Stimulating the Production of Ketones Through the Combined Use of Aerobic Exercise (AE) and Dietary a Medium Chain Triglycerides (MCT) Supplement
Phase: NA
Conditions: Prediabetic State
Sponsor: Université de Sherbrooke

English version – February 2016 
A short-term intervention combining aerobic exercise (AE) with 
medium-chain triglycerides (MCT) on ketonemia: a comparison of 
normoglycemic and prediabetic older women 
 
PROTOCOLE: 2015-508 
 
 
 
 
 
 
PRINCIPAL INVESTIGATORS and CO-INVESTIGATORS: 
 
Stephen Cunnane, PhD 
 
Isabelle Dionne, PhD 
 
CONTRIBUTORS: 
 
Camille Vandenberghe, MSc, research assistant 
 
Mathieu Maltais, PhD, kinesiologist 
 
Christian-Alexandre Castellano, PhD, research assistant 
 
Mélanie Fortier, MSc, research assistant 
 
Valérie St-Pierre, MSc, research assistant 
 
Christine Brodeur-Dubreuil, research nurse 
 
 
 
 
 
 
Centre de recherche sur le vieillissement (CDRV); Centre de santé et de service sociaux- Institut 
universitaire de gériatrie de Sherbrooke (CSSS-IUGS) 

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Participants 
 
This study was conducted with the informed written consent of all the participants and 
was approved by the appropriate ethics committees (Health and Social Services Center – 
Sherbrooke University Geriatrics Institute and the Centre hospitalier universitaire de 
Sherbrooke). Participants underwent a screening visit, including the analysis of a blood sample 
collected after a 12 h overnight fast. Exclusion criteria included smoking, diabetes (fasting 
glucose >7.0 mmol/l and glycated hemoglobin >6.9%), unfit to practice exercise, untreated 
hypertension, dyslipidemia, and abnormal renal, liver, heart or thyroid function. Prediabetic (PD) 
participants needed to have a fasting plasma glucose of 6.0-6.9 mmol/l and/or a plasma glycated 
hemoglobin of 6.1-6.4% in order to be eligible to be included in the study (Golderngerg and 
Punthakee 2013). The Physical Activity Scale for the Elderly (PASE) questionnaire was used as 
an additional tool to characterise participants’ baseline physical activity. To be eligible, 
participants had to be doing structured physical exercise ≤ two times per week. Participants 
taking medications known to affect triglyceride or carbohydrate metabolism (i.e. diuretics, beta-
blockers, steroids, insulin sensitizers) were excluded. Body composition and visceral adipose 
tissue area were measured by dual-energy x-ray absorptiometry (GE Healthcare LUNAR iDXA, 
Madison, WI, USA; (Hangartner et al. 2013). 
 
Experimental design 
 
Participants completed a 4 h metabolic study after each of the four following sequential 
experimental conditions: (i) no treatment control (CTL), (ii) five days of MCT supplementation 
alone (MCT), (iii) 30 min of aerobic exercise alone (AE), and (iv) five days of MCT 
supplementation combined with aerobic exercise (MCT+AE). Participants started with the CTL 
metabolic study during which they received only the lactose-free skim milk vehicle for the MCT 
drink. The CTL metabolic study served to set the baseline ketogenic response for the other three 
experimental conditions. Participants then took the MCT supplement daily for five days; on the 
fifth day, they repeated the metabolic study, this time with a drink containing 15 g MCT at 
breakfast. During the second week of the study, participants repeated the metabolic study with 
vehicle only (such as CTL) but did 30 minutes of AE. This allowed assessment of the effect of a 

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single acute session of AE on the ketone response during the 4 hour metabolic study. They then 
did five days of MCT supplementation simultaneously with five days of AE (30 min/day). On the 
fifth day of the combined intervention, they repeated the metabolic study with a drink containing 
15 g MCT at breakfast but also followed by 30 min AE at 1.0 - 1.5 h. The 5 days period of MCT 
supplementation alone was separated from the MCT+AE period by a 1-3 week washout. 
 
Metabolic studies 
 
For each metabolic study, the participants arrived at 7:30 a.m. after a 12 h overnight fast 
and a minimum of 24 h without alcohol intake. A forearm venous catheter was installed and the 
baseline fasting blood sample withdrawn (Time 0 h). Participants then received a standardized 
breakfast comprised of two pieces of toast with raspberry jam and the MCT drink or the vehicle, 
which was 250 ml lactose-free skim milk. Water was available ad libitum throughout the study 
period. Blood samples were taken every 30 min for 4 h in EDTA collection tubes (BD 
Vacutainer, NJ, USA). Blood samples were centrifuged at 2846 g for 10 min at 4°C and plasma 
stored at -80°C until analyzed. 
 
MCT supplement 
 
The MCT used to make the emulsion was 55% tricaprylin oil (8-carbon MCT) and 35% 
tricaprin oil (10-carbon MCT; Captex 355, Abitec, Columbus, OH, USA). The emulsion was 
manufactured under aseptic conditions at Université Laval Laboratory of Food Technologies 
(Québec, QC, CAN) using our proprietary technology. The emulsion was prepared using 120 g 
MCT per liter of lactose-free skim milk (12%) and provided to participants in 250 ml bottles. 
When taking the supplement, participants consumed 15 g of MCT at breakfast (125 ml of the 
drink) and 15 g again at supper (total MCT 30 g/d; a previously used dose (Courchesne-Loyer et 
al. 2013) for five consecutive days. Compliance was measured by bottle count. 
 
