Revealing the Mechanisms by Which Milk Sugars Exaggerate Postprandial Lipaemia
Phase: NA
Conditions: Cardiovascular Risk Factor, Lipaemia, Metabolic Disease
Sponsor: University of Bath

Lactose & Lipids Protocol
Protocol
Study title: Revealing the mechanisms by which milk sugars exaggerate postprandial lipaemia
Short title: Lactose & Lipids
Principal Investigator:
Co-Investigators:
Dr Javier Gonzalez
Professor Leanne Hodson
Professor James Betts
Professor Dylan Thompson
Dr Francoise Koumanov
Dr Steven Carter
Sponsor:
Funder:
University of Bath
British Heart Foundation
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CONTENTS
Table of Contents
1. SYNPOSIS................................................................................................................................... 3
2. BACKGROUND AND RATIONALE ............................................................................................ 4
2.1. Executive Summary ............................................................................................................ 4
2.2 Relevant Literature and Pilot Data ...................................................................................... 5
3. AIMS, OBJECTIVES AND HYPOTHESIS................................................................................... 8
RO3 - determine the degree to which lactose ingestion alters dietary fat oxidation .............. 8
4. PROGRAMME AND METHODOLOGY....................................................................................... 9
4.1. Study Design........................................................................................................................ 9
4.2. Primary Outcome................................................................................................................. 9
4.3. Secondary Outcomes.......................................................................................................... 9
4.4. Tertiary Outcomes............................................................................................................... 9
4.5. Participants and Eligibility Criteria .................................................................................... 9
4.6. Recruitment........................................................................................................................ 10
4.7. Screening ........................................................................................................................... 11
4.8. Schedule of Measurements .............................................................................................. 11
4.9. Measurements and Analysis ............................................................................................ 13
4.10. Sample Size...................................................................................................................... 14
4.11. Statistical Analyses......................................................................................................... 14
4.12. Storage and Management of Data.................................................................................. 14
4.13. Expenses and Benefits ................................................................................................... 15
5. DISADVANTAGES/RISK AND DISCOMFORT......................................................................... 15
6. REFERENCES........................................................................................................................... 16
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1. SYNPOSIS
Study Title
Short Title
Trial Design
Trial Participants
Intervention
Planned Sample
Size
Primary Objective
Secondary
Objectives
Primary
Outcomes
Secondary
Outcomes
Revealing the mechanisms by which milk sugars exaggerate postprandial
lipaemia
Lactose & Lipids
Randomised Controlled, Crossover Trial in men and women
Men and women aged 18-50 years and premenopausal (for women)
Acute ingestion of three test drinks:
Maltodextrin (Control)
Lactose (Intervention)
Sucrose (Active comparator)
24 (12 men and 12 women)
To determine to what extent ingestion of lactose exaggerates postprandial
triglyceridaemia in men and women.
To determine the effect of lactose ingestion on hepatic fatty acid synthesis (de
novo lipogenesis).
To determine the degree to which lactose ingestion alters dietary fat oxidation.
The effect of lactose versus maltodextrin co-ingestion on postprandial
incremental area under the curve (iAUC) for plasma triglyceride concentrations
(6-hour postprandial period).
The effect of treatment (lactose versus maltodextrin co-ingestion on:
1) Postprandial de novo lipogenesis (DNL)
2) Dietary fat oxidation
Tertiary
Outcomes
Treatment (lactose versus fructose versus maltodextrin) by sex (male versus
female) interaction effect on:
1) Postprandial incremental area under the curve (iAUC) for plasma
triglyceride concentrations (6-hour postprandial period).
