Pro-opiomelanocortin and ACTH-cortisol dissociation during pediatric cardiac surgery.

In critically ill adults, high plasma cortisol in face of low ACTH coincides with high pro-opiomelanocortin (POMC) levels. Glucocorticoids further lower ACTH without affecting POMC. We hypothesized that in pediatric cardiac surgery-induced critical illness, plasma POMC is elevated, plasma ACTH transiently rises intraoperatively but becomes suppressed post-operatively, and glucocorticoid administration amplifies this phenotype. From 53 patients (0-36 months), plasma was obtained pre-operatively, intraoperatively and on post-operative day 1 and 2. Plasma was also collected from 24 healthy children. In patients, POMC was supra-normal pre-operatively (p<0.0001) but no longer thereafter (p<0.05). ACTH was never high in patients. While in glucocorticoid-naive patients ACTH became suppressed by post-operative day 1 (p<0.0001), glucocorticoid-treated patients had suppressed ACTH already intraoperatively (p≤0.0001). Pre-operatively high POMC, not accompanied by increased plasma ACTH, suggests a centrally-activated HPA-axis with reduced pituitary processing of POMC into ACTH. Increasing systemic glucocorticoid availability with glucocorticoid treatment accelerated the suppression of plasma ACTH.

Abnormal DNA methylation within HPA-axis genes years after paediatric critical illness.

BACKGROUND: Critically ill children suffer from impaired physical/neurocognitive development 2 years later. Glucocorticoid treatment alters DNA methylation within the hypothalamus-pituitary-adrenal (HPA) axis which may impair normal brain development, cognition and behaviour. We tested the hypothesis that paediatric-intensive-care-unit (PICU) patients, sex- and age-dependently, show long-term abnormal DNA methylation within the HPA-axis layers, possibly aggravated by glucocorticoid treatment in the PICU, which may contribute to the long-term developmental impairments. RESULTS: In a pre-planned secondary analysis of the multicentre PEPaNIC-RCT and its 2-year follow-up, we identified differentially methylated positions and differentially methylated regions within HPA-axis genes in buccal mucosa DNA from 818 former PICU patients 2 years after PICU admission (n = 608 no glucocorticoid treatment; n = 210 glucocorticoid treatment) versus 392 healthy children and assessed interaction with sex and age, role of glucocorticoid treatment in the PICU and associations with long-term developmental impairments. Adjusting for technical variation and baseline risk factors and correcting for multiple testing (false discovery rate < 0.05), former PICU patients showed abnormal DNA methylation of 26 CpG sites (within CRHR1, POMC, MC2R, NR3C1, FKBP5, HSD11B1, SRD5A1, AKR1D1, DUSP1, TSC22D3 and TNF) and three DNA regions (within AVP, TSC22D3 and TNF) that were mostly hypomethylated. These abnormalities were sex-independent and only partially age-dependent. Abnormal methylation of three CpG sites within FKBP5 and one CpG site within SRD5A1 and AKR1D1 was partly attributable to glucocorticoid treatment during PICU stay. Finally, abnormal methylation within FKBP5 and AKR1D1 was most robustly associated with long-term impaired development. CONCLUSIONS: Two years after critical illness in children, abnormal methylation within HPA-axis genes was present, predominantly within FKBP5 and AKR1D1, partly attributable to glucocorticoid treatment in the PICU, and explaining part of the long-term developmental impairments. These data call for caution regarding liberal glucocorticoid use in the PICU.

Development of muscle weakness in a mouse model of critical illness: does fibroblast growth factor 21 play a role?

BACKGROUND: Critical illness is hallmarked by severe stress and organ damage. Fibroblast growth factor 21 (FGF21) has been shown to rise during critical illness. FGF21 is a pleiotropic hormone that mediates adaptive responses to tissue injury and repair in various chronic pathological conditions. Animal studies have suggested that the critical illness-induced rise in FGF21 may to a certain extent protect against acute lung, liver, kidney and brain injury. However, FGF21 has also been shown to mediate fasting-induced loss of muscle mass and force. Such loss of muscle mass and force is a frequent problem of critically ill patients, associated with adverse outcome. In the present study, we therefore investigated whether the critical illness-induced acute rise in FGF21 is muscle-protective or rather contributes to the pathophysiology of critical illness-induced muscle weakness. METHODS: In a catheterised mouse model of critical illness induced by surgery and sepsis, we first assessed the effects of genetic FGF21 inactivation, and hence the inability to acutely increase FGF21, on survival, body weight, muscle wasting and weakness, and markers of muscle cellular stress and dysfunction in acute (30 h) and prolonged (5 days) critical illness. Secondly, we assessed whether any effects were mirrored by supplementing an FGF21 analogue (LY2405319) in prolonged critical illness. RESULTS: FGF21 was not required for survival of sepsis. Genetic FGF21 inactivation aggravated the critical illness-induced body weight loss (p = 0.0003), loss of muscle force (p = 0.03) and shift to smaller myofibers. This was accompanied by a more pronounced rise in markers of endoplasmic reticulum stress in muscle, without effects on impairments in mitochondrial respiratory chain enzyme activities or autophagy activation. Supplementing critically ill mice with LY2405319 did not affect survival, muscle force or weight, or markers of muscle cellular stress/dysfunction. CONCLUSIONS: Endogenous FGF21 is not required for sepsis survival, but may partially protect muscle force and may reduce cellular stress in muscle. Exogenous FGF21 supplementation failed to improve muscle force or cellular stress, not supporting the clinical applicability of FGF21 supplementation to protect against muscle weakness during critical illness.

