Creatine is one of the best supplements for improving strength and high intensity exercise performance. It works by increasing your capacity to produce ATP energy. Like your muscles, your brain stores phosphocreatine and requires plenty of ATP for optimal function.

Preclinical studies mostly on animals suggest that creatine supplementation may help treat:. In a review , creatine supplements improved brain function in vegetarians. Even in healthy adults, creatine supplementation may improve short-term memory and intelligence.

This effect may be strongest in older adults. Creatine may reduce symptoms and slow the progression of some neurological diseases, although more research in humans is needed.

Research also indicates that creatine may :. Early research suggests that creatine might help treat high blood sugar, fatty liver disease, and heart disease.

The most common and well-researched supplement form is called creatine monohydrate. Many other forms are available, some of which are promoted as superior, though evidence to this effect is lacking. Creatine monohydrate is very cheap and is supported by hundreds of studies.

Until new research claims otherwise, it seems to be the best option. The best form of creatine you can take is called creatine monohydrate, which has been used and studied for decades.

Many people who supplement start with a loading phase, which leads to a rapid increase in muscle stores of creatine. To load with creatine, take 20 grams g per day for 5—7 days.

Split this into four 5-gram servings throughout the day. Eating a carb- or protein-based meal may help your body absorb the creatine. Following the loading period, take 3—5 g per day to maintain high levels within your muscles. As there is no benefit to cycling creatine, you can stick with this dosage for a long time.

If you choose not to do the loading phase, you can simply consume 3—5 g per day. However, it may take 4 weeks to maximize your stores. Since creatine pulls water into your muscle cells, it is advisable to take it with a glass of water and stay well hydrated throughout the day.

To load with creatine, take 5 g four times per day for 5—7 days. Then take 3—5 g per day to maintain levels. Creatine is one of the most well-researched supplements available, and studies lasting up to 4 years reveal no negative effects.

There is also no evidence that creatine harms the liver and kidneys in healthy people who take standard doses. That said, people with preexisting liver or kidney concerns should consult a doctor before supplementing.

Studies suggest it can reduce cramps and dehydration during endurance exercise in high heat. One study linked creatine supplements with an increase in a hormone called DHT, which can contribute to hair loss.

But most available research does not support this link. Creatine exhibits no harmful side effects. Creatine is a leading supplement used for improving athletic performance.

It may help boost muscle mass, strength, and exercise efficiency. It may also reduce blood sugar and improve brain function, but more research is needed in these areas to verify these benefits. Research from has found that creatine supplementation may be beneficial for women across many life stages by helping support both the muscles and the brain.

When combined with resistance training, creatine may help improve body composition and bone density in post-menopausal women.

Earlier research suggested that creatine may not be as effective in women compared to men due to physiological and hormonal differences. But newer research seems to suggest there are still plenty of benefits for women.

More research is needed on larger doses. The effects of creatine are noticeable in as little as 2 weeks. Without a loading phase, it may take you up to 4 weeks to observe the effects. A study noted that taking up to 30 g per day well above the standard dosage of creatine did not have adverse effects on the kidneys of healthy people.

Still, the researchers cautioned that it might be safest for people with pre-existing kidney disease to not use creatine because taking it could metabolize into methylamine and formaldehyde, which could be toxic to the kidneys with pre-existing conditions. It supports quality of life in older adults, brain health, and exercise performance.

Vegetarians — who may not obtain enough creatine from their diet — and older adults may find supplementing particularly useful. A baseline urine sample was collected to measure D 3 -creatinine enrichment.

Participants were then given three 50 ml aliquots of 70 atom percent D 2 O to drink, each taken 25 min apart to minimise the risk of side effects such as dizziness or nausea that are sometimes reported after D 2 O consumption due to disturbance of specific gravity within the vestibular fluid [ 29 ].

Two hours after consumption of the final D 2 O aliquot, participants provided another saliva sample, to be used to determine the plateau D 2 O concentration in body water.

Prior to leaving the laboratory, participants were provided with a container to collect all urine for 24 h, a tube for a spot urine sample at 48 h and three tubes for saliva samples at 12, 24 and 48 h after the final D 2 O aliquot was consumed. These samples were used to measure D 3 -creatine spill-over, urinary D 3 -creatinine enrichment and the size of the creatine pool, respectively.

