Study Setting
The trial will be conducted at the University of Auckland Clinical Research Centre. The study is a randomised crossover trial to capture biological difference in postprandial nutrient dynamics. Research subjects will act as their own controls and will consume each meal on a separate occasion in random order. The study compares exemplars of pasture-fed New Zealand beef, grain-finished New Zealand beef, lamb and a plant-based meat analogue. The pasture-fed beef was obtained from Angus Steers that had been fed exclusively on pastures (ryegrass and white clover mix) whereas the grain-finished beef was Angus steers that had been finished for an average of 122 days on a maize silage, barley wheat and straw ration. Marble score (3-4/9 on the Australian Grading System) [15] and pH (average 5.5) of the meat were consistent between production systems. The pasture-raised Lamb was sourced from free-range New Zealand farms. The meat analogue has been selected from commercial varieties on the basis of its macronutrient profile (protein and fat) and appearance to best match that of meat.
The digestion and metabolism of key nutrients in beef will be measured immediately after the ingestion of a single meal. This experimental setting will also be used to examine some subjective qualities of the meal experience, such as satisfaction score (i.e., liking, satisfaction, enjoyment, satiety, appetite) and gastrointestinal score (i.e., comfort, fullness, bloating, rumbling, flatulence, faecal urgency, diarrhoea).
Eligibility criteria
All participants will be omnivores willing to consume both red meat and plant-base alternatives for the purposes of the trial. Those with chronic health conditions, hyperlipidaemia, obesity (BMI ≥ 30), use of medications (except occasional use of NSAIDs and antihistamines), history of anosmia and ageusia (issues with taste and smell), current dieting or disordered eating pattern and smoking tobacco or recreational drugs will be excluded from participating. Participants will complete an on-line screening which will include the Three-factor Eating Questionnaire-R18 (TFEQ) and a health survey. Participants with a TFEQ score greater than 75% will be excluded from participating on the basis their perception of food is potentially influenced by underlying psychological issues.
Recruitment and Informed Consent
Males will be recruited from the millennial generation (20-34 y), as males typically have a greater post-prandial lipid response than females [16]. In general, Millennials were chosen as this population demographic has been demonstrated to have the greatest variation in meat intake [17]. Recruitment will occur via posters placed around the University of Auckland and using social media sites (FacebookTM). Informed consent will be collected by research staff following participant inquiry and provision of information.
Ethics and dissemination
The trial has been granted ethical approval by the New Zealand Ministry of Health’s Health and Disability Ethics Committees (Ref: 19/STH/226). All results originating from this study will be submitted for publication in scientific journals and presented at meetings.
Sample size calculation
The principal biomarker for calculation of sample size was postprandial change in LCPUFA concentrations in blood chylomicrons (namely 20:4 n-6, 20:5 n-3, 22:5 n-3, 22:6 n-3). We estimate a sample size of 29 would enable the detection of a smallest worthwhile change of 0.5 mmol/L from baseline to 5 hours post meal consumption [19]. Pooled published data indicate an increase of ~3.9 mmol/L (±2.8 mmol/L) in chylomicron fatty acids post meal consumption. An effect size of 0.2 was used to calculate the smallest worthwhile effect. Recruitment will continue until 29 men have successfully completed the trial.
Randomisation and Blinding
The design will be a single blinded crossover. Participants will consume each test meal in random order, using a computer-generated sequence, at least one week, but no more than one month, apart. The randomisation will be conducted centrally, with one research staff responsible for meal preparation aware of the allocation. Staff responsible for blood collection and analysis will be blinded to the intervention, as will participants.
Reminder text messages will be sent to participants to increase attendance to each of the three study appointments. Additionally, participants will receive financial compensation for partaking in each of the three assessment visits. Participants will have the right to withdraw from the study at any time. The principal investigator will have the right to discontinue participants’ involvement in the study if they become ineligible or when significant protocol deviations occur. The data of participants who withdraw will be kept and might be used in exploratory analyses, unless the participant requests for the data to be deleted.
Study Overview
Two test meals will include the same cut and quality of pasture-fed New Zealand beef or grain-fed New Zealand beef of well-defined provenance, which has been specifically slaughtered, packaged and stored for this trial. The third test meal will be a commercial plant-based meat alternative that best resembles in terms of appearance and macronutrient profiles.
