Research Article| Volume 31, ISSUE 2, 106237, February 2022

Plasma Pro-Enkephalin A and Ischemic Stroke Risk: The Reasons for Geographic and Racial Differences in Stroke Cohort


      • Higher circulating PENK-A, an endogenous opioid neuropeptide, was independently associated with ischemic stroke risk in REGARDS
      • PENK-A's association with stroke was non-linear
      • Adjustment for established stroke risk factors did not alter the relationship between PENK-A and stroke
      • PENK-A's association with stroke may be most relevant to White men



      The opioid neuropeptide pro-enkephalin A (PENK-A) may be a circulating marker of cardiovascular risk, with prior findings relevant to heart failure, kidney disease, and vascular dementia. Despite these findings, the association of PENK-A with ischemic stroke is unknown, so we examined this association in a prospective cohort study and analyzed differences by race and sex.

      Materials and Methods

      The REasons for Geographic and Racial Differences in Stroke study (REGARDS) is a prospective cohort study of 30,239 Black and White adults. Plasma PENK-A was measured in 473 participants that developed first-time ischemic stroke over 5.9 years and 899 randomly selected participants. Cox models adjusted for demographics and stroke risk factors were used to calculate hazard ratios (HRs) of stroke by baseline PENK-A.


      PENK-A was higher with increasing age, female sex, White race, lower body mass index, and antihypertensive medication use. Each SD higher increment of PENK-A was associated with an adjusted HR of 1.20 (95% CI 1.01-1.42) for stroke, with minimal confounding by stroke risk factors. Spline plots suggested a U-shaped relationship, particularly in White men, with an adjusted HR 3.88 (95% CI 1.94-7.77) for the 95th versus 50th percentile of PENK-A in White men.


      Higher baseline plasma PENK-A was independently associated with future stroke risk in REGARDS. This association was most apparent among White men. There was little confounding by established stroke risk factors, suggesting a possible causal role in stroke etiology. Further research is needed to understand the role of endogenous opioids in stroke pathogenesis.