Participants were also given 10 mg of D 3 -3MH dissolved in 50 mL of water to take home and store refrigerated.

Participants were instructed to consume the D 3 -3MH immediately after the h samples were collected Fig. Please refer to Fig. Schematic representation of the combined use of D 3 -3MH, D 2 O and D 3 -Cr to measure myofibrillar muscle protein synthesis MPS A , whole-body muscle protein breakdown MPB B , and muscle mass C , simultaneously.

Participants returned to the laboratory 72 h after consumption of the final D 2 O aliquot given on the first study visit, again having fasted overnight.

At this visit, hourly bloods were collected over 6 h between 72 and 78 h after consumption of the final D 2 O aliquot , plus a further spot urine and saliva sample at 72 h.

A muscle biopsy from the m. Participants were not fasted throughout the blood sampling period, with a standardised light meal sandwich, crisps, yoghurt and drink provided at the midpoint of the blood collections for all participants.

Figure 2 illustrates the protocol. One hundred microlitres of each saliva was aliquoted into the cap of auto-sampler vials which were crimped onto inverted vials and placed on a heating block at 90 °C for 4 h. These vials were then immediately cooled upright on ice for 10 min and the water collected transferred to fresh vials.

The mean of the final 3 of 4 repeat injections was taken to control for carry over from previous samples. The mean area under the curve AUC of the atom percent excess APE was used to represent the average precursor labelling of free intramuscular alanine, multiplied by 3.

Between 30 and 50 mg of muscle was used for the analysis of deuterium labelling of alanine in the myofibrillar protein bound fraction, although 10 mg is sufficient, thereby permitting the needle biopsy approach.

The supernatant sarcoplasmic fraction was then collected and the pellet was resuspended in μL mitochondrial extraction buffer MEB and then homogenized by Dounce and centrifuged at g for 5 min.

The myofibrillar pellet was solubilised using 1 mL 0. After the addition of 1 mL 0. The pellet was transferred to a boiling tube containing on dowex with 0. The amino acids AA were derivatized as their N-methoxycarbonyl methyl esters MCME as previously described [ 33 ].

Briefly, the AA were re-suspended in 60 μL of distilled H 2 O and vortex mixed before the addition of 32 μL of methanol and 10 μL of pyridine. The solution was left at room temperature for 5 min to react.

Following the addition of μL of chloroform and μL of 0. Molecular sieve was added to remove any excess water from the sample. The sample was transferred to an autosampler vial, ready for mass spectrometric analysis. Deuterium enrichment into protein-bound alanine was measured using gas-chromatography-pyrolysis-isotope-ratio-mass spectrometry GC-pyrolysis-IRMS, Delta V Advantage, Thermo Scientific [ 34 ].

The calculation of myofibrillar fractional synthetic rate was based on the following product-precursor equation:. where APE Ala is deuterium enrichment of protein-bound alanine, APE P is mean alanine enrichment corrected from D 2 O, and t is the time between the plasma sample to the 72 h muscle biopsy, using previously described equations [ 19 ].

The flow was set to 0. After a 2. The enrichment ratios were log transformed to determine the decay rates k , representative of the rate of whole-body MPB [ 26 ].

The urine samples were prepared using a method based on that described by Leonard et al. Samples were then centrifuged at 17, g for 20 min. The supernatant was filtered through a 0. The estimated creatine pool size is then divided by 4. As the methods have individually been previously validated, here we aimed to measure rates of MPS and MPB using our COSIAM approach, to identify if values obtained were similar to previous literature.

Furthermore, we aimed to confirm if there was a relationship between DXA and D 3 -creatine-derived estimates of muscle mass. All data sets were tested for normality using Shapiro—Wilk test with alpha set at 0.

All statistical analysis was performed using Graphpad Prism software Prism 8, USA. The mean body water enrichment of D 2 O peaked at 2 h 0. The mean myofibrillar FSR over 0—72 h was 0.

A Deuterium enrichment of the body water, measured in APE from saliva samples at baseline, 2, 12, 24, 48 and 72 h post-deuterium oxide ingestion; B Myofibrillar fractional synthetic rates.

The D 3 -3MH enrichment curve of 0—10 APE Fig. Mean plasma D 3 MH enrichment gradually declined from 4. The ratio of labelled to unlabelled 3-MH was log transformed to determine the rate of whole-body MPB.