2) Postprandial de novo lipogenesis (DNL)
3) Dietary fat oxidation
4) Postprandial iAUC for plasma insulin concentrations
5) Postprandial iAUC for plasma glucose concentrations
6) Postprandial iAUC for plasma galactose concentrations
7) Postprandial iAUC for plasma fructose concentrations
8) Postprandial iAUC for plasma VLDL-rich triglyceride [Svedberg flotation
rate (Sf): 20-400] concentrations
9) Postprandial iAUC for plasma chylomicron-rich triglyceride (Sf: >400)
concentrations
10) Postprandial iAUC for plasma lactate concentrations
11) Postprandial iAUC for plasma non-esterified fatty acid concentrations
12) Postprandial iAUC for plasma glycerol concentrations
13) Postprandial iAUC for plasma beta-hydroxybutyrate concentrations
14) Postprandial iAUC for plasma uric acid concentrations
Funder
Sponsor
Principal
Investigator
Chief Investigator
British Heart Foundation (PG/19/43/34432)
The University of Bath
Dr Javier Gonzalez
Dr Javier Gonzalez
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2. BACKGROUND AND RATIONALE
2.1. Executive Summary
Elevated postprandial triglycerides play a causal role in cardiovascular disease (CVD). Restricting
sugar intake can reduce circulating triglycerides and nutrition guidelines recommend restricting free
sugar intakes for health. Importantly, milk sugars are currently omitted from this restriction simply
due to an insufficient evidence-base to draw firm conclusions. Our preliminary data demonstrate
that galactose (the unique component of the milk sugar lactose) increases postprandial triglyceride
concentrations in lean men, produces a similar metabolic milieu to fructose ingestion, and stimulates
triglyceride accumulation in hepatocytes. If galactose exaggerates postprandial triglyceridaemia via
common mechanisms to fructose, then this has important health and policy implications due to the
potential additive effect of consuming milk products containing free sugars. This project will achieve
the following objectives: 1) generate data on the role of lactose in postprandial lipaemia in men and
women; 2) establish if hepatic fatty acid synthesis, VLDL availability, and/or changes in handling of
dietary fat in the postprandial state are the underlying mechanism(s) by which lactose exaggerates
postprandial lipaemia. These data will provide key evidence required to justify longer-term studies,
used to refine nutrition guidelines on sugars and CVD. This is timely, as inclusion of milk-based
drinks in the Soft Drinks Industry Levy is currently being considered.
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2.2 Relevant Literature and Pilot Data
Cardiovascular disease (CVD) is a global health burden and postprandial triglycerides play
a major causal role in CVD risk. Cardiovascular disease is the leading cause of death worldwide,
responsible for ~17 million of the ~54 million deaths in 2013(1). The total cost of CVD is estimated to
rise from $863 billion in 2010 to $1044 billion by 2030(2). With such a huge social and economic
burden, prevention is understandably a global priority. Mendelian randomisation suggests a causal
role of plasma triglyceride concentrations in coronary heart disease(3). Furthermore, it is important to
consider postprandial triglyceride concentrations (as opposed to post-absorptive) for at least two
main reasons. First, in contrast to the fasted state, the fed state captures the total amount of
atherogenic lipoproteins in plasma (from both hepatic and intestinal origin); and second, the
postprandial state predominates most of the 24-h cycle for the majority of populations in developed
countries, thereby better reflecting habitual exposure to atherogenic lipoproteins directly involved in
CVD(4). Individuals with postprandial plasma triglyceride concentrations above 4 mmol´L-1 have a
>5-fold increased CVD mortality compared to those with triglyceride concentrations below 1 mmol´L-
1(5). Therefore, there is an urgent need to uncover strategies that in reduce postprandial plasma
triglyceride concentrations and thus, CVD risk.
