Whey Protein Isolate improves vitamin B12 and

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Molecular Nutrition and Food Research

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Whey Protein Isolate improves vitamin B12 and folate status in elderly Australians with sub-clinical deficiency of vitamin B12

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Research Article 17-Oct-2016

Dhillon, Varinderpal; CSIRO Health and Biosecurity, Genome Health and Nutrigenomics Zabaras, Dimitrios; CSIRO Health and Biosecurity, Genome Health and Nutrigenomics Almond, Theodora; CSIRO Health and Biosecurity, Genome Health and Nutrigenomics Cavuoto, Paul; CSIRO Health and Biosecurity, Genome Health and Nutrigenomics James-Martin , Genevieve ; CSIRO Health and Biosecurity, Genome Health and Nutrigenomics Fenech, Michael; CSIRO Health and Biosecurity

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Complete List of Authors:

mnfr.201600915

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Molecular Nutrition and Food Research

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Cobalamin (Cbl), DNA damage biomarkers, Methylmalonic acid (MMA), Soy protein isolate (SPI), Whey protein isolate (WPI)

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Keywords:

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Molecular Nutrition and Food Research

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Whey Protein Isolate improves vitamin B12 and folate status in elderly Australians with sub-clinical deficiency of vitamin B12

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Varinderpal S. Dhillon*, Dimitrios Zabaras, Theodora Almond, Paul Cavuoto, Genevieve James-Martin and Michael Fenech*

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Commonwealth Scientific and Industrial Research Organisation Health and Biosecurity, Gate 13, Kintore Avenue, Adelaide, South Australia 5000, Australia

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Corresponding Authors:

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Dr Varinderpal S Dhillon, Commonwealth Scientific and Industrial Research Organisation Health and Biosecurity, Gate 13, Kintore Avenue, Adelaide, South Australia 5000, Australia. Telephone: +61 8 8303 8932; Fax: +61 883038899; Email: [email protected]

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Prof Michael Fenech, Commonwealth Scientific and Industrial Research Organisation Health and Biosecurity, Gate 13, Kintore Avenue, Adelaide, South Australia 5000, Australia. Telephone: +61 8 8303 8880; Email: [email protected]

Abbreviations: Whey protein isolate (WPI); Soy protein isolate (SPI); homocysteine (HCY); methylmalonic acid (MMA); micronucleus (MN); nucleoplasmic bridges (NPBs); nuclear buds (NBuds); telomere length (TL); mitochondrial DNA (mtDNA); brain derived neurotrophic factor (BDNF)

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Keywords: Cobalamin (Cbl), DNA damage biomarkers, Methylmalonic acid (MMA), Soy protein isolate (SPI), Whey protein isolate (WPI)

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ABSTRACT

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SCOPE: Whey protein isolate (WPI) contains vitamin B12 and folate. However, the efficacy

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of WPI as a bio-available source of these vitamins in the elderly with low vitamin B12 was

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not previously tested. We investigated the effects of WPI supplementation on vitamin B12 and

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folate status in blood and measured changes in homocysteine, methylmalonic acid and

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genome integrity biomarkers in elderly individuals with low vitamin B12 status. The effect of

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WPI was compared to soy protein isolate (SPI).

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Methods and Results: In this randomized controlled cross-over intervention trial, 56 sub-

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clinically vitamin B12-deficient participants received 50g WPI or 50g SPI as a control for 8

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weeks followed by 16 week wash-out phase and then cross-over to alternative supplement for

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next 8 weeks. Consumption of WPI resulted in significant increase in serum active B12 (p
0.20 µmol/L and serum creatinine concentration of 120 µmol/L or less and willing

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to consume the quantities of WPI and SPI specified for the trial. The exclusion criteria

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include those who were unable to attend an information session and/or read the information

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sheet, current smokers, habitually consume more than two standard alcoholic drinks per day,

treatment

for

any

life-threatening

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diseases,

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BMI 35 Kg/m2 or greater, diagnosed with diabetes, lactose intolerance, history of pernicious

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anaemia or atrophic gastritis and regular users of antacids. Fifty six eligible participants

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attended the information session and read the study information sheet prior to consenting to

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participate. The study was approved by CSIRO Human Ethics Committee, Adelaide,

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Australia. The 45-75 year age was targeted because of a high level of subclinical vitamin B12

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deficiency and DNA damage in this age group [2, 25].