The gradient of the straight line provides the k value rate constant. The mean rate of whole-body MPB was 0. A Enrichment curve of D 3 methylhistidine from 0—10 APE; B Decay curves of D 3 methylhistidine in the plasma by endogenous 3-methylhistidine release; C Rate of whole-body muscle protein breakdown k using D 3 methylhistidine.

This analytical approach was extremely sensitive, accurate and robust, and both standard curves showed good linearity across both the range of concentrations of creatine expected i. In our hands, only very small amounts of D 3 -Cr were excreted during the 0—24 h collection period 0.

D 3 -Cr enrichment in the urine samples increased over time and plateaued at 48 h, there was no significant difference between 48 and 72 h enrichments Fig.

A Creatine concentration curve ranging from 0. expected APE. Values displayed are min to max; C Ratio of labelled creatinine D 3 -creatinine to unlabelled creatinine, corrected to the baseline sample 0 h for 24, 48 and 72 h urine samples.

total arms and legs measured by DXA. We present a minimally invasive study designed to measure rates of MPS, MPB and muscle mass concurrently, using a novel combination of orally ingested stable isotope labelled tracers, and mass spectrometric measures made in saliva, and blood or urine samples, in conjunction with a single muscle biopsy.

In contrast to traditional stable isotope approaches using intravenous tracer infusions, which require extremely well-controlled laboratory conditions and imaging techniques, our protocol has the potential to be applied to a wider range of cohorts and settings. In our protocol, we used a creatine tracer method to quantify muscle mass.

The use of 30 mg D 3 -Cr tracer was based upon the dosing validation by Clark and colleagues in where 30 mg was identified as adequate for quantification of muscle mass, a finding that we support. Our results also corroborate previous relationships between DXA-derived parameters of skeletal muscle and muscle mass measured using D 3 -creatine [ 12 , 36 , 37 ].

A significant correlation was only found between muscle mass measured by D 3 -creatine and AFFM calculated from DXA and not with FFM by DXA , this may be due to the fact that DXA does not measure muscle mass directly. Our findings are supported by those of Proctor et al.

Therefore, if DXA FFM is used to determine muscle mass, it will provide an erroneous overestimation of total muscle [and by extenstion, contractile] mass [ 39 ]. Ideally, the gold-standard approach to determine muscle mass i. Although there is a strong correlation between AFFM using DXA or MRI, and muscle mass measured by D 3 -creatine, a potential limitation that requires further validation, is the widespread use of the 4.

It is important to ensure that the constant used accurately reflects intramuscular creatine in different populations, particularly where the muscle creatine pool size may vary e. vegetarians [ 40 ], athletes, especially following acute or endurance exercise [ 41 ] or when creatine is being supplemented in the diet [ 42 ].

In an average 70 kg young male the creatine pool size is approximately — g [ 43 ] with creatine excretion approximately 1. Future applications of this approach could also look to minimise the urine sampling to a single spot urine, given that the spillover is less than 0. We speculate that the percentage spillover may be related to the creatine pool size and thus, muscle mass and should be investigated further.

Going forward, we suggest that 1 or 2 spot urine samples taken 48—72 h after ingestion of tracer would suffice, making the method even more practicable and widely applicable.

To exemplify, in this data set, removing the correction of the 24 h collection, led to an average increase in muscle mass estimation of 9 g. Alternatively, Shankaran et al. Using D 2 O to study skeletal muscle protein metabolism is now a well-established stable-isotope tracer technique that has been validated for both longer over weeks or months; [ 19 ] and also relatively short study durations i.

over 2—8 days [ 22 ], and even over as little as 3 h with appropriate increases in the pre-loading dose [ 20 ]. For instance, as little as mL 70 AP D 2 O was adequate to measure MPS over 2—8 days [ 22 ]; therefore, we used this dose to measure MPS in this 4-day protocol.

In doing so, we report similar cumulative MPS values to those reported before [ 22 , 48 ] and interference with the other co-consumed tracers D 3 -Cr or D 3 -3MH i. It is unlikely that a deuterium labelling of either arginine or glycine amino acids involved in the initial step of creatine biosynthesis from D 2 O would be incorporated to any extent into D 3 -labelled guanidinoacetate a precursor to creatine , given the very small dose of D 2 O we use in this study, and therefore the probability of D 2 O labelling all three position of the methyl group at this level of dosing is vanishingly small.

