measures
An analysis of HRV parameters for both time domain and frequency domain measures was performed for each recorded epoch. Time domain measures included (i) the SD of the average beat-to-beat intervals (SDANN), which measures total HRV and overall system adaptability; (ii) the square root of mean squared successive differences (RMSSD) of interbeat intervals, considered to reflect mainly vagus nerve pathways; and (iii) the percentage of successive interbeat interval differences greater than 50 ms (pNN50), associated with respiratory sinus arrhythmia and therefore also vagus nerve activity. Frequency domain measures included (i) high-frequency variability (HF) (0.15–0.4 Hz), which correlates with parasympathetic and vagal tone; (ii) low-frequency variability (LF) (0.04–0.15 Hz); and (iii) the LF/HF ratio (LF/HF) used as a measure of sympathetic/parasympathetic balance (25).
Statistical analysis of HRV parameters
Feeding route differences in parameters of HRV were determined by two-way, repeated-measures analysis of variance using Statistica version 6.1 (StatSoft, Inc, Tulsa, Okla). P < 0.05 was considered to be statistically significant. Changes due to diurnal variation were not considered in this pilot study
Characteristics of the subjects completing the study and the nutritional content of these dietary formulations are shown in Table 1. There were no changes in any parameter of biochemical screening during the course of feeding, and all blood glucose levels remained less than 120 mg/dL (data not shown).
Measures of HRV
Time domain parameters of HRV
The results for time domain parameters are shown in Figure 1 as the change (mean ± SEM) from baseline level for the duration of the feeding period. The time domain measures of HRV include one of overall autonomic adaptability (Fig. 1A, SDANN), as well as measures reflecting predominantly parasympathetic activity (Fig. 1B, pNN50; Fig. 1C, RMSSD). During the 72-h observation period, subjects in the oral diet (PO) group experienced neither decreased autonomic adaptability nor diminished parasympathetic function as reflected by the HRV data. In contrast, both the EN and PN groups exhibited a significant decrease (P < 0.001) from baseline in all three time domain parameters (SDANN, pNN50, and RMSSD) over the feeding period. In addition, comparisons between groups (PO, EN, and PN) both of overall variability and of variability over time were made. Comparing PO versus EN group revealed significant decreases in SDANN and pNN50, between the groups (main effect; P < 0.05) and over time (interaction effect; P < 0.05), after 72 h of EN. When the PO and PN groups were compared, all time domain parameters, SDANN, pNN50, and RMSSD, were significantly diminished (main effect; P < 0.02), as were changes over time (interaction effect; P < 0.02) after 72 h of PN. Between EN and PN groups, there were no significant differences in time domain parameters or in changes over time. These HRV findings demonstrate that, compared with oral feeding, both continuous EN and PN lead to diminished autonomic adaptability and decreased parasympathetic function, which worsens over time as nutrition is delivered.
Frequency domain parameters of HRV
The results for frequency domain parameters for each feeding group are presented in Figure 2 as the change from baseline (mean ± SEM). Frequency domain parameters reflect HRV waveform analysis; commonly reported measurements include LF (0.04–0.15 Hz), HF (0.15–0.4 Hz), and their ratio (LF/HF). In general, HF power measurements reflect vagal tone and parasympathetic activity; LF measures combined sympathetic and parasympathetic signaling; LF/HF ratios reflect overall sympathovagal balance. Similar to time domain measurements, subjects in the PO group experienced no differences in any of the frequency domain parameters during the 72-h observation period. However, both continuous EN and PN groups each experienced a decline from baseline in both HF power (P < 0.001) and LF power after 72 h of continuous artificial feeding (Fig. 2B, LF) (P < 0.05). The ratio of LF/HF remained unchanged in all groups in comparison to baseline and over time (Fig. 2C). In comparing the EN to the PO group, HF power decreased significantly both overall (main effect; P < 0.05) and over time (interaction effect; P < 0.05). Comparisons of PN and PO groups demonstrated decreased HF power overall (main effect; P < 0.02) and over time (interaction effect; P < 0.001). No differences were seen in LF. There were no significant differences between EN and PN for any frequency domain measures. The diminished HF power identified in the EN and PN groups indicates a significant decrease in vagal tone and in parasympathetic activity after 72 h of these continuous feeding modalities.