Abnormal DNA methylation within genes of the steroidogenesis pathway two years after paediatric critical illness and association with stunted growth in height further in time.

BACKGROUND: Former critically ill children show an epigenetic age deceleration 2 years after paediatric intensive care unit (PICU) admission as compared with normally developing healthy children, with stunted growth in height 2 years further in time as physical correlate. This was particularly pronounced in children who were 6 years or older at the time of critical illness. As this age roughly corresponds to the onset of adrenarche and further pubertal development, a relation with altered activation of endocrine pathways is plausible. We hypothesised that children who have been admitted to the PICU, sex- and age-dependently show long-term abnormal DNA methylation within genes involved in steroid hormone synthesis or steroid sulphation/desulphation, possibly aggravated by in-PICU glucocorticoid treatment, which may contribute to stunted growth in height further in time after critical illness. RESULTS: In this preplanned secondary analysis of the multicentre PEPaNIC-RCT and its follow-up, we compared the methylation status of genes involved in the biosynthesis of steroid hormones (aldosterone, cortisol and sex hormones) and steroid sulphation/desulphation in buccal mucosa DNA (Infinium HumanMethylation EPIC BeadChip) from former PICU patients at 2-year follow-up (n = 818) and healthy children with comparable sex and age (n = 392). Adjusting for technical variation and baseline risk factors and corrected for multiple testing (false discovery rate < 0.05), former PICU patients showed abnormal DNA methylation of 23 CpG sites (within CYP11A1, POR, CYB5A, HSD17B1, HSD17B2, HSD17B3, HSD17B6, HSD17B10, HSD17B12, CYP19A1, CYP21A2, and CYP11B2) and 4 DNA regions (within HSD17B2, HSD17B8, and HSD17B10) that were mostly hypomethylated. These abnormalities were partially sex- (1 CpG site) or age-dependent (7 CpG sites) and affected by glucocorticoid treatment (3 CpG sites). Finally, multivariable linear models identified robust associations of abnormal methylation of steroidogenic genes with shorter height further in time, at 4-year follow-up. CONCLUSIONS: Children who have been critically ill show abnormal methylation within steroidogenic genes 2 years after PICU admission, which explained part of the stunted growth in height at 4-year follow-up. The abnormalities in DNA methylation may point to a long-term disturbance in the balance between active sex steroids and mineralocorticoids/glucocorticoids after paediatric critical illness, which requires further investigation.

The association of hypoglycemia with outcome of critically ill children in relation to nutritional and blood glucose control strategies.

BACKGROUND: Withholding parenteral nutrition (PN) until one week after PICU admission facilitated recovery from critical illness and protected against emotional and behavioral problems 4 years later. However, the intervention increased the risk of hypoglycemia, which may have counteracted part of the benefit. Previously, hypoglycemia occurring under tight glucose control in critically ill children receiving early PN did not associate with long-term harm. We investigated whether hypoglycemia in PICU differentially associates with outcome in the context of withholding early PN, and whether any potential association with outcome may depend on the applied glucose control protocol. METHODS: In this secondary analysis of the multicenter PEPaNIC RCT, we studied whether hypoglycemia in PICU associated with mortality (N = 1440) and 4-years neurodevelopmental outcome (N = 674) through univariable comparison and multivariable regression analyses adjusting for potential confounders. In patients with available blood samples (N = 556), multivariable models were additionally adjusted for baseline serum NSE and S100B concentrations as biomarkers of neuronal, respectively, astrocytic damage. To study whether an association of hypoglycemia with outcome may be affected by the nutritional strategy or center-specific glucose control protocol, we further adjusted the models for the interaction between hypoglycemia and the randomized nutritional strategy, respectively, treatment center. In sensitivity analyses, we studied whether any association with outcome was different in patients with iatrogenic or spontaneous/recurrent hypoglycemia. RESULTS: Hypoglycemia univariably associated with higher mortality in PICU, at 90 days and 4 years after randomization, but not when adjusted for risk factors. After 4 years, critically ill children with hypoglycemia scored significantly worse for certain parent/caregiver-reported executive functions (working memory, planning and organization, metacognition) than patients without hypoglycemia, also when adjusted for risk factors including baseline NSE and S100B. Further adjustment for the interaction of hypoglycemia with the randomized intervention or treatment center revealed a potential interaction, whereby tight glucose control and withholding early PN may be protective. Impaired executive functions were most pronounced in patients with spontaneous or recurrent hypoglycemia. CONCLUSION: Critically ill children exposed to hypoglycemia in PICU were at higher risk of impaired executive functions after 4 years, especially in cases of spontaneous/recurrent hypoglycemia.

Skeletal Muscle Myokine Expression in Critical Illness, Association With Outcome and Impact of Therapeutic Interventions.