In addition, it is possible to provide an alternatively labelled tracer such as 13 C-Cr for repeat measurements, and this would be recommended in short duration longitudinal studies to avoid any overlap of isotopes given the slow turnover of these pools [ 22 ].

For measurement of whole-body MPB, 10 mg of D 3 -3MH was utilised, as previously described by Sheffield-Moore and colleagues, with the rates of MPB in our older men similar to those previously observed i.

the rate constant k of approximately 0. Sheffield-Moore et al. It is important to note that subjects were not maintained fasted throughout the period of plasma sampling for D 3 -3MH measures, therefore the rates of MPB we observed reflect the changes in MPB over the period of sampling.

Feeding increases insulin secretion which is known to inhibit MPB [ 49 ]; therefore, it is likely that our measures will somewhat reflect MPB in the fed state, thereby explaining why our MPB rates are slightly lower than the cummulative rates of MPS obtained in this study.

One subject had a much lower D 3 -3MH enrichment than the other subjects, which is probably indicative that they did not ingest the full 10 mg or perhaps took the dose much earlier than reported i.

not the morning before; however, the decay rate determined was similar to that of our other participants, due to the high sensitivity and reproducibility of the LC—MS approach employed.

Further investigations could look to reduce the dose of D 3 -3MH given or standardise the dose on a weight or LBM basis to standardise the approach and measurement of enrichment across participants. Furthermore, using timed urine samples, instead of blood samples for the measurement of labelled D 3 -3MH excretion could further minimise the invasiveness of this approach [ 26 ].

Other considerations to the work presented herein are warranted. With regard to selecting which timepoint to take the spot samples, Clark et al. As such, a single spot urine sample taken at any timepoint during this steady-state period 2—4 days post-ingestion of tracer would be sufficient.

For more acute measures, the timepoint for MPS measurement could be altered i. shortened, and with an additional biopsy to measure MPS, with appropriate sampling over the same time frame i.

Although this would require additional biopsies, the use of the micro-needle biopsy technique [ 50 ], which is well tolerated [ 50 ], may be used towards developing a less invasive approach. While this would further reduce the invasiveness of the approach and should be investigated further, the higher dose of deuterium oxide required for this approach may have a greater burden with the potential of increased side effects e.

Furthermore, the relationship of plasma surrogates such as CK-M would require validation across different populations and scenarios. This study has demonstrated the feasibility of concurrent assessment of muscle protein turnover i. MPS and MPB , and muscle mass through a far less invasive approach than using traditional stable isotope infusion approaches.

The COSIAM approach provides rates of protein turnover and estimates of muscle mass that are in agreement with previous individual tracer approaches. The combination of these tracers via a minimally invasive approach has wider applicability than traditional methods and potential utility to provide novel, valuable information about the regulation of muscle mass in difficult to study clinical populations.

Gasier HG, Fluckey JD, Previs SF. The application of 2H2O to measure skeletal muscle protein synthesis. Nutr Metab Lond. Article Google Scholar. Gasier HG, Riechman SE, Wiggs MP, Previs SF, Fluckey JD. A comparison of 2H2O and phenylalanine flooding dose to investigate muscle protein synthesis with acute exercise in rats.

Am J Physiol Endocrinol Metab. Article CAS Google Scholar. Frontera WR, Ochala J. Skeletal muscle: a brief review of structure and function. Calcif Tissue Int. Johnstone AM, Murison SD, Duncan JS, Rance KA, Speakman JR. Factors influencing variation in basal metabolic rate include fat-free mass, fat mass, age, and circulating thyroxine but not sex, circulating leptin, or triiodothyronine.

Am J Clin Nutr. Morley JE, Malmstrom TK, Rodriguez-Mañas L, Sinclair AJ. Frailty, sarcopenia and diabetes. J Am Med Dir Assoc. Rudrappa SS, Wilkinson DJ, Greenhaff PL, Smith K, Idris I, Atherton PJ.

Creatine in supplement form is synthetically produced in a commercial laboratory. The most common form is creatine monohydrate, though other forms exist 1. Whey is one of the primary proteins found in dairy products.

In terms of protein quality, whey is at the top of the list, hence why its supplements are so popular among bodybuilders and other athletes. Consuming whey protein following a bout of exercise has been linked to enhanced recovery and increased muscle mass.

