Recruitment

Subject recruitment started April 2014 and data collection and follow-up was completed by February 2015. The study was not registered on a CT Database at the time of subject recruitment as trial registration in these kinds of studies is not commonly practiced. The trial was subsequently registered in order to comply with publication requirements according to the WHO guidelines. The authors confirm that all ongoing and related trials for this intervention are registered.

Participants

Twenty-five healthy adults (female = 13 male = 12) aged 34.3+/-14.8y, height 171.5+/-11cm, weight 75.9 +/-14 Kg, were recruited from the staff and student population of the University of Hertfordshire and word of mouth to local residents and randomly assigned into either a magnesium cream or placebo cream group by random allocation. Randomisation was determined by allocation to a group selected from a box with equal amounts of paper stating either placebo or magnesium and selected at time of recruitment. The trial was single blind with only the lead investigator being aware of the content of the cream. One participant dropped out of study before completion. Participants were excluded if they were taking magnesium supplementation in any form within a month of recruitment onto the trial, were under the age of 18y or above the age of 60y.There were no height or weight restrictions, Written informed consent was completed and ethical approval was granted by the University of Hertfordshire Health and Human Science Ethics Committee on 14^th April 2014. Blood collection and blood and urine analysis was undertaken at the University of Hertfordshire Human Physiology Laboratory.

Dietary magnesium intake was below the RNI in one of the placebo participants and three of the magnesium participants. This was not considered a bar to inclusion into the study as any change in serum or urinary Mg2⁺ from the cream could still be shown. High levels of physical exercise have been shown to deplete human Mg²⁺ status ([18]. Four participants were considered “athletes” as they engaged in at least 2hrs of physical exercise at least 5 days per week during the study, three who were assigned to the Mg²⁺ intervention group and one assigned to receive placebo. All other 20 participants who completed the study were considered “non-athletes”, i.e. engaging in less than 2 hrs physical exercise per day for no more than 3 days per week during the study. Participants were instructed not to exercise 24h before blood draws, but “athletes” may have disregarded this instruction, so results were statistically analysed in two ways: 1. “all subjects”, including both athletes and non-athletes (n = 24 who completed the study) and 2. “non-athletes” (n = 20) which excluded the 4 athletes. Baseline serum and urine Mg²⁺ concentrations as well as dietary Mg²⁺ recorded in the randomised intervention and placebo groups did not differ significantly from each other (P<0.05) (Tables 1 & 2) in either the “all subjects” or “non-athletes” groupings.

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Table 1. Demographic and baseline serum, urine and dietary magnesium (mean +/- s.d. [range]) for all subjects, athletes plus non-athletes.

https://doi.org/10.1371/journal.pone.0174817.t001

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Table 2. Demographic and baseline serum, urine and dietary magnesium (mean +/- s.d. [range]) for non-athletes only.

https://doi.org/10.1371/journal.pone.0174817.t002

Baseline measurements

Baseline data consisted of a 24 hour urinary collection to assess baseline magnesium excretion and venous bloods to assess serum magnesium levels. (Tables 1 and 2). Urine was collected from the second urinary passing of the day until and including the first urinary passing of the next day. Blood collection could take place at any time of the day to suit the participants but each participant had to return at that same timeslot for the second blood collection. No food was allowed for the 3 hours before blood collection although water was allowed.

Dietary analysis

Each participant recorded a 4-day food diary (3 midweek days and 1 weekend day) prior to the intervention plus a second 4-day food diary at the end of the 12–14 day period, giving a total of 8 days dietary analysis over the period of the intervention for each participant. This was analysed for Mg²⁺ intake using Diet Plan 6 software (Forestfield Software Ltd., West Sussex, UK). (Tables 1 and 2).

Intervention

After baseline measurements were taken, participants were randomly assigned to either the Mg²⁺ Cream or a placebo control cream and were instructed to apply 2 x 5ml spoonfuls of cream per day for two weeks. The resulting daily Mg²⁺ dose received by subjects in the Mg²⁺ group consisted of 56mg of Mg²⁺. This was manufactured, in the course of research and development, for the Center for Magnesium Education & Research by Urist Cosmetics of Vancouver, B.C. Canada. For full ingredients see S1 Text Ingredients list.

The placebo was a commercially available aqueous cream containing no magnesium (by analysis) and creams were packaged identically. Instructions to participants suggested that Mg²⁺ or placebo cream be applied to the stomach and torso in the first instance and then spread down to the legs. Time of day was not important, but no showering or washing could take place for a minimum of 3 hours after application. After 12–14 days, final urine and blood samples were collected. The cream was applied up to and including the day of the final urine and blood collection. Participants were instructed to stop use of the cream if there was any adverse reaction, of which there were none. At the end of the trial compliance was ensured from verbal communication as well of collection of the trans-dermal cream container to ensure full usage.

Sample collection

Serum blood samples were collected by venepuncture from the median cubital, basillic or cephalic vein. Serum separator vacutainers were inverted 10 times before being left to rest for 30 minutes. Subsequently, samples were centrifuged at 3000 rpm for 10 minutes. Serum was checked to be free from haemolysis and was immediately pipetted and frozen at -80°C for subsequent analysis

Urine was collected into 3 litre collection vessels over 24 hours. Urine was then decanted into a measuring vessel and volume of urine was recorded. The measuring vessel was then placed on a magnetic stirrer at 100rpm. To re-suspend the Mg²⁺, the pH was lowered to 3–3.25 by adding 5 M hydrochloric acid. Duplicate 1mL aliquots were frozen at -80°C for batch analysis.

