The Concentration-Dependent Effects of Non-Nutritive Sweeteners on the Phase Transition Temperature and Enthalpy of DOPC-Based Lipid Membranes Using Differential Scanning Calorimetry

Presented by: Maryam Aamir

Introduction:

• As of 2021, approximately 38.4 million Americans, or 11.6% of the population, have diabetes. (American Diabetes Association, 2021)
• 101,209 people died from diabetes in the United States in 2021, which was the eighth leading cause of death. The death rate per 100,000 people was 30.4 (CDC, 2021)

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Healthcare professionals commonly recommend non-nutritive sweeteners (NNS) as sugar alternatives to aid weight loss and diabetes management

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Source: International Diabetes Federation, World Health Organization

Introduction:

• Nonnutritive sweeteners (NNS) provide more intense sweetness with little to no calories since the body is unable to break them down (Pete et al., 2024)

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• The US FDA authority approved six NNS for use in humans, classifying them as generally recognized as safe (GRAS)

(Sharma et al., 2024)

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• The World Health Organization recommended that NNS not be used to achieve weight control due to potential severe risks and the lack of any potential benefits (WHO, 2024)

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• The US food industry dismissed the WHO report, continuing its heavy use of NNS, claiming they have no serious effects at suggested intake levels (WHO, 2023)

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Review of Literature:

• Research contains a lack of quantitative data and cohesive conclusions on a molecular level (Conz et al., 2023)

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• Limited sample size variability as well as significant variability among individuals in each of the groups
• Much of the environment and dietary intake were highly controlled which was not very realistic (Suez J. et al., 2022)

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• It has been found that acesulfame K, aspartame, and sucralose did not disrupt monolayer integrity in the cells. Saccharin was found to disrupt the integrity of cells in vitro (Santos et al., 2018)

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Gap in Research:

None of the studies investigate the effects of other NNS on cell membrane functioning ability

Problem:

There is a current lack of understanding concerning the effects of NNS on human cell membranes

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Hypothesis:

NNS are expected to alter lipid membrane properties by affecting phase transition temperature and enthalpy

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As higher concentrations of the NNS will decrease the phase transition temperature

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Indicating an increased fluidity of the lipid membrane, while increasing the enthalpy, reflecting alterations in lipid packing and membrane stability

Purpose:

Investigate the concentration-

dependent effects of NNS on the phase transition temperature and enthalpy of DOPC-based lipid membranes using Differential Scanning Calorimetry

Methods (Sample Preparation):

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Freezer

Evaporated Sample Films

Films are evaporated and vacuumed

Methods (Sample Preparation):

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Parafilmed samples vortexed: 1min

Sonicator

Sugars were accurately weighed using a balance and added to the film.

Micropipette

Methods (Differential Scanning Calorimetry (DSC)):

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Aluminum Pan

Differential Scanning Calorimetry

Nitrogen Tank

Methods (Data Analysis):

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Sucrose Effect on Phase Transition Temperature:

Sorbitol Effect on Phase Transition Temperature:

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Mannitol Effect on Phase Transition Temperature:

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• Sucrose:
• As concentrations increased, the delta H decreased from 0.6058 J/g to 0.5185 J/g
• Sorbitol:
• The peak was skewed at the 5 mM concentration, indicating some disruption
• As concentrations increased, the delta H increased steadily from 0.5058 J/g to 0.7675 J/g
• There was an outlier of a sharp decrease with the 20 mM concentration as it dropped to 0.6445 J/g
• The phase transition temperature also decreased steadily, staying within -17℃ to -18.6℃, closely reflecting that of the Sucrose control
• Mannitol:
• As concentrations increased, the delta H decreased steadily from 0.7246 J/g to 0.4057 J/g indicating some stress caused the reaction to become exothermic
• 20mM and 0.4 mM concentrations faced some disruption
• The phase transition temperature decreased steadily, staying within -16.66℃ to -17.33℃, except for the 20mM pre transition peak being -24.06℃

