Effect of short-term administration of lipid emulsion on endothelial glycocalyx integrity in ICU patients – A microvascular and biochemical pilot study

Abstract

BACKGROUND:

Endothelial glycocalyx (EG) is a carbohydrate-rich vascular lining of the apical surface of endothelial cells. It has been proved to have an essential role in vascular homeostasis. Lipid emulsions as part of parenteral nutrition (PN) are widely used in patients in the setting of critical care and perioperative medicine. Due to their structure, lipids may potentially interact with EG. The aim of the study was to evaluate the effect of lipid emulsion on EG.

OBJECTIVE:

To assess the influence of lipid emulsion on EG integrity in ICU patients using a videomicroscopic and biochemical methods.

METHODS:

Patients in surgical ICU after major abdominal surgery or cardio surgery and in general ICU were assessed for eligibility for this pilot observational study in University Hospital. The study was performed during the first day of adding lipids as a part of their PN. The patients were given the SMOFlipid 20% for 6 hours in prescribed dose of approx. 1 g/kg of body weight. EG integrity was measured indirectly by automated sublingual videomicroscopy calculating a parameter PBR which describes the amount of lateral deviation of red blood cells from the central column and by levels of syndecan-1 and syndecan-4 in plasma as EG degradational products. Measurements were performed before lipid administration (T0) and 30 minutes after (T6) the infusion of lipid emulsion was completed. The statistical analysis was performed at the level of significance p < 0.05, data are expressed as mean ± standard deviation (SD) and for PBR as median and interquartile range (IQR).

RESULTS:

Fifteen patients were studied, from them 9 included in final analysis. PBR (expressed in μm) increased after the lipid infusion with no statistical significance (T0 = 2.10; 1.97–2.33 vs. 2.28; 2.11–2.45, p = 0.13). At T6 both syndecans showed statistically significant decrease in their particular levels. Syndecan–1 at T0 = 2580±1013 ng/l, resp. at T6 = 2365±1077 ng/l, p = 0.02; syndecan–4 at T0 = 134±29 ng/l, resp. at T6 = 123±43 ng/l, p = 0.04.

CONCLUSION:

In our study, we showed that six hours long SMOFlipid 20% infusion had no detrimental effect on the EG integrity as assessed by PBR value and by syndecan-1 and syndecan-4 plasmatic levels. Observed decrease of syndecans shortly after lipid infusion allows us to hypothesize even possibly protecting effect of lipids on EG.

Keywords

Endothelial glycocalyx lipid emulsion syndecan-1 syndecan-4 perfused boundary region critically ill

List of abbreviations

B.w.

body weight

endothelial glycocalyx

ELISA

enzyme-linked immunosorbent assay

HDL

high-density lipoprotein

ICU

intensive care unit

IRQ

interquartile range

LDL

low-density lipoprotein

PBR

perfused boundary region

parenteral nutrition

RBC

red blood cells

S1P

sphingosine-1-phosphate

standard deviation

SpO₂

pulse oximetry

1 Background

Endothelial glycocalyx (EG) plays an important role in mechanotransduction of shear stress onto the endothelial cells and serves also as a natural barrier forming a protective coating of the inner vessel wall [1 –3]. The shear stress also promotes release of the nitric oxide from red blood cells [4]. The major components of endothelial glycocalyx include: a) glycoproteins, b) proteoglycans like heparan sulfate proteoglycans with core proteins syndecans and glypican-1, and c) glycosaminoglycans side chains [5]. The EG has been firstly indirectly described as a cell free zone in glass tubes [6].

The recent studies focused on the structural changes of EG and its unique role in the pathophysiology of human diseases, including inflammation, sepsis, trauma, cardiovascular diseases, hypertension, diabetes, hypercholesterolemia or even cancer [7 –11]. Endothelial glycocalyx shedding plays also probably an important role in promoting atherosclerosis [12 –14], interestingly, reduction of hypercholesterolemia by rosuvastatin led to partial recovery of damaged EG [15].

There is increasing evidence for EG as a signalling platform integrating several biological inputs, e.g. shear stress, chemokines, cytokines and sphingosine–1–phosphate (S1P) for maintaining vascular homeostasis [16, 17].