PBM gene expression
The number of differentially expressed gene probes detected in PBMs after 72 h of parenteral or enteral feeding compared with baseline expression before nutritional delivery is summarized in Table 2. For each group, differentially expressed probes are shown at the level of initial significance (P < 0.05) and for more highly expressed differences (both P < 0.05 and ≥1.5 fold-change). The complete listing of these gene probes is provided as Tables, Supplemental Digital Content 1 and 2, at http://links.lww.com/SHK/A136 and http://links.lww.com/SHK/A137. Very few gene expression changes met significance and fold-change criteria after 72 h of EN. After 72 h of PN, 471 genes had significant changes with 73 also meeting fold-change criteria.
Between groups, the number of differentially expressed probes detected in PBMs after 72 h of PN as compared with expression after 72 h of EN is shown in Table 3. A detailed listing of these gene probes is provided as Table, Supplemental Digital Content 3, at http://links.lww.com/SHK/A138. There were 854 significant differentially expressed genes between EN and PN groups with 157 changes meeting fold-change criteria.
Innate immune-specific gene expression
Filtering expressed genes for consensus innate immunity probes identified a number of significantly expressed (P < 0.05) genes over 72 h of PN. These differentially expressed innate immunity genes in PBMs are shown in Table 4. A small number of genes met the criteria for significance and fold change of 1.5 or greater, including upregulated genes for STAT3, Toll-like receptor 4 (TLR4), STAT2, and downregulated genes for TRAF5 and KLHDC2.
Enteral and parenteral feeding are crucial adjuncts to care during critical illness, and both have been studied extensively in the intensive care unit population. To our knowledge, this pilot study is the first, however, to assess the influence of common nutritional support modalities on parameters of autonomic activity in healthy human subjects and to correlate them with immune cell gene expression in that population. The present study documents that even relatively brief periods of continuous nutrition, either enteral or parenteral, alter measures of HRV with a reduction in host adaptability and overall parasympathetic outflow. Furthermore, because the subjects in the study were healthy, normal subjects, the data suggest that the use of either continuous EN or PN influences measures of autonomic activity in the absence of disease comorbidities or other inflammatory conditions. Changes in autonomic activity, similar to those observed in the present study, have also been noted in stressed patients and have been correlated to increased morbidity and mortality (26–29). Hence, the current observations may be important to the influence of nutritional support technologies on organ systems that depend on rhythmic autonomic signals for optimal function (30, 31), as well as on the use of these technologies in the critically ill population.
In addition to the influence of altered autonomic activity on organ system function, there is considerable evidence to suggest that the present findings, specifically that continuous EN, not just PN, depresses parasympathetic activity, may negatively impact autonomic regulation of innate immunity. Animal experiments suggest that much of the vagally mediated influence over tumor necrosis factor α (TNF-α) production occurs within tissue-fixed monocyte/macrophage cell populations (32). In the present study, we observed that the in vivo reduction in HRV parameters reflecting parasympathetic/vagus nervous activity (RMSSD, pNN50, HF) during continuous PN or EN did not influence ex vivo whole-blood immune cell responsiveness to endotoxin (data not shown). This observation is consistent with evidence that vagal signaling has a prominent influence, not so much on circulating immunocytes, but rather on splenic or other splanchnic tissue sites of TNF production and on enhanced splanchnic TNF production during PN in humans (12).
How these pathways are influenced by feeding schedule, continuous versus intermittent, is unknown. We postulate that the well-described “cholinergic anti-inflammatory pathway,” mediated by the vagus nerve, is operative involving CCK stimulation of α-7-nicotinic acetylcholine receptors on immune cells (15, 16). In an interesting series of studies, intermittent bolus feeding, but not continuous feeding, created feelings of satiety and caused appetite suppression in healthy volunteers (33, 34). These and other studies (35, 36) demonstrate that the induced neuroendocrine environment may be different between the two feeding modalities. A number of mechanisms, alone or in concert, may be involved. Continuous feeding may lead to tonic stimulation of neuroendocrine pathways and deplete preformed/stored hormones or may cause changes in neurotransmitter receptor numbers or activation thresholds. Alternatively, the lower luminal concentrations of nutrients, as present in continuous feedings compared with large bolus feedings, may not reach threshold levels necessary to stimulate gut hormone release. Also, gastric distention, which is known to stimulate vagal afferents (37), and as experienced with normal oral intake or during intermittent bolus enteral feeding but not during continuous enteral feeding, may be contributory (34). Whatever the mechanism, our preliminary findings using HRV analysis to assess vagal tone clearly demonstrate diminished parasympathetic output over time with continuous feedings, both enteral and parenteral.
