molecularly imprinted polymers synthesized as adsorbents for ergot alkaloids

234 Pages·2017·4.51 MB·English

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Theses and Dissertations--Animal and Food
Animal and Food Sciences
Sciences
2017
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Manoj B. Kudupoje
University of Kentucky, [email protected]
Author ORCID Identifier:
https://orcid.org/0000-0002-5509-5559
Digital Object Identifier: https://doi.org/10.13023/ETD.2017.392
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Kudupoje, Manoj B., "MOLECULARLY IMPRINTED POLYMERS SYNTHESIZED AS ADSORBENTS FOR
ERGOT ALKALOIDS: CHARACTERIZATION AND IN VITRO AND EX VIVO ASSESSMENT OF EFFECTS ON
ERGOT ALKALOID BIOAVAILABILITY" (2017). Theses and Dissertations--Animal and Food Sciences. 76.
https://uknowledge.uky.edu/animalsci_etds/76
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The document mentioned above has been reviewed and accepted by the student’s advisor, on
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Manoj B. Kudupoje, Student
Dr. Eric Vanzant, Major Professor
Dr. D. L. Harmon, Director of Graduate Studies
MOLECULARLY IMPRINTED POLYMERS SYNTHESIZED AS ADSORBENTS FOR ERGOT
ALKALOIDS: CHARACTERIZATION AND IN VITRO AND EX VIVO ASSESSMENT OF EFFECTS
ON ERGOT ALKALOID BIOAVAILABILITY
DISSERTATION
A dissertation submitted in partial fulfillment of the
requirements for the degree of Doctor of Philosophy in the
College of Agriculture, Food and Environment at the University of Kentucky
By
Manoj Bojappa Kudupoje
Lexington, Kentucky
Director: Dr. Eric Vanzant, Associate Professor of Animal and Food Sciences
Lexington, Kentucky
2017
Copyright © Manoj Bojappa Kudupoje 2017
http://orcid.org/0000-0002-5509-5559
ABSTRACT OF DISSERTATION
Alkaloid toxicities negatively impact livestock health and production and are of serious
economic concern to animal industries. To date, few strategies have been developed to evaluate
alkaloid levels in feed or to counteract alkaloid toxicities. The present research evaluated the
applicability of imprinting technology to synthesize polymers that have potential to interact with
ergot alkaloids and therefore reduce their bioavailability in the GIT. The studies also evaluated
applicability of synthesized polymers for use in the ruminal environment using an in vitro
ruminal fermentation model, and for the ability to ameliorate vasoconstriction using ex vivo
myographic evaluations.
In the first experiment, styrene-based molecularly imprinted polymer (MIP) was
synthesized using ergotamine as the imprinting template and evaluated for specificity of
adsorption to various ergot alkaloids. Cross reactivity with related alkaloids exists due to
similarities in structure and functional groups. Both polymers (MIP and NIP) showed strong
adsorption intensity and no difference was observed for estimated maximum adsorption
capacity between MIP and NIP. Morphologically, MIP was highly porous with greater surface
area than NIP. Solid phase extraction indicated stronger adsorption of MIP than NIP to ergot
alkaloids suggesting the potential for MIP as a sorbent material for solid phase extraction (SPE)
columns used for sample clean-up prior to HPLC or LC-MS/MS analysis of complex samples.
In Experiment 2, methacrylic acid-based polymers were synthesized with ergotamine as
a template. Among the 4 alkaloids evaluated for selectivity, adsorption difference between MIP
and NIP interacted with alkaloid concentration, although differences were generally consistent
across concentrations. Imprinting did not affect lysergol and bromocriptine adsorption, but
resulted in higher adsorption to methylergonovine. However, there was no difference between
MIP and NIP for adsorption of ergotamine. Hydrophobic interactions and H-bonding were the
primary interactive forces between polymers and alkaloid adsorbents. Morphologically, MIP had
greater surface area and porosity implying a larger surface for adsorption. In addition to its
application as SPE sorbent, this MIP was a suitable candidate for application as a feed adsorbent
to reduce the bioavailability of certain alkaloid in the gut.
In experiment 3, methacrylic acid-based polymers were evaluated for their effect on in
vitro ruminal fermentation. There were no interactions between polymer type and inclusion
level, and no differences between polymer types for cumulative gas production or rate of gas
production. Total gas production and rate of gas production were unaffected by inclusion level.