Intravenous lipid emulsions are an essential component of parenteral nutrition (PN). Their administration seems to be safe, well tolerated and has been found to reduce inflammatory markers in surgical patients with highly inflamed states [18]. Nevertheless, we have not found in the available literature (searching keywords: parenteral nutrition, lipid emulsion, glycocalyx) any report evaluating effect of parenteral nutrition or lipid emulsion on EG. Thus, we hypothesized, there is no acute detrimental effect of lipid emulsion, as a part of PN, on EG integrity.

2 Objective

The aim of the study was to evaluate the effect of 6 hours lasting administration of lipid emulsion on the integrity of EG in surgical ICU patients by using two distinct methods − videomicroscopy and biochemically, by determination of EG degradation products in plasma: syndecan–1 and syndecan–4.

3 Methods

3.1 Study design and setting

The study was a prospective observational single center study performed at the Department of Anesthesiology and Intensive Care and Department of Cardiac Surgery, general ICU and surgical ICU (University Hospital Hradec Kralove, Czech Republic) in the three-month period between January and March 2018. Written informed consent was obtained from all patients prior to inclusion. The study was approved by the local Ethics Committee of University Hospital Hradec Kralove (approval number: 201408 S15P). The study was performed in accordance with the Declaration of Helsinki, the current version of Good Clinical Practice, and the Clinical Trials Directive. The study was registered in Clinical Trial Database: NCT03216850.

3.2 Participants

Eligible patients were male or female at an age between 18 and 85 years, in–patients unable to sustain an adequate oral/enteral food intake after ICU admission according to ESPEN guidelines for parenteral nutrition in surgery [18]. Exclusion criteria were as follows: previous PN before entering the study, clinical or laboratory signs of organ hypoperfusion, known hypersensitivity to fish, egg, soy, peanut or any of the active ingredients of study treatments, hyperlipidaemia (cholesterol above 5 mmol/l), hypertriglyceridemia (triglycerides above 2 mmol/l), severe liver insufficiency, pancreatitis, chronic renal insufficiency, chemotherapy during or within 4 weeks before study start and administration of propofol within 24 hours prior the study.

Patients who enrolled in the study received PN containing SMOFlipid 20% (Fresenius Kabi, Bad Homburg, Germany). Lipid emulsion was infused via a central venous catheter separately for six hours, followed by all-in-one solutions with amino-acids, glucose, electrolytes, vitamins and trace elements. Each patient was assigned a patient number at the time of inclusion, and the amount of fat for infusion was calculated the target dose of lipids was 1 g/kg/b.w./day in six-hour lasting infusion. Composition of SMOFlipid 20% emulsion used in the study is displayed in Table 1.

Table 1
Composition of SMOFlipid 20%

Soybean oil [g/l] 60

MCT [g/l] 60

Olive oil [g/l] 50

Fish oil [g/l] 30

Vitamin E [mg α-tocopherol/l] Approx. 200

Egg phospholipids [g/l] 12

Glycerol [g/l] 25

Total energy [kcal/l] 2000

pH value 7,5–8,8

Osmolarity [mosmol/l] 273

Legend: MCT (medium-chain triglycerides).

3.3 Data collection and laboratory analysis

At the time of patient's enrolment, demographic data and medical history were recorded. Vital signs including blood pressure, heart rate and pulse oximetry (SpO₂) were monitored continuously and selected laboratory variables were measured at the baseline (time point T0) and 30 minutes after the end of SMOFlipid 20% infusion (time point T6). Blood samples were collected by arterial line as a first line option. If there was no arterial line place, blood samples were taken by peripheral venous access preferentially. All determinations of routine laboratory assessments, including triglycerides, other lipid parameters – total, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, electrolytes, and glycemia were drawn. Levels of syndecans have been used to assess the integrity of EG, the plasma concentration of circulating syndecan-1 and syndecan-4 was determined by enzyme-linked immunosorbent assay (RayBiotech, Norcross, Georgia, USA, Human Syndecan ELISA kit) at the same time points (T0 and T6).

