1H NMR global metabolic phenotyping of acute pancreatitis in the emergency unit.
TL;DR: It is demonstrated that combinatorial biomarkers have a strong diagnostic and prognostic potential in AP with relevance to clinical decision making in the emergency unit.
Abstract: We have investigated the urinary and plasma metabolic phenotype of acute pancreatitis (AP) patients presenting to the emergency room at a single center London teaching hospital with acute abdominal pain using 1H NMR spectroscopy and multivariate modeling. Patients were allocated to either the AP (n = 15) or non-AP patients group (all other causes of abdominal pain, n = 21) on the basis of the national guidelines. Patients were assessed for three clinical outcomes: (1) diagnosis of AP, (2) etiology of AP caused by alcohol consumption and cholelithiasis, and (3) AP severity based on the Glasgow score. Samples from AP patients were characterized by high levels of urinary ketone bodies, glucose, plasma choline and lipid, and relatively low levels of urinary hippurate, creatine and plasma-branched chain amino acids. AP could be reliably identified with a high degree of sensitivity and specificity (OPLS-DA model R2 = 0.76 and Q2Y = 0.59) using panel of discriminatory biomarkers consisting of guanine, hippurate ...
Summary (3 min read)
- Acute pancreatitis (AP) is an inflammatory condition associated with a progressive systemic inflammatory response (SIRS) and, in severe cases, autonecrosis of pancreatic tissue, organ failure, and death, also known as KEYWORDS.
- This lacks sensitivity and specificity, and it is subject to variation in its diagnostic threshold over time after the initial pancreatic insult.
- 6−10 Other generic physiological scoring systems are used in critical care such as the Acute Physiology and Chronic Health Evaluation (APACHE II) score,11 a generic physiological measurement based on 12 parameters, that is designed to measure the severity of disease for adult patients 24 hrs following admission to intensive care units.
- 14−16 The determination of metabolite changes that describe a biological phenotype based on either 1H nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry (MS) has been widely applied for global profiling to define diagnostic or prognostic biofluid profiles for physiological or pathological states.
- A key feature of AP is abdominal pain, a common and often nonspecific presenting complaint for a large array of other surgical pathologies that require urgent treatment.
- This was an observational control study of consecutive patients presenting with acute abdominal pain to a single center “Accident and Emergency” unit at St. Mary’s hospital, London, U.K.
- Patients were excluded if they had been discharged from hospital within 72 h with the same pain, if they were unconscious at presentation, if they were pregnant, if they were under the age of 18, or if they had undergone surgery within the previous 6 weeks or received a blood transfusion.
- Patients with congenital pancreatic malformations or those with cystic fibrosis were also excluded.
- Patients were investigated and managed as per the current British Society of Gastroenterology guidelines.
- Samples were collected from each patient for the first 5 days of their admission.
Sample Collection and Preparation
- If catheterized, a “clean catch” sample was taken.
- Urine samples were kept on ice after collection prior to being stored frozen at −80 °C.
- The mixture was transferred into a 5 mm outer diameter NMR tube.
- 1H NMR spectra of the plasma samples were acquired employing two 1-D NMR experiments.
- All method characteristics and details are detailed elsewhere.
- (OPLS-DA)27 was also carried out to optimize recovery of potential biomarkers, that is, compounds with high correlation and covariance with the class (e.g., disease severity or etiology).
- Modeling was conducted in SIMCA-P v.12.1 using unit variance scaling.
- Metabolite identification was based on chemical shifts published in literature and statistical total correlation spectroscopy of peaks29 (e.g., of urinary 3-hydroxyisovalerate is shown in Supplemental Figure 1, SI).
- Data were analyzed by unpaired two-tailed Student’s t test.
- The etiologies of pain in the non-AP patients group were heterogeneous, reflecting the typical distribution of the causes of acute abdominal pain presenting to a typical accident and emergency department32 (Table 1).
- One of the cholelithiasis AP cohorts had necrotic pancreatitis.
- One patient with cholelithiasis AP and one patient with alcoholic AP diagnosis had a myocardial infarction during their admission, both of whom were treated with a standard acute coronary syndrome protocol.
- The APACHE II score11 varied significantly between the groups, indicating the increased general severity of illness in the AP group compared with the non-AP group.
Characterizing the Urinary Phenotype of AP
- The urinary 1H NMR spectra from this clinical study demonstrated substantial interindividual variation.
- Figure 1, shows urinary 1H NMR spectra from an alcoholic AP patient (A) and a non-AP patient (B).
- Unsurprisingly, large concentrations of acetate, ethanol, acetone, and ethyl glucuronide were also observed in this patient, reflecting the underlying etiology .
- NMR spectral profiles of acetaminophen and ibuprofen and their urinary metabolites have been published previously.
- The drug-related origin of the mannitol was supported by a Pearson correlation test35 using all compounds in the profile and a selected driver peak for the mannitol signal (δ 3.805 ppm, (5-CH)), with an established cutoff of p < 0.05.
Plasma NMR Spectra
- There were few obvious disease-specific metabolites that varied systematically between raw spectra; however, clear quantitative changes in the spectra were observed, particularly in concentrations of VLDL, LDL, and nonesterified fatty acids.