Polymers did not affect total or individual VFA concentrations, ammonia-N or methane
concentration at any inclusion level. However, a logarithmic increase in polymer dose level
decreased the pH linearly with maximum depression of 0.24 units. This study indicated that,
within the range of expected use levels, polymers were essentially inert and would not be
expected to affect ruminal fermentation.
In experiment 4, ex vivo myographic bioassays were used to determine the impact of
polymers on ergotamine bioavailability. Responses measured in the ex vivo myographic studies
had similar trend as the responses generated from in vitro isothermal adsorption studies.
Results of that study also showed that ex vivo myographic responses could be predicted from in
vitro isothermal adsorption studies with more than 80% accuracy. These studies indicate that
synthetic polymers are potentially effective adsorbents to mitigate ergot toxicity with little
evidence of substantial differences between MIP and NIP.
KEYWORDS: ergotamine, adsorbent, myograph, imprinted polymer, isotherms, fescue
Manoj Kudupoje
----------------------------
Author’s Signature
September 7, 2017
----------------------------
Date
MOLECULARLY IMPRINTED POLYMERS SYNTHESIZED AS ADSORBENTS FOR
ERGOT ALKALOIDS: CHARACTERIZATION AND IN VITRO AND EX VIVO ASSESSMENT OF
EFFECTS ON ERGOT ALKALOID BIOAVAILABILITY
By
Manoj Bojappa Kudupoje
Dr. Eric Vanzant
---------------------------------
Director of Dissertation
Dr. D.L.Harmon
---------------------------------------
Director of Graduate Studies
September 7, 2017
--------------------------
Date
This dissertation is dedicated to those who have supported and encouraged me
throughout my life. My parents, Bojappa and Shanthy Kudupoje, who gave me the desire to
never stop learning and taught me to follow my passions. My sister Mamatha and niece
Apeksha, who reminded me to take a break now and then to see where my work and dreams
met the real world. Friends too numerous to name, who kept me laughing and sane through
it all. But, most of all, my wife Sandhya, who married into this wild ride and helped me with
every step of my life. Without her I might still be working. I am grateful for your love and
support.
Most of all I dedicate everything to my kids, my daughter Aishanya and son
Aayushman, who waited for me all these years, waiting eagerly to join them.
ACKNOWLEDGMENTS
First and foremost, I would like to express my deep sense of gratitude to my supervisor
Prof. Dr. Eric Vanzant for giving me an opportunity to pursue a doctoral study in his group. I am
very thankful to him for his, inspiring guidance, many discussions and resourceful ideas. He
continuously supported me and let me guide my own research and make mistakes where it was
acceptable. It was a good learning curve under his guidance during these years. He has laughed
when things went wrong and pushed me to know more and work hard. I will forever be
thankful for his guidance, support, and encouragement.
I am thankful Dr. Lyons for the financial support from Alltech. My sincere gratitude
towards Dr. Karl Dawson who saw potential in me and supported me through the Alltech-
University of Kentucky Alliance program. I am also thankful to Dr. Alexandros Yiannikouris, my
committee member, who initiated the topic of imprinting technology in animal industry for my
PhD, and guiding me in analytical aspects and giving the industrial perspective of my research.
I cannot thank enough my dissertation advisory committee who have helped and
encouraged me over these years. Dr. Brad Anderson provided a non-animal perspective that
often made me think differently about what and why of my research. Dr. Kyle McLeod has been
extremely helpful in furthering my understanding and enjoyment of energy metabolism. Each of
these members has been invaluable in their advice and support through my research. I
acknowledge to Dr. James Klotz for providing the opportunity to work in the FAPRU lab, USDA,
and guiding me during myographic study.
I wish to thank all the other students and lab technicians in the ruminant nutrition
department who helped with animal care, sample collection, and analysis including Adam
Barnes, Mariam Snyder, Adam Bohannon, and Cynthia Roberts. Many thanks beef farm staffs,
most particularly Derrick Wise, Kip Sparrow and Lauren Clark for their assistance in handling
animals and and sample collection. My gratitude to Kirk Vanzant who always kept animals for
my research beyond the regular time frame and also helping me in providing information
regarding the animals. My sincere gratitude to my friend Alex Altman, who spent many hours
helping me collect rumen fluid and in lab through my research, for which I am truly grateful. I
would like to thank all my friends for their enjoyable conversation during these years, who
reminded me how much fun it is to know when the research is finished.