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Intervention Design

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The study design was a randomized controlled cross-over intervention to maximize the power

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of the study (Figure 1). The selected 56 sub-clinically B12-deficient participants were

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randomized to daily intake of 50g whey protein isolate (WPI) or 50g soy-protein isolate (SPI)

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as a control for 8 weeks following which there was a 16 week wash-out phase and then they

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crossed-over to the alternative supplement for the next 8 weeks. The randomization scheme

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was generated using the online resource web site: http://www.randomization.com and the

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intervention trial has been registered ([ACTRN12614000159651) in Australian New Zealand Clinical

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Trials Registry (ANZCTR) http://www.ANZCTR.org.au/ACTRN12614000159651.aspx].

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Participants were asked to maintain their habitual diet during the intervention trial, but to

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refrain from eating food high in vitamin B12 (i.e. foods with B12 levels > 4.9µg/100g) and also

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refrain from taking supplements containing vitamin B12/choline/antioxidants. Dietary

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restrictions also included limiting consumption of vegetables or pulses (2 servings/day), fruits

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or juices (3 servings/day), black tea or coffee (2 cups/day), chocolate (50g/day), wine

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(200ml/day) and/or beer (375ml/day) in order to avoid an excessive intake of antioxidants to

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minimize variation between subjects and groups. Participants were required to complete a 3-

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day food and beverage intake record during the first and last week of each intervention phase

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at which points their BMI was also measured. A specific section requesting information on

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intake of soy-based liquid and solid meals was included in the 3-day food record. A list of

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soy-rich and/or vitamin B12 fortified foods and beverages that are commonly available in

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Australia were provided to each participant to assist with accurate record reporting.

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Instructions on how to record intake and advice on the specific dietary recommendations for

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the study design were given by the dietician. Energy consumption, intake of macronutrients

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and micronutrients was calculated according to Australian food and nutrient databases

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(www.foodstandards.gov.au).

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Compliance with the required dietary restrictions and intake of the WPI or SPI supplement

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were checked on a weekly basis initially (during first two weeks) and every 2 weeks if no

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problems were evident. If a deviation greater than 10% of the intake recommendations was

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detected (for either the dietary restrictions or SPI/WPI supplements) the dietician interviewed

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the participant to identify strategies that could help rectify any reduced compliance and

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facilitate compliance in case of any difficulty.

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The WPI or SPI was consumed daily as 25g blended in 200 ml fruit juice or water 2 times a

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day so that the total daily intake was 50g WPI or 50g SPI each day. The WPI and SPI was

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provided in 25g sachets (two per participant for each day of the intervention phases). A

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compliance checklist for use of the WPI and SPI sachets was completed by the participants.

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During the washout period participants were allowed to return to their pre-intervention

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habitual dietary habits and required to avoid consumption of supplements containing vitamin

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B12 or foods fortified with vitamin B12. Serum B12 was checked mid-way during the washout

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period. Those exceeding 350pmol/L serum B12 concentration were required to restrict intake

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of foods rich in B12 until they achieved a < 350pmol/L serum concentration before starting

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the second treatment phase. Blood samples were collected at the beginning and end of each of

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the intervention phases and half-way during the washout phase i.e. at 0, 8, 16, 24 and 32

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weeks. Blood samples were used fresh or bio-banked frozen at -80oC depending on the

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requirements of each assay. The primary outcome measures include: serum B12, active B12

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(holotranscobalamin) and plasma MMA concentrations. The secondary outcome measures

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include: red cell folate, serum folate, plasma total homocysteine, plasma brain derived

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neurotrophic factor (BDNF), chromosomal damage biomarkers, telomere length and

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mitochondrial DNA damage.