3.4 Videomicroscopy and glycocalyx thickness assessment

The Perfused Boundary Region (PBR) parameter was used to monitor the possible EG change induced by SMOFlipid 20% infusion. PBR was designated to determine the extent of penetration of the flowing red blood cells in the capillary to its luminal border by calculating the radial motion of red blood cell away from central flow in the capillary. Methodology, current techniques and hardware and software possibilities for PBR measurement were in detail described previously [23, 24]. Briefly, during EG damage through various pathologic stimuli, this spreading closer to endothelial cells is more obvious and thus the PBR value becomes higher. Intravital real-time microscopy of sublingual circulation by specialized hand-held video microscope (KK camera, Research Technology Limited, Alliance Court, Honiton, UK) was performed at each time point (T0 and T6). Acquired data were processed with GlycoCheck software version 1.2.5.7211 (GlycoCheck, Maastricht, Netherlands). The software automatically measures PBR in vessels of diameter from 5 to 25μm. Automated signal quality assessment is performed by the software itself displaying direct user-friendly feedback enabling gathering only valid data. The maximal data-sampling period is 5 minutes making the monitoring less prone to interobserver variability. However, the recording stops automatically when 3000 segments in focus and without movement are acquired; hence under normal conditions much shorter periods (i.e., 1–2 minutes) are needed. Then the software selects segments with sufficient contrast with the background and counts the median RBC column width and its distribution from the intensity profile. From this intensity profile, the perfused diameter of the vessel is calculated by linear regression analysis. The PBR stands for the distance between RBC column width and perfused diameter according to the equation: (perfused diameter – median RBC column width)/2, PBR values are expressed in μm.

3.5 Statistical analysis

The study was designed as a pilot prospective observational and feasibility study because there were no existing data regarding the methodical approach to glycocalyx assessment after exposure to lipid emulsion in clinical settings. We planned to enrol in the pilot study 15 patients, such sample size was deemed to be sufficient to obtain pilot data for hypothesis verification. Results were summarized using descriptive statistics and presented as mean (SD) for continuous variables, and as median (IQR) for non-normally distributed data. Shapiro-Wilk test was used for normality tests, Student’s paired t-tests were used to test the difference between parameters obtained at T0 and T6 time points. Pearson correlation and Spearman rank order correlation analysis were performed to test the relationship between PBR values and soluble fraction of syndecans–1 and syndecan–4. The level of significance was set at p < 0.05. MedCalc 7.6.0. (MedCalc Software, Ostend, Belgium) and Prism 5 for Mac OS X (Version 5. Ob, December 19, 2008) was used for statistical analysis.

4 Results

4.1 Demographic, clinical and laboratory data

In total, 15 patients were enrolled in the study, 8 females and 7 males, the final analysis was performed in 9 patients (Fig. 1 – a flowchart of patients). The demographic and basic laboratory data are shown in Table 2. The infused SMOFlipid 20% emulsion was clinically well tolerated by all patients and no adverse events have been reported by ICU staff. All patients who completed the study received the target dose (1 g/kg b.w.) of lipids as a part of their PN prescription. Videomicroscopy and PBR measurement were performed in 13 patients with adequate quality, in two patients we were not able to obtain images in a sufficient quality (controlled automatically by GlycoCheck software) and those patients were excluded from microvascular analysis. Both syndecans levels were successfully measured at selected time points in 9/13 patients (blood samples from 4 patients got lost due to personal reasons).

Fig.1

Flowchart of patients for the final analysis Patients flow chart and reasons for withdrawal from the final analysis. PBR – perfused boundary region.

Table 2

Patients baseline demographic and laboratory data

Age [years]	64 (±12)
Weight [kg]	84 (±13)
Height [cm]	168 (±10)
Hemoglobin [g/l]	111.2 (±18.59)
Hematocrit [% ]	33 (±5)
Sodium [mmol/l]	139.7 (±3.2)
Creatinine [mmol/l]	88.15 (±29.11)

Data are shown as mean±SD, kg – kilograms, cm – centimeter, g – gram, l – liter, mmol/l – millimole per liter.