These benefits can help improve strength, power, and muscular function 3 , 4. Getting in a good source of protein after resistance exercise is important for maximizing muscle-building.

About 20—25 grams of protein is a good amount to aim for 3. Whey protein powder can be an efficient way to meet this recommendation, considering a typical gram serving provides around 20 grams of protein. Creatine is an organic compound that, when taken as a supplement, can help increase muscle mass, strength, and exercise performance.

Whey protein is a dairy protein commonly consumed with resistance exercise to increase muscle mass and strength. Both creatine and whey protein powder have been shown to increase muscle mass when taken in combination with resistance exercise 1 , 3.

Creatine increases exercise capacity during high-intensity exercise. This leads to improved recovery and adaptations such as increased muscle mass 1.

Meanwhile, ingesting whey protein in combination with exercise provides your body a high-quality source of protein , enhancing muscle protein synthesis and leading to increased muscle gains over time 3. While both creatine and whey protein promote muscle gain, they differ in the ways they work. Creatine increases strength and muscle mass by increasing exercise capacity, whereas whey protein does so by stimulating increased muscle protein synthesis.

Both whey protein powder and creatine supplements have been shown to increase muscle mass, though they accomplish this in different ways. Some people have proposed that taking whey protein and creatine together may lead to benefits beyond those associated with taking either one alone.

One study in 42 middle-aged and older men found that participants did not experience any additional training adaptations when they took both whey protein and creatine, compared with taking either supplement alone 5. Additionally, a study in 18 resistance-trained women found that those who took whey protein plus creatine for 8 weeks experienced no difference in muscle mass and strength than those who took whey protein alone 6.

The results seem to suggest there is no added benefit of taking whey protein and creatine together. However, some people may decide to take them together for convenience 7.

Additionally, no evidence suggests that taking creatine and whey protein at the same time causes any negative effects. Choosing whether to take whey protein, creatine, or both comes down to your individual goals.

If you are a recreational gym-goer just looking to stay in shape, whey protein may be a good option to aid muscle building and recovery. On the other hand, if you are looking to maximize muscle mass and strength, it may be beneficial to take both whey protein and creatine.

Studies have observed that taking whey protein and creatine together with exercise offers no additional muscle or strength gains than taking each individually.

Taking either alone likely provides the same benefits. Whey protein powder and creatine are two popular sports supplements that have been shown to increase muscle mass and improve exercise performance, though the ways in which they accomplish this differ.

Taking the two together does not appear to offer additional benefits for muscle and strength gains.

In these methods, deuterium is rapidly equilibrated in the body water pool over 1—2 h , exchanges onto amino acids such as alanine through intermediary metabolic pathways and is subsequently incorporated into protein [ 21 ].

Measurement of these processes permits the quantitation of cumulative MPS over varying time periods from hours or days [ 22 ] to weeks or months [ 9 , 19 ]. The quantification of MPB is arguably more difficult than both muscle mass and MPS.

Traditional in vivo approaches include arterio-venous A-V balance and fractional breakdown methods [ 23 , 24 ], quantifying the dilution of amino acid tracers across the A-V system of an isolated organ or limb following the breakdown of tissue protein.

Another approach has been to measure the concentration of 3-methylhistidine 3-MH in urine. However, this approach lost favour as urinary 3-MH levels may be perturbed by dietary meat intake [ 27 ] an alternative source of 3-MH ; and the assumption that methylated-histidine is exclusively derived from skeletal muscle of the appendicular skeleton may be incorrect, as the gut has also been shown to be a source of 3-MH [ 28 ].

Nevertheless, an adaptation of this approach using tracer dilution was reported recently in which 10 mg of stable isotopically labelled 3-methylhistidine either D 3 or 13 C was administerd orally, and the rate of dilution of this tracer by endogenously released 3-MH was determined in plasma or urine over 6 h to assess the rate of whole-body myofibrillar protein breakdown [ 26 ].

This method offers a simple, attractive alternative which does not require accurate and complete h urinary collection because a much more easily collected series of spot urine or plasma samples can be used. To date, there has been no attempt to create a single protocol to streamline minimally invasive techniques to simultaneously measure muscle protein turnover and muscle mass.

Being able to do so would have considerable clinical research application in non-laboratory settings i. This study was approved by The University of Nottingham Ethics Committee A and conformed to the standards set by The Declaration of Helsinki Before being enrolled into the study, all participants had a full medical screening.