- Likewise, a comparison of CPMG raw spectra from different patients, an AP and non-AP patient diagnosed with diverticulitis, is presented in Figure 4 in the SI, where differences of VLDL were observed.
Multivariate Modeling of Patients with Severe Abdominal Pain
- Three main analyses were performed based on clinical criteria: (1) diagnosis of AP (AP vs non-AP), (2) etiology (alcohol consumption vs cholelithiasis), and (3) AP severity, based on a validated modified Glasgow score comparison of mild (Glasgow score 1) versus moderate to severe (Glasgow score 2 and 3).
- PLS-DA plots for each clinical scenario in urine and plasma (CPMGand 1-D-spectral) data showed positive predictive models for all with the exception of prediction of etiology in the plasma (1-D-plasma and CPMG) data set.
- Discriminant metabolites found in plasma and urine for the three clinical scenarios are summarized in Tables 2−4 and discussed below.
- Because hippurate is a wellestablished gut microbial cometabolite derived from glycine conjugation of benzoic acid,37,38 a subgroup analysis of antibiotic-treated patients (n = 10; 5non-AP/5AP) and nonantibiotic-treated (n = 21; 12non-AP/9AP) patients was performed to determine if antibiotic ingestion in the AP diagnostic model was responsible for the difference in urinary excretion of hippurate between the two groups .
- Moreover, the cross-validated OPLS-DA was also more robust .
Diagnosis of AP
- Multivariate analysis of both urinary and plasma 1H NMR spectra was able to accurately stratify patients presenting with acute abdominal pain in a clinical setting into those with AP and those with other heterogeneous causes of abdominal pain ).
- Urine provided a stronger predictor of AP diagnosis than plasma .
- Thus, it is very likely that the striking rise in plasma VLDLs seen here relates to the generalized severity of the systemic response rather than AP-specific changes.
Etiology of AP Caused by Alcohol Consumption and Cholelithiasis
- The two causes of AP in this series were alcohol (9/15) and cholelithiasis, which is representative of the epidemiology of the condition.
- Metabolites such as acetone, acetoacetate, and 3- hydroxyisovalerate (Table 3) were predictive for alcoholinduced AP.
- It is well established that alcohol consumption influences the regulation of key pathways such as gluconeogenesis.
- Ketone bodies are products of the oxidative pathway of alcohol metabolism53 and are thus concordant with this observation discriminating the alcohol-induced AP patients, although because there were no dietary records for the patients, the possibility that anorexia, associated with severe abdominal pain, may have been systematically different between the groups and therefore a contributor to the ketone body profile.
AP Severity Based on the Glasgow Severity Score
- The raw NMR spectra from severely unwell AP patients were highly variable in structure compared with control patients .
- These data provide a strong metabolic phenotype of the severity of illness and a clear metabolic description of AP disease severity.
- AP patients with moderate to severe AP disease (Glasgow 2−3) had higher levels of glucose and reduced plasma relative concentrations of alanine, valine, choline, the acetyl signal from α1-acid dx.doi.org/10.1021/pr500161w | J. Proteome Res. XXXX, XXX, XXX−XXXI glycoprotein (NAC1), and urinary creatinine (Table 4).
- As previously described, this combined cohort of metabolic changes is suggestive of insulin resistance54 and probable failure of pancreatic function, with the NAC1 most likely reflecting generalized inflammation.
Differentiation of Metabolic Phenotypes within the Non-AP Group
- It was possible to distinguish between subclasses of the non-AP group using the metabolic profiles, which is of importance to the clinical setting.
- High levels of indoleacetate have typically been related to gastrointestinal cancer and hepato-biliary tract cancer, and high concentrations have been observed in patients with cirrhosis, diabetes, and cholelithiasis occasionally.
- Nevertheless, the authors have demonstrated a convincing metabolic phenotype for the diagnosis, etiology, and severity of AP.
- Scores plot of the first versus second component of the principal component analysis using the most discriminant metabolites from urine and CPMG.
- This material is available free of charge via the Internet at http://pubs.acs.org.
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Cites methods from "1H NMR global metabolic phenotyping..."
...This has also been reported by Bro et al. (2015), who used plasma to determine breast cancer biomarkers, or by Villaseñor et al. (2014), who described the global metabolic phenotyping of acute pancreatitis, and by Dumas et al. (2016), who used this technique to study metabolic syndrome and fatty…...
Cites background from "1H NMR global metabolic phenotyping..."
...Their results suggested that combinatorial biomarkers consisting of guanine , 74 hippurate and creatine (urine), and valine, alanine a d lipoproteins (plasma) have a strong diagnostic 75 and prognostic potential in APP ....
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Q1. What are the contributions mentioned in the paper "H nmr global metabolic phenotyping of acute pancreatitis in the emergency unit" ?
The authors have investigated the urinary and plasma metabolic phenotype of acute pancreatitis ( AP ) patients presenting to the emergency room at a single center London teaching hospital with acute abdominal pain using H NMR spectroscopy and multivariate modeling. This work has demonstrated that combinatorial biomarkers have a strong diagnostic and prognostic potential in AP with relevance to clinical decision making in the emergency unit.