More evidence on the health effects of milk sugars in humans is essential to inform nutrition
guidelines. Nutrition is a major determinant of plasma triglycerides and CVD risk. Accordingly,
public health authorities (e.g. British Heart Foundation, the Scientific Advisory Committee on
Nutrition and the World Health Organisation) provide dietary recommendations for health(6; 7; 8),
which consistently advise reducing intakes of free sugars. Indeed, there are metabolic health
consequences to the overconsumption of sugars such as fructose; shown to exaggerate
postprandial lipaemia and hepatic triglyceride synthesis (i.e. de novo lipogenesis; DNL), whilst
suppressing fat oxidation(9; 10; 11). Importantly, milk sugars are specifically omitted from the current
definition of free sugars and thus no upper limit of sugar intake from dairy is provided(7). This decision
is based on “no reported evidence of adverse effects of consumption of intrinsic sugars and sugars
naturally present in milk”(7). Whilst this is an appropriate recommendation based on the extent of
current evidence available, the statement is based on lack of evidence of an effect, rather than
evidence for a lack of an effect. Moreover, sugar intake from dairy products is not negligible, with
dairy products contributing more than 20% of total sugar intake in many countries(12), and more than
95% of the UK population report regularly consuming dairy products(13). Thus, a better
understanding of the health impact of milk sugars should be a priority. The only randomised,
controlled study in humans to measure plasma triglycerides in response to galactose
supplementation did show that fasting plasma triglyceride concentrations were increased by ~30%
with 4 days of galactose supplementation compared to glucose supplementation(14). In the absence of
relevant human data, the best available evidence to date, for the impact of milk sugars on CVD
comes from studies on non-human primates. Baboons fed lactose for 17 months displayed almost a
7-fold increase in aortic sudanophilia (reflective of lipid deposition) compared to baboons fed
equivalent quantities of sucrose(15). Furthermore, gross atheromatous lesions were present in ~83%
of baboons fed lactose, compared to 33% of baboons fed sucrose(15). Therefore, it is remarkable
that no study has assessed the causal effect of milk sugar in postprandial lipaemia and
cardiovascular health in humans.
Galactose ingestion exaggerates postprandial triglyceride concentrations and produces a
metabolic milieu indicating similarities to fructose metabolism. Our preliminary data are the
first to demonstrate that the addition of galactose to a high-fat meal results in almost a two-fold
elevation in postprandial triglyceridaemia compared to glucose ingestion (Figure 1). This
demonstrates that ingesting milk sugars increases the exposure of the vascular system to
atherogenic lipoproteins. Since the triglyceride response to galactose that we observed appears to
be similar in magnitude to that of fructose(10), we explored whether galactose also reflected fructose
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ingestion in other metabolic aspects. Indeed, similar to fructose ingestion(10), our preliminary data
demonstrate that galactose ingestion increases plasma lactate concentrations and lowers plasma
glucose and insulin concentrations compared to glucose ingestion (Figure 2). The increase in
lactate concentrations with fructose compared to glucose ingestion is due to relatively rapid and
unregulated flux of fructose through hepatic metabolic pathways(16). This results in fructose being
rapidly converted into lactate, glucose, hepatic glycogen, and (via de novo lipogenesis)
triglycerides(16). Therefore, the evidence that galactose also increases postprandial plasma lactate
concentrations (Figure 2) and enhances hepatic glycogen synthesis(17), suggests both hepatic
galactose metabolism is more similar to fructose than glucose metabolism, and that hepatic de novo
lipogenesis may be a contributory factor for the increase in triglyceridaemia. Furthermore, to explore
whether hepatocytes are potentially involved in the exaggerated triglyceride response that we
observed (as is the case with fructose), we studied intracellular triglyceride accumulation in
hepatocytes exposed to glucose alone or combined glucose and galactose at physiologically
relevant concentrations for the human liver(18). The partial replacement of glucose with galactose
increased intracellular triglyceride accumulation by ~20% in hepatocytes in vitro (Figure 3). This
shows that human liver cells accumulate more lipid when exposed to galactose, which would be
expected to have detrimental effects on cardiovascular disease risk in humans, and also implies
that the liver could be involved in galactose-induced hypertriglyceridaemia. We therefore
hypothesise that galactose ingestion also stimulates hepatic de novo lipogenesis to a greater extent
than glucose ingestion.
Figure 1. Postprandial triglyceride concentrations following
ingestion of milk & cream (0.94 g fat·kg-1) are exaggerated
when co-ingested with additional galactose compared to
glucose (both 0.75 g·kg-1) in lean adults. n = 10. Data are
means ± SEM. Unpublished data.
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Figure 2. Postprandial glucose (A), lactate (B), insulin (C)
and non-esterified fatty acid (D; NEFA) concentrations after
ingestion of milk & cream (0.94 g fat·kg-1), co-ingested with
galactose versus glucose (both 0.75 g·kg-1) in lean adults. n
= 10. Data are means ± SEM. Unpublished data.