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Sample Collection and Storage

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Blood from each participant was collected into appropriately labelled tubes depending on the

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assay as follows: 9ml in a gel tube for serum B12, serum folate and red cell folate assay, 9ml

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in an EDTA tube for MMA assay, 18ml in lithium-heparinized tube for the cytokinesis-block

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micronucleus cytome (CBMNcyt assay), telomere length, mtDNA deletion and BDNF

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assay), 4ml EDTA tube (on ice) for the HCY assay and 4 ml serum separation tube for the

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active B12 (holotranscobalamin) assay.

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Serum B12, serum folate, red cell folate and HCY assays were performed on the same day

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whereas plasma samples (for MMA and BDNF assays) and isolated serum for active B12

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were stored at -80°C until the assay was performed. CBMNcyt assay was performed using

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fresh whole blood collected on the same day. Lymphocytes were isolated from the remaining

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fresh whole blood lithium heparin samples and stored at -80°C until DNA was isolated to

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carry out telomere and mtDNA deletion assays.

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Cytokinesis-block micronucleus Cytome (CBMNcyt) assay for DNA damage

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Chromosomal DNA damage was measured in lymphocytes using the CBMNcyt assay. In this

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method three biomarkers of chromosomal damage and genomic instability are measured: (i)

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micronuclei (MN), a biomarker of chromosome breakage or loss, (ii) nucleoplasmic bridges

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(NPBs), a biomarker of dicentric chromosome formation caused either by mis-repair of DNA

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breaks or telomere end fusions and (iii) nuclear buds (NBuds), a biomarker of amplified

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DNA or unresolved DNA repair complexes [26-27]. MN frequency measured in this assay is

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one of the best validated biomarkers for measuring the mitigating or exacerbating effects of

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dietary factors on genome damage [28-29]. The CBMNcyt assay was performed as

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previously described [26]. Briefly, venous blood was collected in lithium heparin tubes and

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incubated at 37oC for up to 2 hr. Whole blood cultures (in duplicate and each visit) were set

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up in RPMI-1640 medium (Sigma, Australia) containing 10% fetal bovine serum (Trace

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Scientific, Australia) 2 mM L-glutamine (Sigma, Australia), 1 mM sodium pyruvate (Trace

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Scientific) and phytohaemagglutinin and incubated at 37oC and 5% CO2 in a humidified

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incubator. Cytochalasin-B was added after 44 h of culture and cells were harvested 24 h later

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by density centrifugation with Ficoll (GE Healthcare, Australia), they were transferred to

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slides by cytocentrifugation and then fixed and stained using Hemacolor (Merck, Australia).

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The CBMNcyt assay slides were labelled with the volunteer and treatment code number and

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scored for frequency of binucleated (BN) cells with one or more MN, NPB, and NBUD in

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1000 BN (bi-nucleate) cells from each duplicate culture using previously described scoring

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criteria [26].

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Telomere length assay

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Genomic DNA was extracted from isolated lymphocytes using the QIAamp DNA blood mini

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kit (Qiagen, Australia). Purified DNA samples were quantified using NanoDrop 1000

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spectrophotometer (Thermo Fisher Scientific, Australia) and diluted as per experimental

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requirements (5ng/µl). Telomere length was measured using quantitative real-time PCR as

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described previously [30]. The ratio of the telomere (T) repeat copy number to the single-

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copy gene (S) was determined for each sample using ABI 7300 Real-Time PCR Detection

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System (Life Technologies, USA). The final concentrations of the PCR reagents were 1 ×

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SYBR Green Mix (Life Technologies, USA), 20 ng DNA, 0.2 µmol of telomere specific

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primers (F: 5′-GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3′; R: 5′-

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TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA-3′) and 0.3 µmol of 36B4

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primers (F: 5′-CAGCAAGTGGGAAGGTGTAATCC-3′; R: 5′-CCCATTCTATCATCA