Context: Muscle expresses and secretes several myokines that bring about benefits in distant organs. Objective: We investigated the impact of critical illness on muscular expression of irisin, kynurenine aminotransferases, and amylase; association with clinical outcome; and impact of interventions that attenuate muscle wasting/weakness. Methods: We studied critically ill patients who participated in 2 randomized controlled trials (EPaNIC/NESCI) and documented time profiles in critically ill mice. Included in the study were 174 intensive care unit (ICU) patients (day 8 ± 1) vs 19 matched controls, and 60 mice subjected to surgery/sepsis vs 60 pair-fed healthy mice. Interventions studied included 7-day neuromuscular electrical stimulation (NMES), and withholding parenteral nutrition (PN) in the first ICU week (late PN) vs early PN. The main outcome measures were FNDC5 (irisin- precursor), KYAT1, KYAT3, and amylase mRNA expression in skeletal muscle. Results: Critically ill patients showed 34% to 80% lower mRNA expression of FNDC5, KYAT1, and amylases than controls (P < .0001). Critically ill mice showed time-dependent reductions in all mRNAs compared with healthy mice (P ≤ .04). The lower FNDC5 expression in patients was independently associated with a higher ICU mortality (P = .015) and ICU-acquired weakness (P = .012), whereas the lower amylase expression in ICU survivors was independently associated with a longer ICU stay (P = .0060). Lower amylase expression was independently associated with a lower risk of death (P = .048), and lower KYAT1 expression with a lower risk of weakness (P = .022). NMES increased FNDC5 expression compared with unstimulated muscle (P = .016), and late PN patients had a higher KYAT1 expression than early PN patients (P = .022). Conclusion: Expression of the studied myokines was affected by critical illness and associated with clinical outcomes, with limited effects of interventions that attenuate muscle wasting or weakness.

Long-term impact of paediatric critical illness on the difference between epigenetic and chronological age in relation to physical growth.

BACKGROUND: Altered DNA-methylation affects biological ageing in adults and developmental processes in children. DNA-methylation is altered by environmental factors, trauma and illnesses. We hypothesised that paediatric critical illness, and the nutritional management in the paediatric intensive care unit (PICU), affects DNA-methylation changes that underly the developmental processes of childhood ageing. RESULTS: We studied the impact of critical illness, and of the early use of parenteral nutrition (early-PN) versus late-PN, on "epigenetic age-deviation" in buccal mucosa of 818 former PICU-patients (406 early-PN, 412 late-PN) who participated in the 2-year follow-up of the multicentre PEPaNIC-RCT (ClinicalTrials.gov-NCT01536275), as compared with 392 matched healthy children, and assessed whether this relates to their impaired growth. The epigenetic age-deviation (difference between PedBE clock-estimated epigenetic age and chronological age) was calculated. Using bootstrapped multivariable linear regression models, we assessed the impact hereon of critical illness, and of early-PN versus late-PN. As compared with healthy children, epigenetic age of patients assessed 2 years after PICU-admission deviated negatively from chronological age (p < 0.05 in 51% of bootstrapped replicates), similarly in early-PN and late-PN groups. Next, we identified vulnerable subgroups for epigenetic age-deviation using interaction analysis. We revealed that DNA-methylation age-deceleration in former PICU-patients was dependent on age at time of illness (p < 0.05 for 83% of bootstrapped replicates), with vulnerability starting from 6 years onwards. Finally, we assessed whether vulnerability to epigenetic age-deviation could be related to impaired growth from PICU-admission to follow-up at 2 and 4 years. Multivariable repeated measures ANOVA showed that former PICU-patients, as compared with healthy children, grew less in height (p = 0.0002) and transiently gained weight (p = 0.0003) over the 4-year time course. Growth in height was more stunted in former PICU-patients aged ≥ 6-years at time of critical illness (p = 0.002) than in the younger patients. CONCLUSIONS: As compared with healthy children, former PICU-patients, in particular those aged ≥ 6-years at time of illness, revealed epigenetic age-deceleration, with a physical correlate revealing stunted growth in height. Whether this vulnerability around the age of 6 years for epigenetic age-deceleration and stunted growth years later relates to altered endocrine pathways activated at the time of adrenarche requires further investigation.

Assessment of aberrant DNA methylation two years after paediatric critical illness: a pre-planned secondary analysis of the international PEPaNIC trial.

Critically ill children requiring intensive care suffer from impaired physical/neurocognitive development 2 y later, partially preventable by omitting early use of parenteral nutrition (early-PN) in the paediatric intensive-care-unit (PICU). Altered methylation of DNA from peripheral blood during PICU-stay provided a molecular basis hereof. Whether DNA-methylation of former PICU patients, assessed 2 y after critical illness, is different from that of healthy children remained unknown. In a pre-planned secondary analysis of the PEPaNIC-RCT (clinicaltrials.gov-NCT01536275) 2-year follow-up, we assessed buccal-mucosal DNA-methylation (Infinium-HumanMethylation-EPIC-BeadChip) of former PICU-patients (N = 406 early-PN; N = 414 late-PN) and matched healthy children (N = 392). CpG-sites differentially methylated between groups were identified with multivariable linear regression and differentially methylated DNA-regions via clustering of differentially methylated CpG-sites using kernel-estimates. Analyses were adjusted for technical variation and baseline risk factors, and corrected for multiple testing (false-discovery-rate <0.05). Differentially methylated genes were functionally annotated (KEGG-pathway database), and allocated to three classes depending on involvement in physical/neurocognitive development, critical illness and intensive medical care, or pre-PICU-admission disorders. As compared with matched healthy children, former PICU-patients showed significantly different DNA-methylation at 4047 CpG-sites (2186 genes) and 494 DNA-regions (468 genes), with most CpG-sites being hypomethylated (90.3%) and with an average absolute 2% effect-size, irrespective of timing of PN initiation. Of the differentially methylated KEGG-pathways, 41.2% were related to physical/neurocognitive development, 32.8% to critical illness and intensive medical care and 26.0% to pre-PICU-admission disorders. Two years after critical illness in children, buccal-mucosal DNA showed abnormal methylation of CpG-sites and DNA-regions located in pathways known to be important for physical/neurocognitive development.