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TABLE OF CONTENTS
ACKNOWLEDGMENTS ..................................................................................................................... iii
TABLE OF CONTENTS....................................................................................................................... iv
LIST OF TABLES ................................................................................................................................. x
LIST OF FIGURES ............................................................................................................................ ..xi
1. INTRODUCTION ................................................................................................................... 1
2. LITERATURE REVIEW ........................................................................................................... 3
2.1. History of Fescue toxicosis ................................................................................ 3
2.2. Ergot alkaloids: Structure .................................................................................. 4
2.3. Toxicodynamics ................................................................................................. 5
2.3.1. Altered hormones ............................................................................................. 6
2.3.2. Adrenergic effects ............................................................................................. 7
2.3.3. Dopaminergic effects ........................................................................................ 7
2.3.4. Serotonergic effects .......................................................................................... 8
2.4. Ergokinetics ....................................................................................................... 9
2.5. Signs of Fescue toxicosis in animals ................................................................ 11
2.5.1. Summer slump ................................................................................................ 12
2.5.2. Fescue foot ...................................................................................................... 13
2.5.3. Fat necrosis ..................................................................................................... 14
2.5.4. Reproductive effects ....................................................................................... 14
2.5.5. Blood parameters ........................................................................................... 15
2.5.6. Energy metabolism ......................................................................................... 15
2.6. Mitigation strategies to reduce ergot alkaloids toxicity ................................. 17
2.6.1. Management approach .................................................................................. 17
2.6.2. Mycotoxin adsorbents .................................................................................... 18
2.6.2.1. Molecularly imprinted polymers .......................................................... 19
2.6.2.1.1. Approaches in molecular imprinting ............................................. 19
2.6.2.1.1.1. Covalent imprinting ................................................................ 20
2.6.2.1.1.2. Non-Covalent imprinting ........................................................ 20
2.6.2.1.1.3. Semi-Covalent imprinting ....................................................... 21
2.6.2.1.2. Parameters involved in molecular recognition .............................. 22
2.6.2.1.2.1. Functional monomers ............................................................. 23
iv
2.6.2.1.2.2. Crosslinkers ............................................................................. 24
2.6.2.1.2.3. Initiator ................................................................................... 25
2.6.2.1.2.4. Porogen ................................................................................... 25
2.6.2.1.2.5. Template ................................................................................. 26
2.6.2.1.2.6. Temperature, pressure and thermodynamic processes ......... 27
2.6.2.1.2.7. Template bleeding .................................................................. 28
2.6.2.1.3. Polymer synthesis .......................................................................... 29
2.6.2.1.3.1. Bulk polymerization ................................................................ 29
2.6.2.1.3.2. Precipitation polymerization .................................................. 29
2.6.2.1.3.3. Multi-step swelling and polymerization ................................. 30
2.6.2.1.3.4. Suspension polymerization ..................................................... 30
2.6.2.1.3.5. Surface imprinting .................................................................. 31
2.6.2.1.3.6. In-situ polymerization ............................................................. 31
2.6.2.1.4. Polymer characterization ............................................................... 32
2.6.2.1.4.1. Chemical characterization ...................................................... 32
2.6.2.1.4.2. Morphological characterization .............................................. 32
2.6.2.1.4.2.1. Gas sorption measurements ............................................ 33
2.6.2.1.4.2.2. Particle size distribution .................................................. 34
2.6.2.1.4.2.3. Electron Microscopy ........................................................ 34
2.6.2.1.4.3. Binding characterization ......................................................... 34
2.6.2.1.4.3.1. Scatchard analysis ............................................................ 36
2.6.2.1.4.3.2. Langmuir isotherm ........................................................... 36
2.6.2.1.4.3.3. Freundlich isotherm ......................................................... 37
2.6.2.1.5. Application of imprinting technology in analytical chemistry ....... 38
2.6.2.1.5.1. Chromatography and Capillary Electrophoresis ..................... 38
2.6.2.1.5.2. Sensors .................................................................................... 39
2.6.2.1.5.3. Extraction technology: Solid phase extraction ....................... 39
2.7. Ergot alkaloid analysis ..................................................................................... 40
2.8. Summary ......................................................................................................... 44
3. SYNTHESIS, EVALUATION AND CHARACTERIZATION OF A STYRENE BASED ERGOTAMINE
IMPRINTED POLYMER FOR POTENTIAL USE AS AN ERGOT ALKALOID HIGHLY SELECTIVE
ADSORBENT ...................................................................................................................... 50
3.1. Introduction .................................................................................................... 50
3.2. Material and methods .................................................................................... 52
3.2.1. Chemicals ........................................................................................................ 52
v