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ACGGGTACAA-3′). The reactions were performed using telomere and 36B4 specific

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primers in a 96-well plate, and each plate included a reference DNA sample. A five-point

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serial dilution standard curve of DNA concentration versus T/S ratio using DNA isolated

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from the 1301 cell line (which has a mean telomere length of 23,000 base pairs) was

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established in each plate. The standard curve was then used to convert the T/S ratio into

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telomere length (TL) in base pairs (bp) using following equation: Absolute TL(bp) =

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2433.23X + 3109.51 where X = T/S ratio, 2433.23 is the slope and 3109.51 is the intercept

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of the standard curve [31]. A standard curve with a high correlation factor (R2 ≥ 0.97) was

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required to accept the results from the plate. We calculated intra-assay and inter- assay CVs

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to assess the variation of results within the data set and to ensure plate-to-plate consistency,

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respectively. The intra-assay coefficient between duplicates was 2.3% for telomeres and 2.1%

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for the single-copy gene, whereas the inter-assay CV between plates was 0.5% for telomeres

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and 0.88% for the single-copy gene.

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Intact mitochondrial DNA (mtDNA) assay

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Isolated DNA was used to determine the percentage of mtDNA that is intact i.e. without

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major deletions. The % intact mtDNA was determined by long-range PCR (XL-PCR) using

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Phusion High-Fidelity DNA polymerase kit (Thermo Fisher Scientific, Australia). The

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amplification was carried out in a total volume of 25 µL, containing 15 ng (3µl) of template

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DNA, 1X High-Fidelity master mix containing dNTPs, buffer and High-Fidelity DNA

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polymerase and 0.5 µM of each primer. The amplification involved an initial denaturation of

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30 sec at 98°C, followed by 35 cycles with denaturation at 98°C for 10 seconds and a

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combined annealing and extension at 72°C for 7½ minutes followed by a final extension of 7

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minutes at 72°C. The forward primer 5’-TGAGGCCAAATATCATTCTGAGGGGC-3’

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(position 15149) and reverse primer 5’-TTTCATCATGCGGAGATGTTG GATGG-3’

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(position 14841) were used to amplify 15.2 kb of total mtDNA covering approximately 91%

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of mitochondrial genome [32]. Each sample was amplified in duplicate and PCR products of

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different size (kb) were separated by gel electrophoresis. Amplified mtDNA PCR products

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were visualised and digitised on the Gel-Doc System (BioRad, Australia) after

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electrophoresis in 1% agarose gel for one hour at constant 80 volts at room temperature 22oC.

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The digitised gel image captured using Gel-Doc System was analysed using ImageJ software

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(https://imagej.nih.gov/ij/) to measure the integrated optical density (IOD) of each band in

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each lane. The IOD of all bands including the main 15.2 kb band that represents intact

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mtDNA and the minor bands (i.e. less than 15.2kb) was measured. The IOD values obtained

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for all bands were added together to obtain “IOD of all bands”. The IOD values of all minor

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bands (i.e. 95%),

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precision (inter-assay: 6%, intra-assay: 5%), linearity (0.11-0.75 µmol/L, r2: 0.9987, extended

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range tested up to 10 µmol/L with no loss of linearity). Limit of detection (3 x SD of a 0.11

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µmol/L sample) was 0.05 µmol/L under the conditions used. The concentration of the lowest

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calibrator was assumed to be the limit of quantification each time.

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Clinical Laboratory Assays

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Serum B12 was measured using the ARCHITECT assay system at SA Pathology, a certified

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medical biochemistry laboratory located in Adelaide, South Australia whereas serum active

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B12 (holotranscobalamin), serum folate, red cell folate and plasma HCY were measured at

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Melbourne Pathology, a certified medical laboratory located at Melbourne, Victoria as per

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standard guidelines for the Architect ci8200 integrated serum/plasma analyzer system [35].