      Key Words


      CI (confidence interval), ECG (electrocardiogram), eGFR (estimated glomerular filtration rate), HR (hazard ratio), IQR (interquartile range), NIHSS (National Institutes of Health Stroke Scale), PENK-A (pro-enkephalin A), SD (standard deviation), REGARDS (REasons for Geographic and Racial Differences in Stroke study), TOAST (Trial of ORG 10172 in Acute Stroke Treatment)
      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Journal of Stroke and Cerebrovascular Diseases
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Katan M
        • Luft A.
        Global burden of stroke.
        Semin Neurol. 2018; 38: 208-211
        • Hijazi Z
        • Lindbäck J
        • Alexander JH
        • et al.
        The ABC (age, biomarkers, clinical history) stroke risk score: a biomarker-based risk score for predicting stroke in atrial fibrillation.
        Eur Heart J. 2016; 37: 1582-1590
        • Debora Kamin Mukaz
        • A Zakai Neil
        • Salvador Cruz-Flores
        • D McCullough Louise
        • Mary Cushman
        Identifying genetic and biological determinants of race-ethnic disparities in stroke in the United States.
        Stroke. 2020; 51: 3417-3424
        • Pandian JD
        • Gall SL
        • Kate MP
        • et al.
        Prevention of stroke: a global perspective.
        The Lancet. 2018; 392: 1269-1278
        • Holm H
        • Nägga K
        • Nilsson ED
        • et al.
        High circulating levels of midregional proenkephalin A predict vascular dementia: a population-based prospective study.
        Sci Rep. 2020; 10: 8027
        • Beunders R
        • Struck J
        • Wu AHB
        • et al.
        Proenkephalin (PENK) as a novel biomarker for kidney function.
        J Appl Lab Med. 2017; 2: 400-412
        • Khorashadi M
        • Beunders R
        • Pickkers P
        • Legrand M.
        Proenkephalin: A new biomarker for glomerular filtration rate and acute kidney injury.
        Nephron. 2020; 144: 655-661
        • Schulz C-A
        • Christensson A
        • Ericson U
        • et al.
        High level of fasting plasma proenkephalin-a predicts deterioration of kidney function and incidence of CKD.
        J Am Soc Nephrol. 2017; 28: 291-303
        • Ng LL
        • Squire IB
        • Jones DJL
        • et al.
        Proenkephalin, renal dysfunction, and prognosis in patients with acute heart failure: A great network study.
        J Am Coll Cardiol. 2017; 69: 56-69
        • Arbit B
        • Marston N
        • Shah K
        • et al.
        Prognostic Usefulness of Proenkephalin in Stable Ambulatory Patients With Heart Failure.
        Am J Cardiol. 2016; 117: 1310-1314
        • Hughes J
        • Kosterlitz HW
        • Smith TW.
        The distribution of methionine-enkephalin and leucine-enkephalin in the brain and peripheral tissues.
        Br J Pharmacol. 1977; 61: 639-647
        • Denning GM
        • Ackermann LW
        • Barna TJ
        • et al.
        Proenkephalin expression and enkephalin release are widely observed in non-neuronal tissues.
        Peptides. 2008; 29: 83-92
        • Drolet G
        • Dumont EC
        • Gosselin I
        • Kinkead R
        • Laforest S
        • Trottier JF.
        Role of endogenous opioid system in the regulation of the stress response.
        Prog Neuropsychopharmacol Biol Psychiatry. 2001; 25: 729-741
        • Przewłocki R
        • Hassan AH
        • Lason W
        • Epplen C
        • Herz A
        • Stein C.
        Gene expression and localization of opioid peptides in immune cells of inflamed tissue: functional role in antinociception.
        Neuroscience. 1992; 48: 491-500
        • Salzet M
        • Tasiemski A.
        Involvement of pro-enkephalin-derived peptides in immunity.
        Dev Comp Immunol. 2001; 25: 177-185
        • Moore RH
        • Dowling DA.
        Effects of intravenously administered Leu- or Met-enkephalin on arterial blood pressure.
        Regul Pept. 1980; 1: 77-87
        • Calero A
        • Villanueva H
        • Giri P
        • Tischler AS
        • Powers JF
        • Evinger M.
        Acute hypoxia induces enkephalin production and release in an adrenergic cell line model of neonatal chromaffin cell responses to hypoxic stress.
        Am J Perinatol. 2018; 35: 1100-1106
        • van den Brink OWV
        • Delbridge LM
        • Rosenfeldt FL
        • et al.
        Endogenous cardiac opioids: enkephalins in adaptation and protection of the heart.
        Heart Lung Circ. 2003; 12: 178-187
        • Webster KA
        • Discher DJ
        • Bishopric NH.
        Induction and nuclear accumulation of fos and jun proto-oncogenes in hypoxic cardiac myocytes.
        J Biol Chem. 1993; 268: 16852-16858
        • Takasaki Y
        • Wolff RA
        • Chien GL
        • van Winkle DM.
        Met 5 -enkephalin protects isolated adult rabbit cardiomyocytes via δ-opioid receptors.
        Am J Physiol-Heart Circ Physiol. 1999; 277: H2442-H2450
        • Allen CL
        • Bayraktutan U.
        Oxidative stress and its role in the pathogenesis of ischaemic stroke.
        Int J Stroke. 2009; 4: 461-470
        • Fearon IM
        • Faux SP.
        Oxidative stress and cardiovascular disease: Novel tools give (free) radical insight.
        J Mol Cell Cardiol. 2009; 47: 372-381
        • Thomas Heitzer
        • Titus Schlinzig
        • Karoline Krohn
        • Thomas Meinertz
        • Thomas Münzel
        Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease.
        Circulation. 2001; 104: 2673-2678
        • Howard VJ
        • Cushman M
        • Pulley L
        • et al.
        The reasons for geographic and racial differences in stroke study: objectives and design.
        Neuroepidemiology. 2005; 25: 135-143
        • Howard VJ
        • Kleindorfer DO
        • Judd SE
        • et al.
        Disparities in stroke incidence contributing to disparities in stroke mortality.
        Ann Neurol. 2011; 69: 619-627
        • Adams HP
        • Biller J.
        Classification of subtypes of ischemic stroke: history of the trial of org 10 172 in acute stroke treatment classification.
        Stroke. 2015; 46
        • Jenny NS
        • Callas PW
        • Judd SE
        • et al.
        Inflammatory cytokines and ischemic stroke risk: The REGARDS cohort.
        Neurology. 2019; 92: e2375-e2384
        • Arora P
        • Agarwal Z
        • Venkatraman A
        • et al.
        Galectin-3 and risk of ischaemic stroke: reasons for geographic and racial differences in stroke cohort.
        Eur J Neurol. 2017; 24: 1464-1470
        • Gillett SR
        • Boyle RH
        • Zakai NA
        • McClure LA
        • Jenny NS
        • Cushman M.
        Validating laboratory results in a national observational cohort study without field centers: the reasons for geographic and racial differences in stroke cohort.
        Clin Biochem. 2014; 47: 243-246
        • Ernst A
        • Köhrle J
        • Bergmann A.
        Proenkephalin A 119–159, a stable proenkephalin A precursor fragment identified in human circulation.
        Peptides. 2006; 27: 1835-1840
        • Barlow WE
        • Ichikawa L
        • Rosner D
        • Izumi S.
        Analysis of case-cohort designs.
        J Clin Epidemiol. 1999; 52: 1165-1172
        • Gutierrez OM
        • Judd SE
        • Muntner P
        • et al.
        Racial differences in albuminuria, kidney function, and risk of stroke.
        Neurology. 2012; 79: 1686-1692
        • Therneau TM
        TL (original S->R port and R maintainer, Elizabeth A, Cynthia C.
        Survival: Survival Analysis. 2020; (Accessed December 12, 2020)
      1. R: The R project for statistical computing. Accessed December 12, 2020.