Height, weight, full medical history, blood pressure, an ECG and clinical chemistry full blood count, urea and electrolytes, liver function tests, thyroid function tests and coagulation were used to assess subject eligibility to participate in the study. All participants were informed of the purpose of the study and of all the risks and procedures involved, before providing their written, informed consent.

On the first study day Day 1 , participants were asked to attend the unit at am for a whole-body DXA scan, having fasted overnight.

They provided a baseline saliva sample to identify baseline body water enrichment and then performed the Short Physical Performance Battery Test SPPBT. Leg extensor strength single-leg 1RM and maximal voluntary contraction for leg extension and handgrip strength using Takei T. A baseline urine sample was collected to measure D 3 -creatinine enrichment.

Participants were then given three 50 ml aliquots of 70 atom percent D 2 O to drink, each taken 25 min apart to minimise the risk of side effects such as dizziness or nausea that are sometimes reported after D 2 O consumption due to disturbance of specific gravity within the vestibular fluid [ 29 ].

Two hours after consumption of the final D 2 O aliquot, participants provided another saliva sample, to be used to determine the plateau D 2 O concentration in body water. Prior to leaving the laboratory, participants were provided with a container to collect all urine for 24 h, a tube for a spot urine sample at 48 h and three tubes for saliva samples at 12, 24 and 48 h after the final D 2 O aliquot was consumed.

These samples were used to measure D 3 -creatine spill-over, urinary D 3 -creatinine enrichment and the size of the creatine pool, respectively.

Participants were also given 10 mg of D 3 -3MH dissolved in 50 mL of water to take home and store refrigerated. Participants were instructed to consume the D 3 -3MH immediately after the h samples were collected Fig. Please refer to Fig.

Schematic representation of the combined use of D 3 -3MH, D 2 O and D 3 -Cr to measure myofibrillar muscle protein synthesis MPS A , whole-body muscle protein breakdown MPB B , and muscle mass C , simultaneously. Participants returned to the laboratory 72 h after consumption of the final D 2 O aliquot given on the first study visit, again having fasted overnight.

At this visit, hourly bloods were collected over 6 h between 72 and 78 h after consumption of the final D 2 O aliquot , plus a further spot urine and saliva sample at 72 h.

A muscle biopsy from the m. Participants were not fasted throughout the blood sampling period, with a standardised light meal sandwich, crisps, yoghurt and drink provided at the midpoint of the blood collections for all participants. Figure 2 illustrates the protocol.

One hundred microlitres of each saliva was aliquoted into the cap of auto-sampler vials which were crimped onto inverted vials and placed on a heating block at 90 °C for 4 h. These vials were then immediately cooled upright on ice for 10 min and the water collected transferred to fresh vials.

The mean of the final 3 of 4 repeat injections was taken to control for carry over from previous samples. The mean area under the curve AUC of the atom percent excess APE was used to represent the average precursor labelling of free intramuscular alanine, multiplied by 3.

Between 30 and 50 mg of muscle was used for the analysis of deuterium labelling of alanine in the myofibrillar protein bound fraction, although 10 mg is sufficient, thereby permitting the needle biopsy approach. The supernatant sarcoplasmic fraction was then collected and the pellet was resuspended in μL mitochondrial extraction buffer MEB and then homogenized by Dounce and centrifuged at g for 5 min.

The myofibrillar pellet was solubilised using 1 mL 0. After the addition of 1 mL 0. The pellet was transferred to a boiling tube containing on dowex with 0. The amino acids AA were derivatized as their N-methoxycarbonyl methyl esters MCME as previously described [ 33 ].

Briefly, the AA were re-suspended in 60 μL of distilled H 2 O and vortex mixed before the addition of 32 μL of methanol and 10 μL of pyridine. The solution was left at room temperature for 5 min to react.

Following the addition of μL of chloroform and μL of 0. Molecular sieve was added to remove any excess water from the sample.

The sample was transferred to an autosampler vial, ready for mass spectrometric analysis. Deuterium enrichment into protein-bound alanine was measured using gas-chromatography-pyrolysis-isotope-ratio-mass spectrometry GC-pyrolysis-IRMS, Delta V Advantage, Thermo Scientific [ 34 ].