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Statistical analysis

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Our previous study in older men (aged 50-70y) showed that they had a mean serum total

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vitamin B12 of 283 pmol/l with a standard deviation of 108 pmol/l [36]. With 50 subjects per

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group the intervention had 90% power to detect an increase of 50pmol/L at p < 0.05 which is

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an effect size of 18%. For comparison, the intervention study of Eussen et al (25) showed that

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supplementation with 2.5ug/d of vitamin B12 orally for 16 weeks produced a 20% increase in

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serum B12 in elderly subjects aged 70y or greater (mean 80y). If, in a worst case scenario,

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20% of participants dropped out of the study, it was still be possible to detect an effect size of

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18% at 80% power. Parametric statistical methods were used for biomarkers exhibiting

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Gaussian distribution. Non-parametric methods were employed to analyse the results for

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biomarkers that were not Gaussian in their distribution. Paired t-test or Wicoxon-matched

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pairs test were used to (i) compare the biomarker differences between the base-line and post-

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intervention results within each treatment group and (ii) compare the differences between the

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effect of SPI and WPI (i.e. the difference between treatments for their effects on biomarkers

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measured as the change in the post-intervention result relative to the base-line result,

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measured by subtraction). Correlation analysis was performed by Spearman’s or Pearson’s

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test depending on whether the biomarker data were Gaussian or non-Gaussian in their

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distribution. Two way analysis of variance tests were also performed when required to assess

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interaction effects of two variables (e.g. treatment by time, gender by treatment). All P values

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were for 2-tailed tests unless otherwise stated. Statistical tests were performed using Prism

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6.0 (Graphpad Inc., USA) and SPSS (IBM SPSS version 22).

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RESULTS

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Participation, age, BMI, compliance and biomarkers characteristics at base-line

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The participation of the 56 participants (27 males and 29 females) selected for the

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intervention and reasons for non-completion are summarised in the Consort diagram (Figure

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2). Forty four out of 56 participants (23 males and 21 females) completed both arms of the

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intervention and complied satisfactorily with the requirements of the study design. The

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percent compliance rate (mean + S.E.) for adhering to the guideline of consuming 50g day of

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the WPI or SPI supplement was 95.3 ± 7.08 % for SPI and 95.6 ± 4.48 % for WPI.

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The mean age (range) and BMI (range) of the females was 60.49 (42.53 - 75.12) y and 26.56

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(18.44 - 33.11) Kg/m2 respectively and for males mean age (range) was 63.31 ± 8.57 (50.09 -

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76.06) y and mean BMI (range) was 26.67 (20.58 - 34.9) Kg/m2.

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Base-line values for all the biomarkers measured before the SPI and before WPI intervention

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were similar and not significantly different (p values ranged from 0.15 to 0.87 with a mean of

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0.587) (Table 2).

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Effects of intervention on primary outcome measures

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The results of the intervention are explained below and summarised in Figures 3-9 and

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Tables 2-4. Table 4 provides complete summary of the percentage change after SPI and WPI

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intervention relative to base-line for each biomarker.

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Serum Total B12 and Active B12

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Serum B12 concentration declined during both the SPI and WPI intervention phases (-9.0%, p

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= 0.0001; -4%, p = 0.0177 respectively) but to a greater extent in SPI, however the difference

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in change between treatments was not statistically significant (p = 0.1267; Figures 3 A-C).

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The reduction in serum B12 during the intervention phases was likely due to good adherence

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by participants to avoid foods rich in vitamin B12 because intake of dietary vitamin B12

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declined by approximately 18% during the intervention phases relative to the base-line and

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washout phases (Table 3). At base-line, before the interventions commenced, and during the

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washout phase, dietary intake of B12 was 6.68µg per day and 6.66µg per day respectively. In

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contrast dietary intake of B12 was 5.68 µg/day during the SPI phase and 5.53 µg/day during

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the WPI phase (note: the latter figure excludes intake of B12 from WPI).