Meals will include a 220 g raw serving of beef (approximately 160 g cooked), which is in line with the World Cancer Research Fund / International Agency for Research on Cancer recommendation on red meat consumption, which suggests limiting the weekly red meat consumption to 350-500 g cooked. A minimum quantity of 100 grams cooked meat is required to ensure adequate fat intake to allow assessment of post-meal lipid dynamics [19]. The test meals will be provided in the morning after an overnight fast. The meals will look identical in appearance and participants will not be told which meal they have received.
In all cases the cut of meat used was the full tenderloin (M. psoas major + M. psoas minor + M. iliacus). This is typically removed in one piece from the full rump and loin. To accommodate between-animal variation, tenderloins were collected from 12 Grass steers, 15 Grain steers and 40 lambs. The intact meat was aged for at least 21 days at -1.5°C. In preparation for the trial, the Grain tenderloins were trimmed of excess fat, ground together to a homogenous 4 mm mince, then vacuum packed in 500 g aliquots and frozen until needed for the trial. The Grass tenderloins were not trimmed and so included intermuscular fat. They were processed as per Grain. The Lamb was closely trimmed of fat and similarly processed.
Blood samples will be collected immediately prior to (baseline) and 0, 60, 120, 180 and 240 minutes following the meal for serum and EDTA plasma. Bloods will be taken in the phlebotomy room in the research facility, by a phlebotomist using via an indwelling catheter. Blood samples will be processed and frozen at -80°C for subsequent analysis.
During this time participants will remain in the research unit. Appetite, digestive comfort and palatability scores will be self-reported using manual questionnaires.
The SPIRIT reporting guidelines for the accurate documentation of clinical trial protocols has been followed [16]. See Table 1 for trial schedule.
Table 2
SPIRIT Chart. Schedule of enrolment, interventions, and assessments
| STUDY PERIOD |
| Enrolment | Allocation | | | Post-allocation | Close-out |
TIMEPOINT | -t1 | 0 | tpre | t0 | t30 | t60 | t120 | t180 | t240 | +t1 |
ENROLMENT: | | | | | | | | | | |
Eligibility screen | X | | | | | | | | | |
Informed consent | X | | | | | | | | | |
Allocation | | X | | | | | | | | |
INTERVENTION MEALS: | | | | | | | | | | |
Grain-fed Beef | | Random order | | X | | | | | | |
Grass-fed Beef | | Random order | | X | | | | | | |
Meat Alternative | | Random order | | X | | | | | | |
ASSESSMENTS: | | | | | | | | | | |
Amino acids/ Neurotransmitters | | | X | | | X | X | X | X | |
Chylomicron fatty acids | | | X | | | X | X | X | X | |
Glucose/ Insulin | | | X | | | X | X | X | X | |
Iron/ Haemoglobin | | | X | | | | | | X | |
Questionnaire-Fullness | | | X | X | X | X | X | X | X | |
Questionnaire-Digestive Symptoms | | | X | X | X | X | X | X | X | |
Questionnaire-Palatability | | | | | X | | | | | |
Questionnaire- Participant Insight | | | | | | | | | | X |
Intervention
All meals will be prepared according to standardised recipes by the Research Dietitians and served at the test kitchen site (The University of Auckland Clinical Research Centre). The meat will be minced to ensure homogeneity between participants. All non-meat food items will be purchased at a local supermarket. The test diets will be protein and fat matched, and the meals will be balanced for energy, carbohydrate and fibre. A panel of meat scientists, a nutritionist and research dietitian reviewed available options to choose the plant-based meat analogue. Their criteria included 1) nutrition should approximate beef, 2) appearance should approximate beef, and 3) readily available in the retail space. Nutrient content data for the beef mince was based on New Zealand composition tables, and for the analogue was taken from the NIP.
All participants will be fasted for 10 hours prior to consuming the test meal. All test meals will be given in the morning, as postprandial lipid responses are greatest at this time [17].