The calculation of myofibrillar fractional synthetic rate was based on the following product-precursor equation:. where APE Ala is deuterium enrichment of protein-bound alanine, APE P is mean alanine enrichment corrected from D 2 O, and t is the time between the plasma sample to the 72 h muscle biopsy, using previously described equations [ 19 ].

The flow was set to 0. After a 2. The enrichment ratios were log transformed to determine the decay rates k , representative of the rate of whole-body MPB [ 26 ]. The urine samples were prepared using a method based on that described by Leonard et al.

Samples were then centrifuged at 17, g for 20 min. The supernatant was filtered through a 0. The estimated creatine pool size is then divided by 4. As the methods have individually been previously validated, here we aimed to measure rates of MPS and MPB using our COSIAM approach, to identify if values obtained were similar to previous literature.

Furthermore, we aimed to confirm if there was a relationship between DXA and D 3 -creatine-derived estimates of muscle mass. All data sets were tested for normality using Shapiro—Wilk test with alpha set at 0.

All statistical analysis was performed using Graphpad Prism software Prism 8, USA. The mean body water enrichment of D 2 O peaked at 2 h 0.

The mean myofibrillar FSR over 0—72 h was 0. A Deuterium enrichment of the body water, measured in APE from saliva samples at baseline, 2, 12, 24, 48 and 72 h post-deuterium oxide ingestion; B Myofibrillar fractional synthetic rates. The D 3 -3MH enrichment curve of 0—10 APE Fig.

Mean plasma D 3 MH enrichment gradually declined from 4. The ratio of labelled to unlabelled 3-MH was log transformed to determine the rate of whole-body MPB.

The gradient of the straight line provides the k value rate constant. The mean rate of whole-body MPB was 0. A Enrichment curve of D 3 methylhistidine from 0—10 APE; B Decay curves of D 3 methylhistidine in the plasma by endogenous 3-methylhistidine release; C Rate of whole-body muscle protein breakdown k using D 3 methylhistidine.

This analytical approach was extremely sensitive, accurate and robust, and both standard curves showed good linearity across both the range of concentrations of creatine expected i. In our hands, only very small amounts of D 3 -Cr were excreted during the 0—24 h collection period 0.

D 3 -Cr enrichment in the urine samples increased over time and plateaued at 48 h, there was no significant difference between 48 and 72 h enrichments Fig. A Creatine concentration curve ranging from 0. expected APE. Values displayed are min to max; C Ratio of labelled creatinine D 3 -creatinine to unlabelled creatinine, corrected to the baseline sample 0 h for 24, 48 and 72 h urine samples.

total arms and legs measured by DXA. We present a minimally invasive study designed to measure rates of MPS, MPB and muscle mass concurrently, using a novel combination of orally ingested stable isotope labelled tracers, and mass spectrometric measures made in saliva, and blood or urine samples, in conjunction with a single muscle biopsy.

In contrast to traditional stable isotope approaches using intravenous tracer infusions, which require extremely well-controlled laboratory conditions and imaging techniques, our protocol has the potential to be applied to a wider range of cohorts and settings.

In our protocol, we used a creatine tracer method to quantify muscle mass. The use of 30 mg D 3 -Cr tracer was based upon the dosing validation by Clark and colleagues in where 30 mg was identified as adequate for quantification of muscle mass, a finding that we support.

Our results also corroborate previous relationships between DXA-derived parameters of skeletal muscle and muscle mass measured using D 3 -creatine [ 12 , 36 , 37 ]. A significant correlation was only found between muscle mass measured by D 3 -creatine and AFFM calculated from DXA and not with FFM by DXA , this may be due to the fact that DXA does not measure muscle mass directly.

Our findings are supported by those of Proctor et al. Therefore, if DXA FFM is used to determine muscle mass, it will provide an erroneous overestimation of total muscle [and by extenstion, contractile] mass [ 39 ].

Ideally, the gold-standard approach to determine muscle mass i. Although there is a strong correlation between AFFM using DXA or MRI, and muscle mass measured by D 3 -creatine, a potential limitation that requires further validation, is the widespread use of the 4.

It is important to ensure that the constant used accurately reflects intramuscular creatine in different populations, particularly where the muscle creatine pool size may vary e. vegetarians [ 40 ], athletes, especially following acute or endurance exercise [ 41 ] or when creatine is being supplemented in the diet [ 42 ].

In an average 70 kg young male the creatine pool size is approximately — g [ 43 ] with creatine excretion approximately 1. Future applications of this approach could also look to minimise the urine sampling to a single spot urine, given that the spillover is less than 0.