Development and validation of clinical prediction models for acute kidney injury recovery at hospital discharge in critically ill adults.

PURPOSE: Acute kidney injury (AKI) recovery prediction remains challenging. The purpose of the present study is to develop and validate prediction models for AKI recovery at hospital discharge in critically ill patients with ICU-acquired AKI stage 3 (AKI-3). METHODS: Models were developed and validated in a development cohort (n = 229) and a matched validation cohort (n = 244) from the multicenter EPaNIC database to create prediction models with the least absolute shrinkage and selection operator (Lasso) machine-learning algorithm. We evaluated the discrimination and calibration of the models and compared their performance with plasma neutrophil gelatinase-associated lipocalin (NGAL) measured on first AKI-3 day (NGAL_AKI3) and reference model that only based on age. RESULTS: Complete recovery and complete or partial recovery occurred in 33.20% and 51.23% of the validation cohort patients respectively. The prediction model for complete recovery based on age, need for renal replacement therapy (RRT), diagnostic group (cardiac/surgical/trauma/others), and sepsis on admission had an area under the receiver operating characteristics curve (AUROC) of 0.53. The prediction model for complete or partial recovery based on age, need for RRT, platelet count, urea, and white blood cell count had an AUROC of 0.61. NGAL_AKI3 showed AUROCs of 0.55 and 0.53 respectively. In cardiac patients, the models had higher AUROCs of 0.60 and 0.71 than NGAL_AKI3's AUROCs of 0.52 and 0.54. The developed models demonstrated a better performance over the reference models (only based on age) for cardiac surgery patients, but not for patients with sepsis and for a general ICU population. CONCLUSION: Models to predict AKI recovery upon hospital discharge in critically ill patients with AKI-3 showed poor performance in the general ICU population, similar to the biomarker NGAL. In cardiac surgery patients, discrimination was acceptable, and better than NGAL. These findings demonstrate the difficulty of predicting non-reversible AKI early.

Impact of duration of critical illness and level of systemic glucocorticoid availability on tissue-specific glucocorticoid receptor expression and actions: A prospective, observational, cross-sectional human and two translational mouse studies.

BACKGROUND: Reduced glucocorticoid-receptor (GR) expression in blood suggested that critically ill patients become glucocorticoid-resistant necessitating stress-doses of glucocorticoids. We hypothesised that critical illness evokes a tissue-specific, time-dependent expression of regulators of GR-action which adaptively guides glucocorticoid action to sites of need. METHODS: We performed a prospective, observational, cross-sectional human study and two translational mouse studies. In freshly-isolated neutrophils and monocytes and in skeletal muscle and subcutaneous adipose tissue of 137 critically ill patients and 20 healthy controls and in skeletal muscle and adipose tissue as well as in vital tissues (heart, lung, diaphragm, liver, kidney) of 88 septic and 26 healthy mice, we quantified gene expression of cortisone-reductase 11ß-HSD1, glucocorticoid-receptor-isoforms GRα and GRß, GRα-sensitivity-regulating-co-chaperone FKBP51, and GR-action-marker GILZ. Expression profiles were compared in relation to illness-duration and systemic-glucocorticoid-availability. FINDINGS: In patients' neutrophils, GRα and GILZ were substantially suppressed (p≤0·05) throughout intensive care unit (ICU)-stay, while in monocytes low/normal GRα coincided with increased GILZ (p≤0·05). FKBP51 was increased transiently (neutrophils) or always (monocytes,p≤0·05). In patients' muscle, 11ß-HSD1 and GRα were low-normal (p≤0·05) and substantially suppressed in adipose tissue (p≤0·05); FKBP51 and GILZ were increased in skeletal muscle (p≤0·05) but normal in adipose tissue. GRß was undetectable. Increasing systemic glucocorticoid availability in patients independently associated with further suppressed muscle 11ß-HSD1 and GRα, further increased FKBP51 and unaltered GILZ (p≤0·05). In septic mouse heart and lung, 11ß-HSD1, FKBP51 and GILZ were always high (p≤0·01). In heart, GRα was suppressed (p≤0·05), while normal or high in lung (all p≤0·05). In diaphragm, 11ß-HSD1 was high/normal, GRα low/normal and FKBP51 and GILZ high (p≤0·01). In kidney, 11ß-HSD1 transiently increased but decreased thereafter, GRα was normal and FKBP51 and GILZ high (p≤0·01). In liver, 11ß-HSD1 was suppressed (p≤0·01), GRα normal and FKBP51 high (p≤0·01) whereas GILZ was transiently decreased but elevated thereafter (p≤0·05). Only in lung and diaphragm, treatment with hydrocortisone further increased GILZ. INTERPRETATION: Tissue-specific, time-independent adaptations to critical illness guided GR-action predominantly to vital tissues such as lung, while (partially) protecting against collateral harm in other cells and tissues, such as neutrophils. These findings argue against maladaptive generalised glucocorticoid-resistance necessitating glucocorticoid-treatment. FUNDING: Research-Foundation-Flanders, Methusalem-Program-Flemish-Government, European-Research-Council, European-Respiratory-Society.