Figure
3.
Intracellular
triglyceride     accumulation
in
hepatocytes 
is 
exaggerated
when half of the glucose in
incubation media is replaced
with
galactose.
Importantly,
these       concentrations       are
physiologically      relevant
to
hepatocytes in the postprandial
state. Data are means ± SEM.
Unpublished data.
If lactose exaggerates lipaemia in similar ways to fructose-containing sugars, the public
health consequences are substantial. Our preliminary data indicate that galactose ingestion
exaggerates postprandial lipaemia via similar mechanisms to fructose ingestion(10). If galactose and
fructose exaggerate lipaemia via common mechanisms, an additive effect would have broad public
health implications due to the common co-ingestion of sucrose (glucose-fructose) and milk products
containing lactose. Furthermore, developing our understanding of milk sugars is timely, since milk
drinks are currently not subject to the Soft Drinks Industry Levy, but former Chancellor of the
Excequer, George Osbourne. has recently advocated the inclusion of milk-based drinks in this levy: "I
was already, before I left office, looking at whether you could extend it to sugar added to milk
products like sugary milkshakes. I think it'll be for others to take further steps forward and I would
predict those steps will be taken."(19). Therefore, an additive effect of milk sugars and fructose-
containing sugars on postprandial lipaemia would provide the necessary support for inclusion of
milk-based drinks in the sugar levy, and would also inform whether intrinsic milk sugars should
contribute to calculation of threshold(s) for the levy. The current thresholds are ³5 g of free sugar
per 100 mL and ³8 g per 100 mL(20). If lactose exaggerates lipaemia via common mechanisms to
sucrose, then it would be logical to include milk sugars in the calculation of free sugars content for
the determination of the levy threshold.
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3. AIMS, OBJECTIVES AND HYPOTHESIS
PRIMARY AIM: To understand the effect and mechanism of lactose on postprandial lipaemia in
men and women by addressing the following research objectives (RO):
RO1 - determine to what extent ingestion of lactose exaggerates postprandial triglycridaemia
RO2 - determine the effect of lactose ingestion on hepatic fatty acid synthesis (de novo lipogenesis)
RO3 - determine the degree to which lactose ingestion alters dietary fat oxidation
HYPOTHESIS: Lactose co-ingestion with a high-fat meal will exaggerate postprandial lipaemia and
stimulate hepatic fatty acid synthesis (de novo lipogenesis) to a similar extent to sucrose ingestion,
and a greater extent than glucose polymer co-ingestion, whilst suppressing dietary fat oxidation.
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4. PROGRAMME AND METHODOLOGY
4.1. Study Design
This research will involve a randomised, double-blind, crossover study that will assess the acute
postprandial metabolic responses to three different test drinks. All three test drinks will be comprised of
a high-fat milkshake with the addition of either maltodextrin, sucrose or lactose. Individuals will be
required to attend the laboratories for a preliminary visit, followed by the three laboratory trial days,
with a washout period of 28±7 days.
4.2. Primary Outcome
The effect of lactose versus maltodextrin co-ingestion on postprandial incremental area under the
curve (iAUC) for plasma triglyceride concentrations (6-hour postprandial period).
4.3. Secondary Outcomes
The effect of treatment (lactose versus maltodextrin co-ingestion on:
1) Postprandial de novo lipogenesis (DNL), using heavy water
2) Dietary fat oxidation, using a stable isotope-labelled palmitate
4.4. Tertiary Outcomes
Treatment (lactose versus fructose versus maltodextrin) by sex (male versus female) interaction
effect on:
1)
Postprandial incremental area under the curve (iAUC) for plasma triglyceride
concentrations (6-hour postprandial period).