Despite nearly identical patterns of altered autonomic activity between EN and PN subjects, differing patterns of PBM gene expression were observed between groups. Continuous EN, using normal gastrointestinal nutrient absorption, differentially influenced the expression of only a few genes when compared with the prefeeding samples. However, for those subjects who received continuous PN, a direct infusion of nutrients into the systemic circulation that bypasses the gut and normal hepatic metabolism, many genes (n = 471) were differentially expressed compared with prefeeding samples.
This study is the first to use PBM gene expression profiles as a means to compare enteral and parenteral feeding, and the differences between them are profound. Distinct patterns of circulating mononuclear cell gene expression have also been identified acutely in response to individual oral dietary constituents (38) as well as more chronic interventions with potentially immunomodulatory diets (39). Interestingly, the limited change in PBM gene expression after 72 h of continuous EN suggests a rapid adaptation, from a metabolic perspective, to this continuous feeding modality in healthy subjects. The findings of divergent PBM gene expression contrast sharply with the nearly identical alterations noted in HRV/autonomic outflow, between the EN and PN groups, over the study period and suggest that very different processes are responsible for such disparate results.
Previous animal studies document tissue-specific changes in local innate immune gene expression based on route of feeding—including upregulation of TLRs (40), which are critical to normal immune surveillance and tolerance(41, 42). That our parenterally fed subjects also demonstrated upregulation of monocyte TLR-4 transcripts suggests that PN influences expression of pattern recognition receptors. This finding along with those from a previous study by our group in humans demonstrating increased monocyte cell surface TNF receptor expression after PN (43) suggests that PN promotes a state of enhanced immune cell surveillance for some pattern recognition receptor and DAMP (damage-associated molecular pattern) ligands.
Specialized nutritional support, as delivered in the critical care setting, imposes systemic and tissue substrate fluxes and nutrient entrainment cues very different from those experienced with normal oral feeding (44–46). These unnatural nutrient signaling patterns, leading to altered autonomic signaling as documented by the HRV findings of the present study, may represent an additional mechanism of nutrition support risk (47, 48). Based on these findings and others, we believe it is likely that both the route (enteral versus parenteral) and timing (intermittent vs. continuous) of substrate administration influence organ system functions in complex clinical scenarios (11–13, 49, 50). During this study, the profound alterations in monocyte gene expression after PN, compared with enteral, do not appear to be related to the changes in HRV/vagal activity and likely represent separate consequences of the direct delivery of intravenous nutrition.
The present preliminary study has identified a heretofore-undocumented influence of specialized nutritional support modalities on the regulation of HRV and on autonomic activity. As investigators continue to explore the influence of vagal tone on limiting destructive hyperinflammation (14, 15), the depression of parasympathetic tone during continuous enteral feeding may be shown to contribute to overall inflammatory risk different from those associated with PN. Nutrient delivery regimens that consider both the route and timing of delivery and therefore long-evolved circadian, neurohumoral, and inflammatory mechanisms may be more physiologic and should more widely studied in critically ill patients requiring specialized nutritional support.
The author thank Dr. John Nosher, chair of the Department of Radiology, who performed the intravenous catheter insertions, for the invaluable assistance and support; Ashwini Kumar for performance of cytokine assays; M. T. Reddell for analysis of HRV and gene expression data; Drs. Paul Lehrer and Maria Katsamanis Karavidas of the Department of Psychiatry who provided guidance in the early performance and interpretation of the HRV data; and Eileen Duffy, RPh, who assisted with preparation of nutrient formulations.
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