Study objective and design
The objective of this prospective observational cohort study is to determine the predictive value of D-lactate, I-FABP and other more common serological markers of ischemia (L-lactate, lactate dehydrogenase (LDH), creatine kinase (CK) and alanine aminotransferase (ALAT)) in intensive care patients suspected to have intestinal ischemia. The results of L-lactate, LDH, CK and ALAT were readily available for the attending clinician. D-lactate and I-FABP were stored and measured later in one batch at the end of the study. D-lactate levels were additionally measured in a group of healthy volunteers to determine reference levels. At the start, this study did not fall under the Dutch research legislation (WMO) because of its observational character, despite the blood sampling. In virtually all patients a blood sample drawn for other measurements could be used for this study, which limited the number of extra blood sampling. An informal consultation of the local ethic and scientific review board (Medical Ethical Committee on research, Onze Lieve Vrouwe Gasthuis, Amsterdam, The Netherlands) was performed confirming that at that time informed consent was not needed. However, the Dutch ethical committee on research (CCMO) changed their policy concerning blood sampling in 2013, after completion of the study.
Setting
The ICU where the study was performed is a closed format highest-level mixed medical, surgical and cardiac surgery department with 20 beds in a teaching hospital.
Patients and sample collection
Over a 24-month period intensive care patients were included when the attending intensivist considered intestinal ischemia in the diagnostic workup. This consideration may have risen on admission or at any moment during intensive care stay. Intestinal ischemia was considered when physical examination and observation of bowel function might be congruent with ischemia. Exclusion criteria other than age below 18 were not defined. As soon as the consideration of intestinal ischemia was raised, a single blood sample of 4 ml was taken to determine serological data.
From each patient only the first blood sample was used in the analysis. Repeated samples were excluded.
Data collection
Baseline data of all admitted patients in the ICU are recorded in a structured and uniform way in the Patient Data Management System (Metavision®, Tel Aviv, Israel). Data from included patients were retrieved from this database including sex, age, the diagnosis at admission, mortality at the ICU and severity of illness. The Acute Physiologic and Chronic Health Evaluation (APACHE) IV and SOFA scores determined illness severity. Validated data from pathological studies, endoscopy, radiological studies and laparotomies were collected from the hospital information system xCare® (McKesson, Nieuwegein, The Netherlands).
Patient classification
Patients were classified as ‘proven intestinal ischemia’ , ‘ischemia likely’ , ‘ischemia unlikely’ or ‘no intestinal ischemia’. Classification was based on reports from the operating surgeon, pathology department, endoscopy reports and CT-scan. All available study results were independently analysed by 2 researchers (B.W and P.V.) and categorization was made without prior knowledge of serological markers including plasma D-lactate and I-FABP levels. In case of disagreement between the two researchers consensus was reached by reviewing the data together. Proven intestinal ischemia was defined as the gross disturbance of blood flow in the bowel, regardless of extent and grade. Terms used by pathologist or surgeon like ‘necrotic changes’ , ‘ischemic colitis’ , ‘transmural ischemia’ , ‘ischemic changes in the resected tissue’ , were considered decisive.
Blood samples
Lactate dehydrogenase (LDH), creatine kinase (CK), alanine aminotransferase (ALAT) and L-lactate were analysed according to instructions of the manufacturer (Roche diagnostics systems, Basel, Switzerland).
The remaining blood of the blood samples were stored in −80°C and analysed for D-lactate and I-FABP in one run after closure of the study inclusion.
D-lactate concentration was spectrophotometrically measured in heparin-plasma. To this end, heparinized blood was centrifuged at 3200 rpm for 10 min in a clinical centrifuge. 500 μL of plasma was deproteinized with 50 μL perchloric acid, mixed on a Vortex for 20 s, and kept on ice for 10 min. Next, the denatured protein solution was centrifuged at 3200 rpm for 10 min. To 350 μL of the supernatant 20 μL KOH was added, mixed for 20 s and kept on ice for 10 min. After centrifugation for 10 min the neutralized-protein-free plasma (NPFP) was used for analysis according to Herrera and co-workers [19]. In brief, D-lactate is oxidized to pyruvate by NAD+ in the presence of the D-lactate dehydrogenase. Then pyruvate is enzymatically converted by D-alanine aminotransferase (D-ALT) to D-alanine and 2-oxoglutarate. The latter reaction shifts the equilibrium to the formation of NADH. The amount of NADH formed during the reaction is stoichiometric to the amount of D-lactate in the sample, and it is measured by the increase in absorbance at 340 nm.
The assay mixture contained, in a final volume of 1000 μL: 111 mmol/L glycylglycine pH 10.0, 111 mmol/L glutamate, 4.65 mmol/L NAD+, 11.6 U/mL D-alanine aminotransferase and 50 μL heparin-plasma. After a preincubation of 30 s, the reaction was started by addition of D-LDH. The production of NADH was followed in time (10s, 100 s and 200 s) on a Shimadzu spectrophotometer at 340 nm using a molar extinction coefficient of 6300 L/mol/cm. A sample blank to correct the non-specific NAD+ transformation was processed using the same analytical conditions as for the analysis of D-lactate but without adding the enzyme D-LDH and was subtracted on all samples.
Literature has shown that D-lactate concentrations will decrease with increasing activities of L-LDH. Indeed, an activity of LDH greater than 1500 IU/L will result in a more than 10% deviation. Hence, in plasma of patients with an LDH activity >1500 the LDH was removed. A reagent blank to compensate for the small, but continuous, non-enzymatic transformation of NAD+at alkaline pH was performed in every run and was subtracted from the calibrators, QCs and samples.
Intestinal fatty acid binding protein (I-FABP) was measured in plasma using a commercially available enzyme-linked immunosorbent assay (ELISA) (Hycult Biotechnology b.v., Uden, The Netherlands). The wells of the EIA plate were coated with the monospecific polyclonal antibody (10 μg/ml). First, the samples were diluted twice. To this end, 150 μL diluent buffer was added to 150 μL sample. Next, 100 μL of the standard solution and 100 μL of the diluted sample were added to the plate and incubated for 60 min at 20 C. This was done in duplo. After incubation, 100 μL of the conjugate tracer was added and incubated for 60 min. Next 100 μL of the conjugate Streptavidin-peroxidase was added and incubated for another 60 min. The wells were washed three times with washing buffer (10 mL washing buffer with 390 mL aqua mill) after each incubation. Finally, the reaction was started by adding 100 μL of the tetramethylbenzidine (TMB) substrate (x) every 15 sec to each strip and incubated for another 20 min, and stopped by the addition of citric acid (x) every 15 sec to each strip (and mixed between). The absorbance at 450 nm was measured spectrophotometrically. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the human I-FABP standards (log). The human I-FABP concentrations of samples, which are run concurrently with the standards, were determined from the standard curve.
Statistical analysis
Continuous variables such as CK, LDH, ALAT, I-FABP, D-lactate and L-lactate were expressed as median and interquartile range (IQR) because of their skewed distribution. The comparison of groups was performed with the Mann–Whitney U-test or the Kruskal-Wallis test were appropriate. Data is shown as absolute numbers and percentage (%). Differences in proportions were evaluated using the Fisher exact test for nominal variables. Sensitivity, specificity, positive predictive value and negative predictive value were calculated according to standard methods. ROC analysis with 95% confidence interval (CI) was performed for the best performing marker, L-lactate, with a positive diagnosis of ischemia tested against the L-lactate level. P-values less than 0.05 were considered statistically significant. Analyses were performed using the statistical software SPSS version 18.0 (SPSS Inc, Chicago, Illinois, USA).