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Serum active B12 (holotranscobalamin) increased significantly by 19% during the WPI

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intervention (p < 0.0001), it declined slightly by 2% in the SPI intervention (p = 0.56), and

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the difference in change in active B12 between treatments was highly significant (p = 0.0001;

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Figures 3 D-F).

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Plasma methylmalonic acid (MMA) and homocysteine (HCY)

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Plasma MMA increased by 12% during the SPI intervention (p = 0.05), declined by 4%

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during the WPI intervention, and the difference in MMA change between treatments

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approached statistical significance (p = 0.09; Figures 4 A-C).

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Plasma HCY increased significantly during SPI intervention by 3% (p = 0.029) and reduced

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by 2% in the WPI phase but the difference in change in plasma HCY between treatments did

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not achieve statistical significance (Figures 4 D-F).

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Serum folate and red blood cell folate

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Serum folate concentration increased significantly during the WPI phase by 13% (p = 0.009)

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but did not change during the SPI intervention phase (p = 0.68) and the difference between

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treatments was highly significant (p = 0.009; Figures 5 A-C). A similar trend was observed

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for red blood cell folate which increased significantly during the WPI phase by 7% (p =

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0.023) but did not change during the SPI phase (Figures 5 D-F).

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Mitochondrial DNA (mtDNA) integrity and Telomere length assay

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Mitochondrial DNA integrity was measured as the percentage (%) of intact mtDNA. During

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the WPI and SPI intervention phases % intact mtDNA increased by 1.5% (p = 0.025) and

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1.4% (p = 0.115) respectively but there was no significant difference between treatments

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(Figures 6 A-C). There was no significant effect of WPI or SPI on telomere length (Figures

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6 D-F).

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Cytogenetic DNA damage biomarkers measured by CBMNcyt assay

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There was a tendency for micronuclei (MN) to be diminished after the WPI intervention (-

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9%) and to increase (+3%) during the SPI phase, however, none of these effects or the

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relative changes between treatments were statistically significant. Nucleoplasmic bridges

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(NPBs) increased sharply by 85% after consumption of SPI (p = 0.0009) compared to only a

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9% increase during the WPI phase and the difference in these changes between treatments

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was statistically significant (p = 0.0197). There was a similar trend for an increase (+11%) in

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nuclear buds (NBuds) during SPI phase but no marked change during the WPI phase. These

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results are illustrated in Figures 7 A-F and Figures 8 A-C.

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Brain-derived neurotrophic factor (BDNF)

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There was no significant change in plasma BDNF concentration as a result of the WPI or SPI

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intervention (Figure 8 D-F).

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DISCUSSION

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It is important to discover alternative sources of bio-available vitamin B12 because the ability

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to absorb and utilise this vitamin may decline with age. The ongoing trend to vegetarianism

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also increases the need to find alternative food sources that contain natural vitamin B12 that is

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bio-available and bio-efficacious. The main purpose of this investigation was to test whether

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consuming 50 grams of WPI improves vitamin B12 status in healthy adults with sub-clinical

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vitamin B12 deficiency. Prevention of sub-clinical vitamin B12 deficiency is increasingly

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recognised as a risk factor for degenerative diseases of older age such as Stroke and

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Alzheimer’s disease [11] and is also associated with increased DNA damage which is a

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fundamental pathology that accelerates the ageing process [2].

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The current intervention trial was designed to compare the effects of WPI with SPI because

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these are two of the most commonly used protein supplements and apart from other

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micronutrients/bio-actives (e.g. folate, isoflavones) they also differ entirely with respect to

460

vitamin B12 content. During the intervention phases dietary vitamin B12 intake declined by

461

17% from 6.7 µg/d down to 5.6 µg/d due to high compliance of participants to avoid foods

462

rich in B12. Therefore, the results of the intervention reflect outcomes that may be expected in

463

a group of subjects who are already sub-clinically deficient in vitamin B12 and whose vitamin

464

B12 intake is declining at a rate of approximately 17% over 8 weeks, the latter period being

465

the duration of each intervention phase.