DNA methylation alterations in muscle of critically ill patients.

BACKGROUND: Intensive care unit (ICU)-acquired weakness can persist beyond ICU stay and has been associated with long-term functional impairment of ICU survivors. Recently, DNA methylation alterations were found in the blood of ICU patients, partially explaining long-term developmental impairment of critically ill children. As illness-induced aberrant DNA methylation theoretically could also be involved in long-term weakness, we investigated whether the DNA methylation signature in muscle of adult critically ill patients differs from that in muscle of healthy controls. METHODS: Genome-wide methylation was determined (Infinium® HumanMethylationEPIC BeadChips) in DNA extracted from skeletal muscle biopsies that had been collected on Day 8 ± 1 in ICU from 172 EPaNIC-trial patients [66% male sex, median age 62.7 years, median body mass index (BMI) 25.9 kg/m2 ] and 20 matched healthy controls (70% male sex, median age 58.0 years, median BMI 24.4 kg/m2 ). Methylation status of individual cytosine-phosphate-guanine (CpG) sites of patients and controls was compared with F-tests, using the Benjamini-Hochberg false discovery rate to correct for multiple comparisons. Differential methylation of DNA regions was assessed with bump hunting, with 1000 permutations assessing uncertainty, expressed as family-wise error rate. Gene expression was investigated for 10 representative affected genes. RESULTS: In DNA from ICU patients, 565 CpG sites, associated with 400 unique genes, were differentially methylated as compared with controls (average difference 3.2 ± 0.1% ranging up to 16.9%, P < 0.00005). Many of the associated genes appeared highly relevant for muscle structure and function/weakness, including genes involved in myogenesis, muscle regeneration, nerve/muscle membrane excitability, muscle denervation/re-innervation, axon guidance/myelination/degeneration/regeneration, synapse function, ion channelling with especially calcium signalling, metabolism (glucose, protein, and fat), insulin signalling, neuroendocrine hormone regulation, mitochondrial function, autophagy, apoptosis, oxidative stress, Wnt signalling, transcription regulation, muscle fat infiltration during regeneration, and fibrosis. In patients as compared with controls, we also identified two hypomethylated regions, spanning 18 and 3 CpG sites in the promoters of the HIC1 and NADK2 genes, respectively (average differences 5.8 ± 0.01% and 12.1 ± 0.04%, family-wise error rate <0.05). HIC1 and NADK2 play important roles in muscle regeneration and postsynaptic acetylcholine receptors and in mitochondrial processes, respectively. Nine of 10 investigated genes containing DNA methylation alterations were differentially expressed in patients as compared with controls (P ≤ 0.03). CONCLUSIONS: Critically ill patients present with a different DNA methylation signature in skeletal muscle as compared with healthy controls, which in theory could provide a biological basis for long-term persistence of weakness in ICU survivors. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00512122, registered on 31 July 2007.

Obesity attenuates inflammation, protein catabolism, dyslipidaemia, and muscle weakness during sepsis, independent of leptin.

BACKGROUND: Muscle weakness is a frequently occurring complication of sepsis, associated with increased morbidity and mortality. Interestingly, obesity attenuates sepsis-induced muscle wasting and weakness. As the adipokine leptin is strongly elevated in obesity and has been shown to affect muscle homeostasis in non-septic conditions, we aimed to investigate whether leptin mediates the protective effect of obesity on sepsis-induced muscle weakness. METHODS: In a mouse model of sepsis, we investigated the effects of genetic leptin inactivation in obese mice (leptin-deficient obese mice vs. diet-induced obese mice) and of leptin supplementation in lean mice (n = 110). We assessed impact on survival, body weight and composition, markers of muscle wasting and weakness, inflammation, and lipid metabolism. In human lean and overweight/obese intensive care unit (ICU) patients, we assessed markers of protein catabolism (n = 1388) and serum leptin (n = 150). RESULTS: Sepsis mortality was highest in leptin-deficient obese mice (53% vs. 23% in diet-induced obese mice and 37% in lean mice, P = 0.03). Irrespective of leptin, after 5 days of sepsis, lean mice lost double the amount of lean body mass than obese mice (P < 0.0005). Also, irrespective of leptin, obese mice maintained specific muscle force up to healthy levels (P = 0.3) whereas lean mice suffered from reduced specific muscle force (72% of healthy controls, P < 0.0002). As compared with lean septic mice, both obese septic groups had less muscle atrophy, liver amino acid catabolism, and inflammation with a 50% lower plasma TNFα increase (P < 0.005). Conversely, again mainly irrespective of leptin, obese mice lost double amount of fat mass than lean mice after 5 days of sepsis (P < 0.0001), showed signs of increased lipolysis and ketogenesis, and had higher plasma HDL and LDL lipoprotein concentrations (P ≤ 0.01 for all). Muscle fibre type composition was not altered during sepsis, but a higher atrophy sensitivity of type IIb fibres compared with IIa and IIx fibres was observed, independent of obesity or leptin. After 5 days of critical illness, serum leptin was higher (P < 0.0001) and the net waste of nitrogen (P = 0.006) and plasma urea-to-creatinine ratio (P < 0.0001) was lower in overweight/obese compared with lean ICU human patients. CONCLUSIONS: Leptin did not mediate the protective effect of obesity against sepsis-induced muscle wasting and weakness in mice. Instead, obesity-independent of leptin-attenuated inflammation, protein catabolism, and dyslipidaemia, pathways that may play a role in the observed muscle protection.