No significant differences in blood pressure, heart rate, and SpO₂ were observed between time points T0 and T6. Levels of triglycerides, total and LDL, resp. HDL cholesterol and glycemia are presented in the Table 3, at T6, there was a significant increase in triglycerides levels and also LDL–cholesterol decreased significantly.

Table 3

Selected laboratory values before (T0) and after (T6) SMOFlipid 20% infusion

Time point	T0	T6	P
Total cholesterol [mmol/l]	3.26±0.79	3.15±0.89	p = 0.43
HDL cholesterol [mmol/l]	1.1±0.35	1.01±0.23	p = 0.21
LDL cholesterol [mmol/l]	1.75±0.88	1.35±0.9*	p = 0.0007
TAG [mmol/l]	0.91±0.34	3.9±2.4	p = 0.006
Glycemia [mmol/l]	7.2±2.0	8.3±1.4	p = 0.0003

Legend: A summary of baseline biochemistry data. HDL – high-density lipoprotein, LDL – low-density lipoprotein, TAG – triacylglycerol. Data are presented as mean±SD.

4.2 Perfused boundary region

High quality real-time video images were obtained in all but 2 patients (Fig. 2). There was no statistically significant difference between PBR (expressed in μm as median, IQR) values at T0 compared to T6 (2.10; 1.97–2.33 vs. 2.28; 2.11–2.45, p = 0.13).

Fig.2

Original image of the sublingual microcirculation. SDF image of the sublingual microcirculation. Surface area: 915×686μm. Magnification: 325×. Recorded by SDF camera.

4.3 Syndecan–1 and syndecan–4 plasma levels

Similarly, as for PBR parameter, syndecan-1 values (expressed as mean±SD) statistically different inside the investigated group (p < 0.0001), the same values of difference inside the group was observed in syndecan-4. The plasmatic level of soluble portion of syndecan-1 was significantly lower after SMOFlipid 20% (2580±1014 ng/l vs. 2366±1077 ng/l, p = 0.02), the same trend was observed for syndecan-4 at the T0 compared to T6 (134±29 ng/l vs. 123±43 ng/l, p = 0.04) (Fig. 3a and 3b). No correlation between PBR and syndecans–1, resp. syndecan–4 was observed throughout the study.

Fig.3

a. Results of syndecan–1. Syndecan–1 levels in ng/l before (T0) and after (T6) lipid infusion. Data are presented as a mean±SD. b. Results of syndecan–4. Syndecan–4 levels in ng/l before (T0) and after (T6) lipid infusion. Data are presented as a mean±SD.

5 Discussion

The key finding of our study was the absence of detectable injury to EG after SMOFlipid 20% infusion in ICU patients as assessed by the use of PBR parameter and syndecans plasma levels. To put our main findings in broader clinical context, EG represents one of the earliest sites of injury (e.g. trauma of surgery) [19, 20], thus avoiding any intervention possibly harming EG integrity should be considered as the key part of any therapeutic step, including organ supporting therapies where PN belongs to.

Perfused Boundary Region remained unchanged after lipid infusion, that could be interpreted in the first line as a mean of absence of any effect of EG thickness after lipid emulsion, however, at least two alternative explanations should be considered. Firstly, there is substantial variability in PBR in critically ill patients described by others and by our group as well [21 –25], in the presented study baseline PBR values differed inside the investigated group significantly. Secondly, PBR values may be affected by several other factors, e.g. age, type of anesthesia, perioperative fluid status, types of fluids and blood products given, blood loss, previous chronic conditions of the patients etc. [26].

Also decreased levels of both syndecans after lipid load must be interpreted very cautiously, despite reaching statistically significant difference and therefore suggesting possibly protective effect of SMOFlipid 20% short term infusion on EG. The syndecans are important transmembrane proteoglycans regulating pathophysiological processes such as endothelial permeability, inflammation, immune cells activation, and lipoprotein uptake. Syndecan–1 plays a role in endothelial cells survival, proliferation and in capillary bed formation, syndecan–4 participates in the endothelial control of blood flow [27, 28]. Increased serum levels of syndecans were observed in many pathological conditions and serum syndecan–1 level represents currently one of the most used and reliable serum EG biomarkers. In our study, the levels of syndecan–1 were comparable to majority of other studies evaluating syndecan levels as a marker of EG degradation in trauma and surgery [29 –31]. Moreover, there are also other possible causes of syndecan elevation in observed population – liver or kidney dysfunction, therapy with vasopressors, ischemia and reperfusion (e.g. due to sudden hypotension), the use of propofol, hypervolemia, pulmonary embolism or infection [30 –41]. Clinical studies evaluating syndecan–4 as a marker of EG degradation are much less frequent and have conflicting results so far [34].