466 467

The outcomes of our intervention indicate that daily consumption of 50g WPI (containing

468

2.8-3.0 µg vitamin B12) relative to 50 g SPI (containing no B12) mitigates against a decline in

469

serum total B12, attenuates the increase in MMA and HCY relative to SPI intervention and,

470

importantly, increases active B12 (holotranscobalamin) by 19%. Our results can be compared

471

to those of a dose-finding intervention with vitamin B12 (cyanocobalamin) supplementation,

472

performed in Holland, in elderly healthy people who were sub-clinically B12 deficient in

473

whom the lowest dose tested was 2.5 µg/d [25]. Similar to our study they observed an

474

increase in active B12 of 18% and no reduction in MMA and HCY with the 2.5 µg/d dose.

475

However, unlike our study they also observed a 20% increase in total B12 possibly because

476

the total dietary intake of B12 did not decline during intervention. The results from our

477

intervention when compared to the study in Netherlands, suggest that the B12 in WPI may be

478

as highly bioavailable as a cyanocobalamin supplement, however, this will need to be

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479

verified in another intervention that directly compares WPI with cyanocobalamin

480

supplement.

481 482

An important advantage of WPI over SPI, apart from the bioavailable B12, is its high amount

483

of natural folate (458µg per 100g). It is likely that folate in WPI is bound to the folate-

484

binding protein found in milk which may influence its bioavailability [37]. The

485

bioavailability of folate from WPI was not previously explored and, although not the primary

486

purpose of this study, this intervention provides important preliminary promising data on the

487

bio-availability of folate from WPI. It was evident that both plasma folate and red blood cell

488

folate increased significantly (by 13% and 7% respectively) in the WPI intervention but not

489

with SPI indicating that the folate contributed by 50g WPI per day makes a substantial

490

contribution to folate status. The bioavailability of natural folate from WPI relative to folic

491

acid will need to be determined in an independent and appropriately designed intervention in

492

subjects who are deficient or sub-clinically deficient in this vitamin. The fact that WPI is not

493

only an important bioavailable source of B12 and folate but also provides a high amount of

494

protein highlights the possibility of exploring its use in preventing stunting, motor and

495

problem solving skills in malnourished children and increasing lean body mass and

496

preventing hyperhomocysteinemia in adults [38-42].

497 498

A consequence of folate and B12 deficiency is an increase in DNA damage because these two

499

vitamins are required for the supply and conversion of the circulating form of folate, 5-

500

methyltetrahydrofolate, to the 5, 10-methylenetetrahydrofolate form required for the

501

synthesis of thymidine and for the synthesis of S-adenosylmethionine required for the

502

maintenance of DNA methylation both of which are essential for maintenance of

503

chromosomal stability [2]. WPI also provides substantial amounts of vitamins B2 and B6

504

(Table 1) which are also needed as cofactors in the metabolism of folate to the forms required

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505

for genome maintenance [2]. The chromosomal DNA damage biomarkers MN, NPB and

506

NBUD tended to be reduced in the WPI intervention relative to the SPI intervention in which

507

a striking 85% increase in NPB was evident. NPB are a biomarker of asymmetrical

508

chromosomal rearrangement leading to formation of dicentric chromosomes from which NPB

509

originate [27]. The increase in NPBs following SPI consumption could be explained by

510

genistein, a bioflavonoid enriched in soy products. High levels of maternal soy consumption

511

have been linked to the development of infant leukemia which may be due to genistein-

512

induced inhibition of topoisomerase function leading to DNA double-strand breaks from

513

which dicentric chromosomes and NPB originate [43]. In contrast other studies suggest that

514

soy isoflavones may prevent oxidative damage to DNA which may be unrelated to the

515

topoisomerase-mediated

516

rearrangements [44-45]. There is considerable debate about whether soy isoflavones

517

consumed in vivo could achieve a high enough concentration to cause chromosomal

518

instability [46] but it might be that it could become a real risk in populations who are B12

519

deficient because this deficiency alone can cause loss of genome stability and might make

520

cells more susceptible to additional genotoxic stress.