Outcomes (Primary and Secondary)
Following the consumption of a single meal:
Primary outcome variable
- Change in LCPUFA (18:2 n-6, 18:3 n-3, 20:4 n-6, 20:5 n-3, 22:5 n-3, 22:5 n-3, 22:6 n-3) concentrations in the chylomicron fraction over 5 hours. Difference in the change between pasture-, grain- and plant-based alternative meat meals.
Secondary outcome variables
- Differences in other fatty acid (14:0, 16:0, 16:1 n-7, 18:0, 18:1 n-9, others) concentrations in the chylomicron fraction over 5 hours between pasture-, grain- and plant-based alternative meat meals.
- Differences in the serum amino acid (isoleucine, leucine, valine, histidine, lysine, methionine, phenylalanine, threonine, alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, tyrosine, 3-methyhistidine, citruline, hydroxyproline, ornithine, taurine, others) concentration over the 5 hours
between pasture-, grain- and plant-based alternative meat meals.
- Differences in plasma neurotransmitters (phenylethyl amine, 3,4-dihydroxyphenylalanine, dopamine, 3-methoxytyramine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, norepinephrine, 3,4-dihydroxyphenylglycol, 3-methoxy-4-hydroxyphenylglycol, normetanephrine, epinephrine, metanephrine, vanillylmandelic acid, tryptophan, kynurenine, 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid, alpha-aminobutyric acid, gamma-aminobutyric acid) concentrations at 60 minutes post consumption, between pasture-, grain- and plant-based alternative meat meals.
- Differences in blood iron and haemoglobin concentration pre and 5 hours post meal between pasture-, grain- and plant-based alternative meat meals.
- Differences in subjective experience to the meal including fullness, satisfaction, gastrointestinal comfort scores between pasture-, grain- and plant-based alternative meat meals.
- Qualitative differences in the participant opinions regarding future red meat consumption (“Participant insights”)
- Adverse events, count score differences between meals.
Measurements
Blood and Plasma Analyses
Serum samples will be kept at room temperature for 15 minutes prior to centrifugation and storage of supernatant at -800C until analysis. An aliquot of plasma will be maintained at 4°C for the chylomicron-rich fraction (CMRF) separation to occur within 6 h, with the remaining stored at −80°C until analysis.
Fatty acid composition of the CMRF will be analysed by the fatty acid methyl esters (FAME) assay as previously described [21] and by lipidomics, as previously described [22]. Plasma samples will be sent to LabPlus certified laboratory to assess haemoglobin and iron of the baseline and 240 minute sample only.
Serum amino acid analysis will be conducted using ultra performance liquid chromatography (UPLC) according to previously published methods [23, 24]. The three test meals will also be analysed for their final amino acid content.
Neurotransmitters will be assessed by mass spectroscopy. The methodology utilises a MS-probe and stable isotope coding LCMS method developed by Plant & Food Research (New Zealand) which has been optimised for plasma samples.
Plasma glucose will be measured using a Roche Cobas c311 by enzymatic colorimetric assay (Roche, Mannheim, Germany) and insulin by an electrochemiluminescence immunoassay.
Subjective Analyses
Satisfaction and gastrointestinal comfort scores will be analysed using a 10 cm visual analogue scale, previously validated for use in single meal investigations [25]. Satisfaction scores will be completed by the participants upon arrival, once immediately prior to meal ingestion (two baseline assessments to account for individual variation), immediately following ingestion, and then timed with blood sampling for 5 hours.
Gastrointestinal comfort scores will be analysed by a questionnaire consisting of a series of visual analogue scales using intensity anchors (“no symptom”, “the most severe symptom imaginable”). Gastrointestinal comfort scores questionnaires will be completed by the participants upon arrival, immediately prior to meal ingestion (two baseline assessments to account for individual variation), immediately following ingestion of the meal, and then timed with blood sampling for 5 hours.
Participant insights will be assessed via a closed and open ended questionnaire which asks the participant to guess which meat-type they were provided the day prior and the degree to which they are likely to consume red meat in the future, with reasoning. The survey will also specifically ask for any adverse reactions to the test meal. The participants will be asked to complete a survey the day following the final test meal trial, a link to the Qualtrics (SAP, Utah, US) survey will be emailed.
Qualitative Analysis
Results from the open-ended survey will be transcribed into NVivo (QRS International, Victoria, AUS) software and analysed using qualitative techniques for emergent themes.