Phasing out DEHP from plastic indwelling medical devices used for intensive care: Does it reduce the long-term attention deficit of critically ill children?

BACKGROUND: Children who have been critically ill face long-term developmental impairments. Iatrogenic exposure to di(2-ethylhexyl)phthalate (DEHP), a plasticizer leaching from plastic indwelling medical devices used in the pediatric intensive care unit (PICU), has been associated with the pronounced attention deficit observed in children 4 years after critical illness. As concerns about DEHP toxicity increased, governmental authorities urged the phase out of DEHP in indwelling medical devices and replacement with alternative plasticizers. We hypothesized that exposure to DEHP decreased over the years, attenuating the pronounced long-term attention deficit of these vulnerable children. METHODS: We compared plasma concentrations of 3 oxidative DEHP metabolites (5cx-MEPP, 5OH-MEHP, 5oxo-MEHP) on the last PICU day in 216 patients who participated in the Tight Glucose Control study (2004-2007) and 334 patients who participated in the PEPaNIC study (2012-2015) and survived PICU stay. Corresponding minimal exposures to these metabolites (plasma concentration multiplied with number of days in PICU) were also evaluated. In patients with 4-year follow-up data, we compared measures of attention (standardized reaction times and consistency). Comparisons were performed with univariable analyses and multivariable linear regression analyses adjusted for baseline risk factors. RESULTS: In the PEPaNIC patients, last PICU day plasma concentrations of 5cx-MEPP, 5OH-MEHP, 5oxo-MEHP and their sum, and corresponding minimal exposures, were reduced to 17-69% of those in the Tight Glucose Control study (p < 0.0001). Differences remained significant after multivariable adjustment (p ≤ 0.001). PEPaNIC patients did not show better attention than patients in the Tight Glucose Control study, also not after multivariable adjustment for risk factors. CONCLUSION: Exposure of critically ill children to DEHP in the PICU decreased over the years, but the lower exposure did not translate into improved attention 4 years later. Whether the residual exposure may still be toxic or whether the plasticizers replacing DEHP may not be safe for neurodevelopment needs further investigation.

C-reactive protein rise in response to macronutrient deficit early in critical illness: sign of inflammation or mediator of infection prevention and recovery.

PURPOSE: Withholding parenteral nutrition (PN) early in critical illness, late-PN, has shown to prevent infections despite a higher peak C-reactive protein (CRP). We investigated whether the accentuated CRP rise was caused by a systemic inflammatory effect mediated by cytokines or arose as a consequence of the different feeding regimens, and whether it related to improved outcome with late-PN. METHODS: This secondary analysis of the EPaNIC-RCT first investigated, with multivariable linear regression analyses, determinants of late-PN-induced CRP rise and its association with cytokine responses (IL-6, IL-10, TNF-α) in matched early-PN and late-PN patients requiring intensive care for ≥ 3 days. Secondly, with multivariable logistic regression and Cox proportional-hazard analyses, we investigated whether late-PN-induced CRP rises mediated infection prevention and enhanced recovery or reflected an adverse effect counteracting such benefits of late-PN. RESULTS: CRP peaked on day 3, higher with late-PN [216(152-274)mg/l] (n = 946) than with early-PN [181(122-239)mg/l] (n = 946) (p < 0.0001). Independent determinants of higher CRP rise were lower carbohydrate and protein intakes (p ≤ 0.04) with late-PN, besides higher blood glucose and serum insulin concentrations (p ≤ 0.01). Late-PN did not affect cytokines. Higher CRP rises were independently associated with more infections and lower likelihood of early ICU discharge (p ≤ 0.002), and the effect size of late-PN versus early-PN on these outcomes was increased rather than reduced after adjusting for CRP rise, not confirming a mediating role. CONCLUSIONS: The higher CRP rise with late-PN, explained by the early macronutrient deficits, did not relate to cytokine responses and thus did not reflect more systemic inflammation. Instead of mediating clinical benefit on infection or recovery, the accentuated CRP rise appeared an adverse effect reducing such late-PN benefits.

Persisting neuroendocrine abnormalities and their association with physical impairment 5 years after critical illness.

BACKGROUND: Critical illness is hallmarked by neuroendocrine alterations throughout ICU stay. We investigated whether the neuroendocrine axes recover after ICU discharge and whether any residual abnormalities associate with physical functional impairments assessed 5 years after critical illness. METHODS: In this preplanned secondary analysis of the EPaNIC randomized controlled trial, we compared serum concentrations of hormones and binding proteins of the thyroid axis, the somatotropic axis and the adrenal axis in 436 adult patients who participated in the prospective 5-year clinical follow-up and who provided a blood sample with those in 50 demographically matched controls. We investigated independent associations between any long-term hormonal abnormalities and physical functional impairments (handgrip strength, 6-min walk distance, and physical health-related quality-of-life) with use of multivariable linear regression analyses. RESULTS: At 5-year follow-up, patients and controls had comparable serum concentrations of thyroid-stimulating hormone, thyroxine (T4), triiodothyronine (T3) and thyroxine-binding globulin, whereas patients had higher reverse T3 (rT3, p = 0.0002) and lower T3/rT3 (p = 0.0012) than controls. Patients had comparable concentrations of growth hormone, insulin-like growth factor-I (IGF-I) and IGF-binding protein 1 (IGFBP1), but higher IGFBP3 (p = 0.030) than controls. Total and free cortisol, cortisol-binding globulin and albumin concentrations were comparable for patients and controls. A lower T3/rT3 was independently associated with lower handgrip strength and shorter 6-min walk distance (p ≤ 0.036), and a higher IGFBP3 was independently associated with higher handgrip strength (p = 0.031). CONCLUSIONS: Five years after ICU admission, most hormones and binding proteins of the thyroid, somatotropic and adrenal axes had recovered. The residual long-term abnormality within the thyroid axis was identified as risk factor for long-term physical impairment, whereas that within the somatotropic axis may be a compensatory protective response. Whether targeting of the residual abnormality in the thyroid axis may improve long-term physical outcome of the patients remains to be investigated. Trial registration ClinicalTrials.gov: NCT00512122, registered on July 31, 2007 ( https://www.clinicaltrials.gov/ct2/show/NCT00512122 ).