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Discussion:

• Mannitol, sucrose, and sorbitol exhibited stable phase transition trends, suggesting minimal disruption to lipid membrane integrity

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• Sorbitol and mannitol appear safe for human intake at lower concentrations, but 5mM sorbitol & 20mM/0.4mM mannitol caused minor disruption

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• The decrease in Tm suggests that NNS increase membrane fluidity, which could alter protein function and transport across cell membranes

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• This could affect nutrient absorption, cell signaling, and even gut membrane integrity, impacting metabolism and inflammation

Limitations:

• Only 1 method was used
• The DOPC based lipid membrane could have some limitations in comparison to human membranes
• Limited trials were completed

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Further Research:

• Conduct Nuclear Magnetic Resonance (NMR) to study how NNS alter lipid packing at a molecular level
• Test NNS with different lipid based membranes
• Explore the 20mM and 5mM concentration for sorbitol as well as the 0.4 and 20mM for mannitol
• Test NNS effects on live cells using Live Cell Imaging (Fluorescence Microscopy)
• Survey diabetic patients directly based on NNS intake

Conclusion:

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• Artificial sweeteners are not as disruptive to the cellular membrane on a molecular level.

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• Further investigation is still required

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• Explore why NNS are unable to be broken down by the body

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• Need to test its effectiveness in combating diabetes
• 1.5 million people die every year due to diabetes (WHO, 2016)

Citations:

• Azad, M.B. et al. (2017) ‘Nonnutritive sweeteners and Cardiometabolic Health: A systematic review and meta-analysis of randomized controlled trials and prospective cohort studies’, Canadian Medical Association Journal, 189(28). doi:10.1503/cmaj.161390.
• Center for Food Safety and Applied Nutrition (no date) Aspartame and other sweeteners in food, U.S. Food and Drug Administration. Available at: https://www.fda.gov/food/food-additives-petitions/aspartame-and-other-sweeteners-food
• Conz, A., Salmona, M. and Diomede, L. (2023) ‘Effect of non-nutritive sweeteners on the gut microbiota’, Nutrients, 15(8), p. 1869. doi:10.3390/nu15081869.
• Gill, P., Moghadam, T.T. and Ranjbar, B. (2010) Differential scanning calorimetry techniques: Applications in biology and Nanoscience, Journal of biomolecular techniques : JBT. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2977967/
• Lee, S. (2023) Aspirin interacts with cholesterol-containing membranes in a ph-dependent manner. doi:10.1021/acs.langmuir.3c02242.s001.
• Lee, S. (2024) Concentration-dependent effects of curcumin on membrane permeability and structure. doi:10.1021/acs pci.4c00093.s001.
• Petre, A. (2024) Artificial sweeteners: Good or bad?, Healthline. Available at: https://www.healthline.com/nutrition/artificial-sweeteners-good-or-bad#:~:text=A%202023%20review%20 concluded%20that,and%20 risks%20of%20these%20products.
• Richardson, I.L. and Frese, S.A. (2022) Non-nutritive sweeteners and their impacts on the gut microbiome and host physiology, Frontiers in nutrition. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9453245/
• Sharma, A. et al. (2016) Artificial sweeteners as a sugar substitute: Are they really safe?, Indian journal of pharmacology. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899993/
• Suez, J. et al. (2022) ‘Personalized microbiome-driven effects of non-nutritive sweeteners on human glucose tolerance’, Cell, 185(18). doi:10.1016/j.cell.2022.07.016.
• van Meer, G., Voelker, D.R. and Feigenson, G.W. (2008) Membrane lipids: Where they are and how they behave, Nature Reviews. Molecular cell biology. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2642958/
• WHO advises not to use non-sugar sweeteners for weight control in newly released guideline (2023) World Health Organization. Available at: https://www.who.int/news/item/15-05-2023-who-advises-not-to-use-non-sugar-sweeteners-for-weight-control-in-newly-released-guideline

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(Medical University of South Carolina, 2023)