We speculate that the percentage spillover may be related to the creatine pool size and thus, muscle mass and should be investigated further. Going forward, we suggest that 1 or 2 spot urine samples taken 48—72 h after ingestion of tracer would suffice, making the method even more practicable and widely applicable.

To exemplify, in this data set, removing the correction of the 24 h collection, led to an average increase in muscle mass estimation of 9 g. Alternatively, Shankaran et al. Using D 2 O to study skeletal muscle protein metabolism is now a well-established stable-isotope tracer technique that has been validated for both longer over weeks or months; [ 19 ] and also relatively short study durations i.

over 2—8 days [ 22 ], and even over as little as 3 h with appropriate increases in the pre-loading dose [ 20 ]. For instance, as little as mL 70 AP D 2 O was adequate to measure MPS over 2—8 days [ 22 ]; therefore, we used this dose to measure MPS in this 4-day protocol.

In doing so, we report similar cumulative MPS values to those reported before [ 22 , 48 ] and interference with the other co-consumed tracers D 3 -Cr or D 3 -3MH i. It is unlikely that a deuterium labelling of either arginine or glycine amino acids involved in the initial step of creatine biosynthesis from D 2 O would be incorporated to any extent into D 3 -labelled guanidinoacetate a precursor to creatine , given the very small dose of D 2 O we use in this study, and therefore the probability of D 2 O labelling all three position of the methyl group at this level of dosing is vanishingly small.

In addition, it is possible to provide an alternatively labelled tracer such as 13 C-Cr for repeat measurements, and this would be recommended in short duration longitudinal studies to avoid any overlap of isotopes given the slow turnover of these pools [ 22 ]. For measurement of whole-body MPB, 10 mg of D 3 -3MH was utilised, as previously described by Sheffield-Moore and colleagues, with the rates of MPB in our older men similar to those previously observed i.

the rate constant k of approximately 0. Sheffield-Moore et al. It is important to note that subjects were not maintained fasted throughout the period of plasma sampling for D 3 -3MH measures, therefore the rates of MPB we observed reflect the changes in MPB over the period of sampling.

Feeding increases insulin secretion which is known to inhibit MPB [ 49 ]; therefore, it is likely that our measures will somewhat reflect MPB in the fed state, thereby explaining why our MPB rates are slightly lower than the cummulative rates of MPS obtained in this study.

One subject had a much lower D 3 -3MH enrichment than the other subjects, which is probably indicative that they did not ingest the full 10 mg or perhaps took the dose much earlier than reported i.

not the morning before; however, the decay rate determined was similar to that of our other participants, due to the high sensitivity and reproducibility of the LC—MS approach employed. Further investigations could look to reduce the dose of D 3 -3MH given or standardise the dose on a weight or LBM basis to standardise the approach and measurement of enrichment across participants.

Furthermore, using timed urine samples, instead of blood samples for the measurement of labelled D 3 -3MH excretion could further minimise the invasiveness of this approach [ 26 ]. Other considerations to the work presented herein are warranted.

With regard to selecting which timepoint to take the spot samples, Clark et al. As such, a single spot urine sample taken at any timepoint during this steady-state period 2—4 days post-ingestion of tracer would be sufficient. For more acute measures, the timepoint for MPS measurement could be altered i.

shortened, and with an additional biopsy to measure MPS, with appropriate sampling over the same time frame i.

Although this would require additional biopsies, the use of the micro-needle biopsy technique [ 50 ], which is well tolerated [ 50 ], may be used towards developing a less invasive approach. While this would further reduce the invasiveness of the approach and should be investigated further, the higher dose of deuterium oxide required for this approach may have a greater burden with the potential of increased side effects e.

Furthermore, the relationship of plasma surrogates such as CK-M would require validation across different populations and scenarios.

This study has demonstrated the feasibility of concurrent assessment of muscle protein turnover i. MPS and MPB , and muscle mass through a far less invasive approach than using traditional stable isotope infusion approaches.

The COSIAM approach provides rates of protein turnover and estimates of muscle mass that are in agreement with previous individual tracer approaches. The combination of these tracers via a minimally invasive approach has wider applicability than traditional methods and potential utility to provide novel, valuable information about the regulation of muscle mass in difficult to study clinical populations.

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