Altered cholesterol homeostasis in critical illness-induced muscle weakness: effect of exogenous 3-hydroxybutyrate.

BACKGROUND: Muscle weakness is a complication of critical illness which hampers recovery. In critically ill mice, supplementation with the ketone body 3-hydroxybutyrate has been shown to improve muscle force and to normalize illness-induced hypocholesterolemia. We hypothesized that altered cholesterol homeostasis is involved in development of critical illness-induced muscle weakness and that this pathway can be affected by 3-hydroxybutyrate. METHODS: In both human critically ill patients and septic mice, the association between circulating cholesterol concentrations and muscle weakness was assessed. In septic mice, the impact of 3-hydroxybutyrate supplementation on cholesterol homeostasis was evaluated with use of tracer technology and through analysis of markers of cholesterol metabolism and downstream pathways. RESULTS: Serum cholesterol concentrations were lower in weak than in non-weak critically ill patients, and in multivariable analysis adjusting for baseline risk factors, serum cholesterol was inversely correlated with weakness. In septic mice, plasma cholesterol correlated positively with muscle force. In septic mice, exogenous 3-hydroxybutyrate increased plasma cholesterol and altered cholesterol homeostasis, by normalization of plasma mevalonate and elevation of muscular, but not hepatic, expression of cholesterol synthesis genes. In septic mice, tracer technology revealed that 3-hydroxybutyrate was preferentially taken up by muscle and metabolized into cholesterol precursor mevalonate, rather than TCA metabolites. The 3-hydroxybutyrate protection against weakness was not related to ubiquinone or downstream myofiber mitochondrial function, whereas cholesterol content in myofibers was increased. CONCLUSIONS: These findings point to a role for low cholesterol in critical illness-induced muscle weakness and to a protective mechanism-of-action for 3-hydroxybutyrate supplementation.

Early neuromuscular electrical stimulation reduces the loss of muscle mass in critically ill patients - A within subject randomized controlled trial.

PURPOSE: To investigate the effect of Neuromuscular Electrical Stimulation (NMES) on muscle thickness, strength and morphological and molecular markers of the quadriceps. MATERIALS AND METHODS: Adult critically ill patients with an expected prolonged stay received unilateral quadriceps NMES sessions for 7 consecutive days. Before and after the intervention period, quadriceps thickness was measured with ultrasound. After the intervention period, strength was assessed in cooperative patients and muscle biopsies were taken. Multivariable regression was performed to identify factors affecting muscle thickness loss. RESULTS: Muscle thickness decreased less in the stimulated leg (-6 ± 16% versus -12 ± 15%, p = 0.014, n = 47). Strength was comparable. Opioid administration, minimal muscle contraction and more muscle thickness loss in the non-stimulated muscle were independently associated with better muscle thickness preservation. Stimulated muscles showed a shift towards larger myofibers and higher MyHC-I gene expression. NMES did not affect gene expression of other myofibrillary proteins, MuRF-1 or atrogin-1. Signs of myofiber necrosis and inflammation were comparable for both muscles. CONCLUSIONS: NMES attenuated the loss of muscle mass, but not of strength, in critically ill patients. Preservation of muscle mass was more likely in patients receiving opioids, patients with a minimal muscle contraction during NMES and patients more prone to lose muscle mass. TRIAL REGISTRATION: clinicaltrials.govNCT02133300.

Time course of altered DNA methylation evoked by critical illness and by early administration of parenteral nutrition in the paediatric ICU.