521 522

The intervention found no evidence that WPI or SPI substantially affected telomere length or

523

% intact mtDNA. The reason for a lack of an effect on telomere length could be due to the

524

relatively short duration of the intervention given that the large majority of peripheral blood

525

lymphocytes are non-dividing and therefore the opportunity for replication-stress induced

526

telomere shortening in an 8-week intervention is diminished. Although, % intact mtDNA

527

increased significantly in the WPI intervention, the increase was miniscule (1.4%) and it did

528

not differ significantly from the effect with SPI. There is no obvious plausible explanations

529

why % intact mtDNA should increase in both arms of the study and it is unlikely to be related

530

to the increased protein intake given that protein restriction, not its increase, decreases

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mechanism

by

which

genistein

induces

DNA

sequence

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531

mitochondrial oxygen radical production [47] which might induce mtDNA strand breaks and

532

deletions. There is evidence that mtDNA deletions increase with folate deficiency, and it is

533

therefore plausible that the increase in intact mtDNA in the WPI intervention could be partly

534

explained by the improved folate status [48-49].

535 536

The intervention also explored effects of the intervention on BDNF which is associated with

537

brain ageing and cognitive function [50]. Although previous studies suggest that whey

538

proteins and soy phytoestrogens may increase BDNF expression in mammals [51-52] we

539

could find no evidence of an increase in plasma BDNF in our intervention.

540 541

In conclusion it is evident that WPI improves vitamin B12 and folate status in adults with sub-

542

clinical vitamin B12 deficiency. The intervention also provides suggestive evidence that with

543

longer duration of intervention WPI may exhibit substantial effects on maintenance of

544

genome integrity relative to SPI. Overall WPI appeared to be of more benefit than SPI in

545

those who have sub-clinical vitamin B12 deficiency.

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547

Author Contributions

548 549 550 551 552 553 554 555 556

The authors’ responsibilities were as follows – MF (Principal investigator) for planning, designing the study trial, interpreted the data and compiling the manuscript, and had primary responsibility for final content of the manuscript; VSD: co-ordination of the trial, telomere, mtDNA assay, analysing the data and writing the manuscript; DZ: MMA assay; PC: BDNF assay and analysing the data; TA: (CBMNcyt assay); GJM (Dietician); and all authors: conducted the research, and read and approved the final manuscript before submission.

557 558 559 560 561 562 563

We thank Anne McGuffin (Clinical Trials Manager) and Lindy Lawson (Clinical nurse) for their role in the planning and collection of samples and other data during the intervention trial. We also thank Dairy Research Institute (National Dairy Council) for grant to MF to carry out the intervention trial.

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Acknowledgement

The authors have declared no conflict of interest related to the study.

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22

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phosphatase activity to alleviate early weaning diarrhoea in postnatal piglets. J. Nutr. Biochem. 2014, 25, 834-842.

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27 Table 1. Nutritional composition of Whey Protein isolate (WPI) and Soy Protein isolate (SPI) as per manufacturer’s* product specifications

Composition Protein (percent)

Type Total Proteins β-Lactoglobulin α-Lactalbumin GMP (Glycomacropeptide) Minor components Immunoglobulins BSA (Bovine Serum Albumin) Lactoferrin

Amount in WPI 93.1 (%) Dry basis 45% 15% 16% 3-5% 4% 1%

Amount in SPI 91.0 (%) Dry basis -

0.1%

-

1% -

2.5% 0.8% 1.6% 0.6% 0.5%

1.2% 1.2%

1%

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Fat (percent)

Total Fats Saturated fat Polyunsaturated Monounsaturated Trans Fatty acids

Carbohydrate (percent)

Total Carbohydrates Lactose Sucrose

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Minerals (per 100gm)

Potassium Calcium Phosphorus Sodium Chloride

Isoflavones Polyphenols *: Both products were procured from MyoPureTM

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