2)
Postprandial de novo lipogenesis (DNL)
3)
Dietary fat oxidation
4)
Postprandial iAUC for plasma insulin concentrations
5)
Postprandial iAUC for plasma glucose concentrations
6)
Postprandial iAUC for plasma galactose concentrations
7)
Postprandial iAUC for plasma fructose concentrations
8)
Postprandial iAUC for plasma VLDL-rich triglyceride [Svedberg flotation rate (Sf): 20-
400] concentrations
9)
Postprandial iAUC for plasma chylomicron-rich triglyceride (Sf: >400) concentrations
10)
Postprandial iAUC for plasma lactate concentrations
11)
Postprandial iAUC for plasma non-esterified fatty acid concentrations
12)
Postprandial iAUC for plasma beta-hydroxybutyrate concentrations
13)
Postprandial iAUC for plasma uric acid concentrations
4.5. Participants and Eligibility Criteria
Twenty-four men (n = 12) and women (n = 12) will be recruited from the local area and matched for
age and body mass index.
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Inclusion Criteria:
  Age: 18-50 years and premenopausal (for women);
  Body mass index: 18.5 – 40 kg·m2
Exclusion criteria:
  weight instability (>5 kg change in body mass within last 6 months)
  diagnosis of any form of diabetes
  intolerances or allergies to any of the study procedures (e.g. lactose intolerance)
  Galactose disorders (e.g. galactokinase deficiency, UDPgalactose-4-epimerase deficiency,
galactose-1-phosphate uridyl transferase deficiency)
  Fructose malabsorption
  Inborn errors of fructose metabolism (e.g. fructokinase deficiency, aldolase B deficiency,
fructose-1,6-bisphosphatase deficiency)
  pregnant or lactating
  any condition that could introduce bias to the study (e.g. diagnoses of lipid disorders,
including cardiovascular disease, or therapies that alter lipid or glucose metabolism, such as
statins or niacin).
4.6. Recruitment
Participants will be recruited through two different routes (1) direct advertisement and (2) letters sent
to relevant patients in GP databases.
Direct Advertisement: Information about the study will be distributed via the placement of adverts
and posters. Examples include: local newspapers and magazines; community noticeboards (e.g. in
the public library, local market and cafes), community centres (e.g. leisure centres, churches, and
art and music venues); larger local industry/establishments of employment (e.g. Wessex water and
the University of Bath); healthcare centres (e.g. GP practices, pharmacies and dental clinics). In
addition to printed adverts/posters, we will also utilise electronic routes: e.g. via E-mail bulletins,
local community forums, university website/newsletters as well as social media (e.g. Twitter and
Facebook). Potentially interested participants will be asked to contact the research team by email or
phone if they want to find out more about the study. Potentially interested and eligible participants will
be invited to a meeting to further discuss the study and what it entails. After reading the
participant information sheets, if they decide to take part, they will be asked to sign an informed
consent form.
Letters from GP database searches: Recruitment via this route will be conducted with the support of
the Clinical Research Network (CRN) in BANES and Wiltshire. We will ask GP practices to search
databases for patients who potentially meet the inclusion criteria. All patient identification and
database searching will be conducted by the patient’s existing care team (GPs, nurses and IT
managers in study practices). Potentially eligible patients will be approached by a letter from their
GP, jointly signed by the chief investigator. The recruitment letter will emphasise that participation is
entirely voluntary and will not have any impact on their health care or their relationships with NHS
staff.
Potentially interested participants will be asked to contact the research team via email or phone.
Potentially interested and eligible participants will be invited to a meeting to further discuss the study
and what it entails. After reading the participant information sheets, if they decide to take part, they
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will be asked to sign an informed consent form. GP's will send out reminder letters to all eligible
participants after two weeks, with an acknowledgement in the letter to ignore the reminder for those
individuals who have already responded.
4.7. Screening
Participants will be invited to the University of Bath to provide informed consent, and thereafter will be
invited to undertake the assessments of eligibility listed below. Given the range of screening
measurement required, these measurements may take place over more than one visit. As all
potential participants will be offered feedback from the screening, in the event that a potential
participant be deemed ineligible for the study based on a single measurement, the individual will be
informed immediately and the subsequent screening measures will only be carried out at the
participant’s request.
General health status: Suitability to participate (e.g. menopausal status, pregnancy etc.) and other
practical screening questions (e.g. any reason why participants could not complete the study within a
reasonable time-frame etc.) will be self-reported by the participant via a health screen
questionnaire. Food allergies and intolerances will be reported in this questionnaire to ensure that
participants are able and willing to consume the test drinks.