BACKGROUND: A genome-wide study identified de novo DNA methylation alterations in leukocytes of children at paediatric intensive care unit (PICU) discharge, offering a biological basis for their impaired long-term development. Early parenteral nutrition (early-PN) in PICU, compared with omitting PN in the first week (late-PN), explained differential methylation of 23% of the affected CpG-sites. We documented the time course of altered DNA methylation in PICU and the impact hereon of early nutritional management. RESULTS: We selected 36 early-PN and 36 late-PN matched patients, and 42 matched healthy children. We quantified DNA methylation on days 3, 5 and 7 for the 147 CpG-sites of which methylation was normal upon PICU admission in this subset and altered by critical illness at PICU discharge. Methylation in patients differed from healthy children for 64.6% of the 147 CpG-sites on day 3, for 72.8% on day 5 and for 90.5% on day 7 as revealed by ANOVA at each time point. Within-patients methylation time course analyses for each CpG-site identified different patterns based on paired t test p value and direction of change. Rapid demethylation from admission to day 3 occurred for 76.2% of the CpG-sites, of which 67.9% remained equally demethylated or partially remethylated and 32.1% further demethylated beyond day 3. From admission to day 3, 19.7% of the CpG-sites became hypermethylated, of which, beyond day 3, 34.5% remained equally hypermethylated or partially demethylated again and 65.5% further hypermethylated. For 4.1% of the CpG-sites, changes only appeared beyond day 3. Finally, for the CpG-sites affected by early-PN on the last PICU day, earlier changes in DNA methylation were compared for early-PN and late-PN patients, revealing that 38.9% were already differentially methylated by day 3, another 25.0% by day 5 and another 13.9% by day 7. CONCLUSIONS: Critical illness- and early-PN-induced changes in DNA methylation occurred mainly within 3 days. Most abnormalities were at least partially maintained or got worse with longer time in PICU. Interventions targeting aberrant DNA methylation changes should be initiated early.

The clinical potential of GDF15 as a "ready-to-feed indicator" for critically ill adults.

BACKGROUND: Circulating growth-differentiation factor-15 (GDF15), a cellular stress marker, abruptly increases during critical illness, but its later time course remains unclear. GDF15 physiologically controls oral intake by driving aversive responses to nutrition. Early parenteral nutrition (PN) in ICU patients has overall been shown not beneficial. We hypothesized that low GDF15 can identify patients who benefit from early PN, tolerate enteral nutrition (EN), and resume spontaneous oral intake. METHODS: In secondary analyses of the EPaNIC-RCT on timing of PN initiation (early PN versus late PN) and the prospective observational DAS study, we documented the time course of circulating GDF15 in ICU (N = 1128) and 1 week post-ICU (N = 72), compared with healthy subjects (N = 65), and the impact hereon of randomization to early PN versus late PN in propensity score-matched groups (N = 564/group). Interaction between upon-admission GDF15 and randomization for its outcome effects was investigated (N = 4393). Finally, association between GDF15 and EN tolerance in ICU (N = 1383) and oral intake beyond ICU discharge (N = 72) was studied. RESULTS: GDF15 was elevated throughout ICU stay, similarly in early PN and late PN patients, and remained high beyond ICU discharge (p < 0.0001). Upon-admission GDF15 did not interact with randomization to early PN versus late PN for its outcome effects, but higher GDF15 independently related to worse outcomes (p ≤ 0.002). Lower GDF15 was only weakly related to gastrointestinal tolerance (p < 0.0001) and a steeper drop in GDF15 with more oral intake after ICU discharge (p = 0.05). CONCLUSION: In critically ill patients, high GDF15 reflected poor prognosis and may contribute to aversive responses to nutrition. However, the potential of GDF15 as "ready-to-feed indicator" appears limited. TRIAL REGISTRATION: ClinicalTrials.gov , NCT00512122, registered 31 July 2007, https://www.clinicaltrials.gov/ct2/show/NCT00512122 (EPaNIC trial) and ISRCTN, ISRCTN 98806770, registered 11 November 2014, http://www.isrctn.com/ISRCTN98806770 (DAS trial).

Towards a fasting-mimicking diet for critically ill patients: the pilot randomized crossover ICU-FM-1 study.

BACKGROUND: In two recent randomized controlled trials, withholding parenteral nutrition early in critical illness improved outcome as compared to early up-to-calculated-target nutrition, which may be explained by beneficial effects of fasting. Outside critical care, fasting-mimicking diets were found to maintain fasting-induced benefits while avoiding prolonged starvation. It is unclear whether critically ill patients can develop a fasting response after a short-term nutrient interruption. In this randomized crossover pilot study, we investigated whether 12-h nutrient interruption initiates a metabolic fasting response in prolonged critically ill patients. As a secondary objective, we studied the feasibility of monitoring autophagy in blood samples. METHODS: In a single-center study in 70 prolonged critically ill patients, 12-h up-to-calculated-target feeding was alternated with 12-h fasting on day 8 ± 1 in ICU, in random order. Blood samples were obtained at the start of the study, at the crossover point, and at the end of the 24-h study period. Primary endpoints were a fasting-induced increase in serum bilirubin and decrease in insulin requirements to maintain normoglycemia. Secondary outcomes included serum insulin-like growth factor I (IGF-I), serum urea, plasma beta-hydroxybutyrate (BOH), and mRNA and protein markers of autophagy in whole blood and isolated white blood cells. To obtain a healthy reference, mRNA and protein markers of autophagy were assessed in whole blood and isolated white blood cells of 23 matched healthy subjects in fed and fasted conditions. Data were analyzed using repeated-measures ANOVA, Fisher's exact test, or Mann-Whitney U test, as appropriate. RESULTS: A 12-h nutrient interruption significantly increased serum bilirubin and BOH and decreased insulin requirements and serum IGF-I (all p ≤ 0.001). Urea was not affected. BOH was already increased from 4 h fasting onwards. Autophagic markers in blood samples were largely unaffected by fasting in patients and healthy subjects. CONCLUSIONS: A 12-h nutrient interruption initiated a metabolic fasting response in prolonged critically ill patients, which opens perspectives for the development of a fasting-mimicking diet. Blood samples may not be a good readout of autophagy at the tissue level. TRIAL REGISTRATION: ISRCTN, ISRCTN98404761. Registered 3 May 2017.