Hypothetically possible protective effect of lipid emulsion infusion on EG may be partially related to the content of S1P in SMOFlipid 20% emulsion, however we did not measure the content of S1P in our emulsion that may be variable depending on many factors during manufacturing lipid emulsions (e.g. type of fish oil used, phytosterols content etc.). Ceramides are at the center of sphingolipid metabolism, [18] and their metabolites, as sphingosine, S1P, and ceramide–1–phosphate are well-known biological compounds implicated in the regulation of a variety of metabolic processes, although their complex role has not been fully understood yet. S1P is reported to protect EG and to induce reparation of the heparan sulfate [16 , 42–44]. S1P is a kind of membrane phospholipid metabolite presenting as an important signalling molecule in vascular endothelial cells with an impact on cell survival, protection, thrombosis and inflammation [13]. Experimental data focusing on EG modulation, regeneration and synthesis have shown the S1P as a bioactive agent with the potential to induce recovery of the heparan sulfate component of the glycocalyx [42], furthermore, exogenous heparan sulfate and S1P restore inter-endothelial gap junctions and related regulation of transendothelial permeability [16 , 43]. The complete mechanism of S1P role in EG restoration is still not clear and other mechanisms may contribute as well. Further contributing protective factor could be the decrease of LDL cholesterol level observed at the end of lipid emulsion [45] and also HDL cholesterol levels may play the partial role. According to recent study, HDL cholesterol ranging between 71 and 101 mg/dl can be moderately protecting EG and endothelial function in hypertensive patients [46].

Unchanged EG thickness assessed by PBR together with decreased levels of syndecan–1 and syndecan–4 after infusion of SMOFlipid 20% suggests, despite the complexity of EG and its role in maintaining vascular integrity, there could be modulating effect of lipids emulsion on the EG. The role of S1P is not clear and also other components of lipid emulsions commercially available may exert some effect.

The result of our pilot study brings another small piece to the current knowledge on the safety of lipid emulsions with respect to EG integrity in ICU patients requiring lipids as a part of parenteral nutritional support. Research agenda focusing on the importance of acute endotheliopathy in determining clinical outcome of acute care patients, on avoiding iatrogenic EG injury and on maintaining EG integrity at the bedside is obviously expanding and definitely non-negligible [47]. We need urgently to know that our currently used interventions (including nutritional support) are safe also in term of effect on EG. We have found no previous study described similar findings and we hope that our work will accelerate further research focusing on the possibly modulating role of nutritional components or regimens on EG integrity in the various clinical setting.

6 Conclusion

Six hours lasting administration of lipid emulsion was not associated with significant changes of EG as assessed by microvascular PBR values and syndecan plasma levels.

Authors’ contributions

DA acquired part of the data, collected all data, evaluated the results, was responsible for manuscript editing, images, and tables preparation.

ZT acquired part of the data. Evaluated the results, performed statistics and prepared the core of the manuscript.

PD acquired part of the data, was responsible for manuscript editing and evaluation of the results.

RH performed biochemical analysis and was responsible for manuscript editing.

AT performed biochemical analysis and was responsible for manuscript editing.

MK was responsible for conduction of the study in surgical ICU.

ZZ was responsible for manuscript editing and study design.

RS was responsible for manuscript editing.

CL was responsible for manuscript editing and language check.

VC set up the study protocol, evaluated the results and was responsible for manuscript writing, editing, and submission.

All authors read and approved the final manuscript.

Conflict of interest

All the authors declare that they have no conflict of interest.

Funding

Footnotes

Acknowledgments

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