Anthropometrics: Body mass will be measured using digital scales and height using a stadiometer.
Body mass index (BMI) will be calculated by dividing body mass (kg) by height (m) squared. Waist
circumference (cm) will be measured at the narrowest point between the lowest rib and iliac crest,
and hip circumference (cm) will be measured at the widest point of the gluteal using a tape measure.
Body composition (DXA): The participant will be positioned supine in a dual-energy x-ray
absorptiometry (DXA) scanner with their extremities within limits indicated and not touching their
torso if possible. Fat mass (kg), lean mass (kg), body fat percentage (%) and bone mineral density
(g/cm2) will be measured. Fat mass index will be calculated by dividing fat mass (kg) by height (m)
squared.
4.8. Schedule of Measurements
Following screening participants will undertake the three main laboratory trial visits in a random
order (randomized by JG using www.randomizer.org). Men and women will be allocated to separate
randomization plans to allow for sex-difference comparisons to be made. The washout period
between trials will be between 28±7 days.
Pre-trial standardisation
To assist in producing a similar metabolic state on the morning of laboratory trial days, participants
will be asked to record diet and physical activity for 48 h before the first trial, and to replicate these for
the 48 h before each subsequent trial. Physical activity levels will be confirmed by objective
measures using an Actiheart physical activity monitor (Cambridge Neurotechnology Ltd). This is
attached to the chest either using ECG pads or a chest strap for measurement of free-living physical
activity(21).
Laboratory trial days
On the evening before trial days, a 5-mL blood sample will be taken (via venepuncture) for analysis of
baseline 2H enrichment of the body water pool. Participants will then consume a standardised meal
with 3 g·kg body water-1 of 2H2O for the assessment of fasting hepatic DNL(22). Participants will
continue to consume 2H2O throughout trial days for the measurement of postprandial DNL(22).
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On postprandial study days, participants will arrive at the laboratories in a rested, overnight fasted
state, after 24-hours of diet and physical activity standardisation.
Body mass will first be recorded, and then a cannula will be inserted retrograde into a pre-heated
dorsal hand vein for arterialised blood sampling, and baseline blood and expired breath samples
will be taken for analysis of plasma metabolite and hormone concentrations, plasma tracer
enrichments, and indirect calorimetry, respectively. Participants will then consume a high-fat test
drink containing 50 g of fat, plus 100 g of either lactose, sucrose or maltodextrin (Table 1). The
test drink will also contain 200 mg [U-13C]palmitate in order to trace the fate of the dietary fat(22).
This quantity of lactose is roughly equivalent to 400 mL of milk and 400 g plain yoghurt, which is
very high but not supraphysiological in light of typical UK intakes.
Table 1. Main laboratory trials and drink composition.
Trial
Carbohydrate type mixed with fat source
Fat Source
(100 g)                                                       (50 g)
Maltodextrin
Glucose polymer; a common sugar substitute (control)
30 g palm oil and
20 g olive oil
Lactose
Galactose-glucose disaccharide (intervention)
30 g palm oil and
20 g olive oil
Sucrose
Fructose-glucose disaccharide (active comparator)
30 g palm oil and
20 g olive oil
Low-calorie-sweetened chocolate flavouring will be added to all drinks.
Participants will be advised that dairy products are used in this study and people following a vegan
diet may therefore choose not to participate.
Blood samples will be drawn at 30, 60, 120, 180, 240, 300 and 360 min after ingestion of the test
drink and will be centrifuged for plasma collection and determination of metabolite and insulin
concentrations (Figure 4). A portion of plasma will undergo density gradient ultracentrifugation to
obtain the VLDL-rich [Svedberg flotation rate (Sf): 20-400] and chylomicron-rich (Sf: >400)
fractions(10). Expired breath samples will be taken at 60, 120, 180, 240, 300 and 360 min after
ingestion for indirect calorimetry (Douglas bag method) and [13C] appearance in expired CO2 (by
isotope ratio mass spectrometry).