 
 
 

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Aerobic exercise 
 
During the AE phase of the study, participants exercised for 30 minutes in the morning for 
five consecutive days, a simple, short protocol feasible for older women. Exercise sessions were 
supervised by a kinesiologist and were performed on a treadmill or on a stationary bicycle at 55-
75% of heart rate reserve, which was calculated as maximum heart rate - resting heart rate using 
the Karvonen equation (Karvonen and Vuorimaa 1988). Once the participant selected one of the 
two exercise methods (treadmill or stationary bicycle), it was kept throughout the 5 days of AE. 
Theoretical maximum heart rate was estimated as 206.9-(0.67 × age) (Tanaka et al. 2001) and the 
resting heart rate was measured before exercise. During the aerobic exercise sessions, heart rate 
was monitored with a Polar FT2 watch and a T31 heart rate sensor strap (Polar Electro, Kempele, 
Finland). A physician was on hand in case of a medical emergency. 
 
Plasma metabolite analyses 
 
Plasma acetoacetate (AcAc) and β-hydroxybutyrate (BHB) were measured by an 
automated colorimetric assay as previously described (Courchesne-Loyer et al. 2013). Briefly, for 
AcAc, 25 µL of plasma was mixed with 330 µL of fresh reagent (Tris buffer, pH 7.0, 100 
mmol/L, 20 mmol/L sodium oxamate; 0.15 mmol/L NADH and 1U/mL β-hydroxybutyrate 
dehydrogenase [BHBDH]). For BHB, the reagent was Tris buffer (pH 9.0; 20 mM sodium 
oxamate, 1 mmol/L NAD, and 1U/mL BHBDH). Tris, oxamic acid, DL-BHB sodium salt, Li-
AcAc standard, and NAD were purchased from Sigma (St. Louis, MO, USA), NADH, from 
Roche (Mannheim, Germany), and BHBDH from Toyobo (Osaka, Japan). The change in 
absorbance at 340 nm between 15 and 120 s after the addition of the reagent was measured on an 
automated clinical chemistry analyzer (Dimension Xpand Plus; Siemens, Deerfield, IL, USA). 
Plasma glucose, lactate, triglycerides (Siemens Medical Solutions USA, Inc., Deerfield, IL, USA) 
and FFA (Randox Laboratories Limited, West Virginia, USA) were analysed using commercial 
kits. Plasma insulin was analyzed by enzyme-linked immunosorbent assay (Alpco Diagnostics 
Ltd., Salem, NH, USA) with a microplate reader (Victor multi-label plate reader 2030; Perkin 
Elmer, MA, USA). Glycated hemoglobin was measured by HPLC-723G7, a fully automated high 

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performance liquid chromatography instrument-reagent system (Tosoh Bioscience, King of 
Prussia, PA, USA). 
 
Statistical analysis 
 
The sample size calculation was based on a previous study in which 9 participants were 
sufficient to measure a significant difference (β= 0.80) in plasma ketones after consuming 20 g of 
MCT during a study with the same 4-hour metabolic study period (Vandenberghe et al. 2017b). 
We recruited n=10/group for the present study in case of a dropout during the two weeks. When 
plasma ketones are present, this refers to the total of AcAc and BHB combined. For all 
metabolites, the area-under-the-curve (AUC) was calculated according the trapezoid method from 
0-4 h during the metabolic study (Gagnon and Peterson 1998). The Shapiro-Wilk test 
demonstrated that the plasma metabolite data were not normally distributed, so the results of the 
four conditions were compared using Friedman’s test, and the effect of the treatments was 
determined in each group using Wilcoxon’s signed rank test. All statistical analyses were carried 
out using SPSS 23.0 software (SPSS Inc., Chicago, IL, USA). NG and PD were treated as 
independent groups and compared using Mann-Whitney’s test. Differences were considered 
statistically significant at P≤0.05. Graphs were prepared using Prism version 6.0 (GraphPad 
Software Inc., San Diego, CA, USA). 
 
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Gagnon, R.C. and Peterson, J.J. 1998. Estimation of confidence intervals for area under the curve 
from destructively obtained pharmacokinetic data. J. Pharmacokinet. Biopharm. 26: 87-102. 
PMID:9773394. 
 

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Golderngerg, R. and Punthakee, Z. 2013. Lignes directrices de pratique clinique. Canadian 
Journal of Diabetes, 37: S36-S372. doi:10.1016/j.jcjd.2013.07.031. 
 
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Vandenberghe, C., St-Pierre, V., Pierotti, T., Fortier, M., Castellano, C., and Cunnane, S. 2017b. 
Tricaprylin alone increases plasma ketone response more than coconut oil or other medium chain 
triglycerides: an acute crossover study in healthy adults. Current Developments in Nutrition, 
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