        • Rosenberger J
        • Petrovics G
        • Buzas B.
        Oxidative stress induces proorphanin FQ and proenkephalin gene expression in astrocytes through p38- and ERK-MAP kinases and NF-κB.
        J Neurochem. 2001; 79: 35-44
        • Islam MR
        • Yang L
        • Lee YS
        • Hruby VJ
        • Karamyan VT
        • Abbruscato TJ.
        Enkephalin-Fentanyl Multifunctional Opioids as Potential Neuroprotectants for Ischemic Stroke Treatment.
        Curr Pharm Des. 2016; 22: 6459-6468
        • Wang S
        • Cao X
        • Duan Y
        • Zhang G.
        Delta Opioid Peptide [d-Ala2, d-Leu5] Enkephalin (DADLE) Exerts a Cytoprotective Effect in Astrocytes Exposed to Oxygen-Glucose Deprivation by Inducing Autophagy.
        Cell Transplant. 2019; 28: 775-782
        • Stefano GB
        • Salzet M
        • Hughes TK
        • Bilfinger TV.
        δ2 opioid receptor subtype on human vascular endothelium uncouples morphine stimulated nitric oxide release.
        Int J Cardiol. 1998; 64: S43-S51
        • Arendt RM
        • Schmoeckel M
        • Wilbert-Lampen U
        • Plasse A
        • Heucke L
        • Werdan K.
        Bidirectional effects of endogenous opioid peptides on endothelin release rates in porcine aortic endothelial cell culture: mediation by delta opioid receptor and opioid receptor antagonist-insensitive mechanisms.
        J Pharmacol Exp Ther. 1995; 272: 1-7
        • Balasubramanian S
        • Ramakrishnan S
        • Charboneau R
        • Wang J
        • Barke RA
        • Roy S.
        Morphine sulfate inhibits hypoxia-induced vascular endothelial growth factor expression in endothelial cells and cardiac myocytes.
        J Mol Cell Cardiol. 2001; 33: 2179-2187
        • Vidal EL
        • Patel NA
        • Wu G
        • Fiala M
        • Chang SL.
        Interleukin-1 induces the expression of μ opioid receptors in endothelial cells.
        Immunopharmacology. 1998; 38: 261-266
        • Siti HN
        • Kamisah Y
        • Kamsiah J.
        The role of oxidative stress, antioxidants and vascular inflammation in cardiovascular disease (a review).
        Vascul Pharmacol. 2015; 71: 40-56
        • Sesso HD
        • Buring JE
        • Christen WG
        • et al.
        Vitamins E and C in the Prevention of Cardiovascular Disease in Men: The Physicians’ Health Study II Randomized Trial.
        JAMA J Am Med Assoc. 2008; 300: 2123-2133
        • Cook NR
        • Albert CM
        • Gaziano JM
        • et al.
        A Randomized Factorial Trial of Vitamins C, E, and Beta-Carotene in the Secondary Prevention of Cardiovascular Events in Women.
        Arch Intern Med. 2007; 167: 1610-1618
        • Doehner W
        • von Haehling S
        • Suhr J
        • et al.
        Elevated Plasma Levels of Neuropeptide Proenkephalin A Predict Mortality and Functional Outcome in Ischemic Stroke.
        J Am Coll Cardiol. 2012; 60: 346-354
        • Gruber P
        • Fluri F
        • Schweizer J
        • et al.
        Proenkephalin A Adds No Incremental Prognostic Value After Acute Ischemic Stroke.
        Clin Appl Thromb. 2020; 261076029619895318
        • Bai X
        • Zhang X
        • Li Y
        • Lu L
        • Li B
        • He X.
        Sex differences in peripheral mu-opioid receptor mediated analgesia in rat orofacial persistent pain model.
        PloS One. 2015; 10e0122924
        • Saloman JL
        • Niu KY
        • Ro JY.
        Activation of peripheral delta-opioid receptors leads to anti-hyperalgesic responses in the masseter muscle of male and female rats.
        Neuroscience. 2011; 190: 379-385
        • Sarton E
        • Teppema L
        • Dahan A.
        Sex Differences in Morphine-induced Ventilatory Depression Reside within the Peripheral Chemoreflex Loop.
        Anesthesiology. 1999; 90: 1329-1338