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4.9. Measurements and Analysis
Plasma metabolite and hormone concentrations
Plasma concentrations of triglycerides, glucose, fructose, galactose, lactate, non-esterified fatty
acids, glycerol, beta-hydroxybutyrate and uric acid concentrations will be determined by
commercially-available enzymatic/colourimetric assays. Plasma insulin, estradiol and
progesterone concentrations will be determined by commercially-available enzyme-linked,
immunosorbent assays. A portion of plasma will undergo density gradient ultracentrifugation to
obtain the VLDL-rich [Svedberg flotation rate (Sf): 20-400] and chylomicron-rich (Sf: >400)
fractions(10).
Fasting and postprandial de novo lipogenesis
s
Postprandial DNL will be determined by the incorporation of deuterium from 2H2O in plasma water
into VLDL-TG palmitate using gas chromatography-mass spectrometry (Finnigan GasBench II
Thermo Fisher Scientific, Paisley, U.K.) with monitoring ions with mass-to-charge ratios of 270
(M+0) and 271 (M+1) as we have previously performed(22). In this respect, DNL refers to the
proportion of newly synthesized palmitate in VLDL-TG, and this represents the synthesis of fatty
acids from non-lipid precursors. The benefit of this method over tracing labelled lactose is that
labelling the ingested sugar only captures the conversion of that specific precursor to fatty acids.
The 2H2O method on the other hand, captures DNL from all precursors. Therefore, if lactose
ingestion stimulates the conversion of lactate, galactose and glucose to lipid, this would be captured
by our proposed method, but would be partially missed if using lactose labelling.
Substrate oxidation
Whole-body carbohydrate and fat utilisation rates will be determined in the fasted and postprandial
state using indirect calorimetry (10-min samples every 60 minutes). Dietary fat oxidation will be
determined by the appearance of [13C] in breath CO2 in line with our previous work(22). Specifically,
13C-to-12C ratios in breath samples, and the relative rate of whole-body meal-derived fat oxidation
will be calculated. First, the rate of expiration of 13CO2 in breath will be calculated by multiplying
CO2 production rate as determined by indirect calorimetry (mmol·min-1), by the expired CO2
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enrichment of 13C (tracer-to-tracee ratio). To allow for sequestration of label into the bicarbonate
pool, a dietary acetate recovery factor of 51% will be applied(23), and data will be corrected for fat-
free mass as determined by DXA.
4.10. Sample Size
Based on our preliminary data, co-ingestion of galactose increased postprandial plasma triglyceride
incremental area under the curve by a further 46.51 ± 0.28 mmol·L-1 x 300 min (mean ± SD) above
glucose. Using this effect size (d = 0.75), 24 participants would provide a >90% probability (power =
0.94) of detecting such an effect with an α-level of 0.05 using a one-way, repeated-measures
ANOVA and accounting for post-hoc comparisons of three factors by the principle of closed testing.
Accordingly, 24 adults will be recruited. Dropouts will be replaced in a rolling fashion to ensure
a complete sample size of 24. Based on our previous experience of similar studies, we expect
a dropout rate of ~20% and thus anticipate recruitment of ~28 people to achieve this target.
4.11. Statistical Analyses
All of the analyses subsequently described will first be conducted on the entire sample as a primary
analysis (males and females combined). A secondary analysis will include the analysis of subgroups
(males only and females only), in addition to exploring sex-by-treatment interaction effects. The
distribution of data will be assessed by visual inspection of Q-Q plots in addition to the Shapiro-Wilk
test.
Primary analyses
Summary statistics (e.g. iAUC, AUC, peak concentrations) will be analysed by one-way ANOVA.
Time-course data will be assessed by a two-way (time x treatment) ANOVA. Paired t-tests will be
used for post-hoc comparisons, which will be corrected for multiple comparisons using the Holm-
Bonferonni adjustment.
Secondary analyses
The analysis of each subgroup (males only and females only), will be analysed by one-way ANOVA,
for summary statistics. Time-course data will be assessed by a two-way (time x treatment) repeated
measures ANOVA. Paired t-tests will be used for post-hoc comparisons, which will be corrected for
multiple comparisons using the Holm-Bonferonni adjustment.
To assess sex-by-treatment interaction effects, summary statistics will be analysed by a two-way
(sex x treatment) mixed-model ANOVA. Time-course data will be analysed by three-way (sex x
treatment x time) mixed-model ANOVA. Unpaired t-tests will be used for post-hoc comparisons,
which will be corrected for multiple comparisons using the Holm-Bonferonni adjustment.
4.12. Storage and Management of Data
Blood samples will be partially processed on the day of collection before freezing in preparation for
analysis. Throughout this period and during the subsequent analysis, blood and breath samples will
be kept in a locked laboratory and will be inaccessible to anyone outside of the research team.
Samples will be coded and identified only using anonymized sample identifier numbers. After
analysis, samples will be stored until the work has been published. We will ask participants to
consent to their samples being used in any other ethically approved project during the lifetime of the
current project.
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Some analyses will be required to be performed at other sites (e.g. University of Oxford for Mass
Spectrometry). These samples will be coded and identified only using anonymized sample identifier
numbers for shipment.
If a participant wishes to withdraw from the study, they will be permitted to do so, however their
samples will only be destroyed by specific request of the participant. Withdrawing participants will be
reminded of this at the point of withdrawal, and should they not wish that their samples not be
analysed or further stored, said samples will be destroyed immediately.
Any results published from this study will be anonymous and include no identifiable information. An
Excel data sheet maintained on a University computer will be used to record participant information
and results. The computer will be password protected to ensure only the designated researchers
can access the data.
Whilst all measurements included in the protocol provide insight into physiology and health, of those
that have recognised clinical significance and established normal ranges, only bone mineral density
from DEXA can be provided in a clinically meaningful timeframe. As such, should a participant’s
data for this measure fall outside clinically normal ranges, we will inform the participant’s GP directly
by way of a letter, provided the participant has consented for us to do so.
4.13. Expenses and Benefits
Reasonable travel expenses for visits to the University will be reimbursed on production of receipts or
via claims for mileage. Participants will also be offered a voucher (e.g. Amazon/Love2shop gift
vouchers) with a value of £150 upon completion of the study. Participants will be provided with an
information pack containing their results for relevant outcomes along with reference ranges (e.g.,
physical activity level, diet, body composition, blood tests).
5. DISADVANTAGES/RISK AND DISCOMFORT
Participants will be asked to give up their time for repeated visits to undertake various tests. For
some people this may be seen as an inconvenience. Blood samples will be collected using an
intravenous catheter, which might cause minor discomfort during the procedure and may result in
minor bruising. More serious complications are the very small risk of infection. However, the
occurrence of such events is very rare and risks are further minimised by our strict adherence to
best practice and standard operating procedures.
We will measure fat mass, fat-free mass, and bone mineral density, using dual-energy x-ray
absorptiometry (DXA). DXA scans are non-invasive radiologic projection scans that use very low
radiation, often compared to the exposure to background radiation experienced on daily basis in the
UK. For the sake of the present study, this equates to less than one day of background radiation
(and a tiny fraction of the amount of radiation experienced during a typical X-ray). These techniques
are routinely used in hospitals and with elite athletes but nonetheless they do represent some
exposure to a small amount of radiation.
We will utilise stable isotope tracers to determine key aspects of postprandial metabolism. These
stable isotopes are non-radioactive and therefore pose no long-term risk to the health of the
participants. On the evening prior to the study day, the participants will consume a loading dose of
2H2O (heavy water). The loading dose of 2H2O given is the amount to achieve a plasma water
enrichment of 0.3%. This will be split into two equal doses with a ~2 h gap between consumption.
This will decrease the likelihood of the participant feeling “dizzy” which can occur within ~1 hour
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after consuming the water. Individuals prone to travel sickness appear to be more likely to get
“dizziness” after consumption of water, but this dizzy feeling is transient and not harmful.
The above will be explained to the potential participant verbally and in the participant information
sheet to ensure that they are fully informed before giving consent.
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