Analyses

Urine and serum samples were analysed by colorimetric assay for magnesium (RX MONZA, Randox Laboratories Limited, County Antrim, United Kingdom). The machine was calibrated according to the manufacturer's instructions and results were calculated from the standard concentration curve generated using the manufacturer’s calibration standard. Low urinary and serum Mg²⁺ were determined to be below 3.0mmol/24h and 0.65mmol/l respectively [19].

All urine and serum samples were frozen and stored at -80°C for between 4–12 weeks.

Data analysis

Serum and urinary Mg²⁺ data for both pre and post intervention were analysed for skewness and normality prior to statistical analysis. All data passed the normality test so a standard two tailed, paired t-test was used to compare baseline to post-intervention values (serum and urinary Mg) for within group analysis.

Statistical analysis was conducted using SPSS V22 (IBM, New York, USA), with P value value of ≤0.05 accepted as statistically significant.

Urinary and serum response to trans-dermal magnesium application

Mean serum and 24-hour urinary Mg²⁺ concentrations were obtained for all participants before and after a 2-week application period of the magnesium or placebo trans-dermal cream. Baseline (Tables 1 & 2) serum Mg²⁺ values were below normal reference values (0.65mmol/l) in one magnesium intervention participant and one placebo participant (both non-athletes), whilst the baseline 24-hr urinary Mg²⁺ excretion was below normal (3.0mmol/24h) in two placebo participants (one non-athlete, one athlete) and three magnesium participants (two non-athletes, one athlete); these were different participants for both the serum and urine. Of the five participants with low urinary excretion, three had below the RNI for dietary Mg²⁺ intake however this was not the case for the low serum Mg²⁺ subjects.

All participants, Table 3

There was no statistically significant effect of Mg²⁺ cream on either serum or urinary Mg²⁺ status. However, after the Mg²⁺ cream intervention there was a clinically relevant increase [11, 19] in serum magnesium (0.82 to 0.89 mmol/l) that was not seen in the placebo group (0.77 to 0.79 mmol/L), but this was not statistically significant (p = 0.29). Similarly, there was a slight increase in magnesium urinary excretion in the Mg²⁺ group but again no statistical significance (p = 0.48). A percentage increase of 8.54% for serum Mg²⁺ and 9.1% in urinary Mg²⁺ was seen in the Mg²⁺ group while for the placebo group these figures were smaller, i.e. +2.6% for serum Mg²⁺ and -32% for urinary Mg²⁺. In the placebo group both serum and urine concentrations showed no statistically significant change after the application of the placebo cream.

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Table 3. Influence of Trans-dermal Magnesium vs placebo cream application on Serum and Urinary Magnesium Concentrations (mean +/- S.D.) for all subjects, athletes plus non-athletes.

https://doi.org/10.1371/journal.pone.0174817.t003

Non-athletes, Table 4

For non-athlete participants (n = 11 in Mg group plus n = 9 in placebo group), there was a statistically significant rise (P = 0.02) in serum Mg²⁺ in the intervention group which was not seen in the placebo group. Serum Mg²⁺ in the intervention showed a % change of +22.7% while that in the placebo group showed a +4.11% rise. Urinary Mg²⁺ did not show any significant change in either Mg²⁺ or placebo group although a large non-significant negative change could be seen in the placebo group (-32.50%) when compared with the 11.3% increase in the intervention group.

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Table 4. Influence of Trans-dermal Magnesium vs placebo cream application on Serum and Urinary Magnesium Concentrations (mean +/- S.D.) for non-athlete subjects.

https://doi.org/10.1371/journal.pone.0174817.t004

Transdermal Mg²⁺ effect on serum Mg²⁺

Previous study of humans have shown that serial determinations of serum Mg²⁺ can prove useful as an indicator of changes in whole body Mg²⁺ status[17]. From this study, 56 mg/day Mg²⁺ applied as transdermal cream for 12–14 days had no statistically significant effect on serum concentration in this small (n = 25) human study when both athletes and non-athletes were included in the statistical analyses. However, a trend towards a rise in serum Mg²⁺ in the Mg²⁺ group could be seen with an increase of 0.07mmol/l, a clinically relevant rise in a measurement that is greater than many previous studies and a rise that would take months to show change with oral Mg²⁺ therapy [11, 19]. Zhang et al., [11] reported that in 41 trials, 941 participants receiving a mean oral Mg supplement of 365 mg/day for a median of 12 weeks showed a mean rise of 0.05 mmol/L circulating Mg (0.78 to 0.83 mmol/L). A meta-analysis of 27 trials by Zhang et al [8] investigating oral Mg²⁺ supplementation, showed a significant (p<0.001) rise in serum Mg with 200 mg/day oral Mg supplement or one month supplement duration but that higher doses (≥ 300 mg/day) or durations of supplementation (≥2 months) were required to achieve a mean rise of 0.05 mmol/l in serum Mg²⁺. Additionally, studies included in the Zhang et al [8] meta-analysis show a baseline mean C.V. for the 27 serum Mg²⁺ studies of 9.3% for Mg groups and 10.8% for placebo groups, i.e. as little as half of the variance for baseline serum Mg²⁺ measurements in this study (C.V. = 21.9% and 17.3% for Mg²⁺ groups, all subjects and non-athletes respectively; C.V. = 20.7% and 12.3% for placebo groups, all subjects and non-athletes respectively). This pilot study of all subjects, both athletes and non-athletes, shows that transdermal Mg²⁺ may possibly influence serum Mg²⁺ in a relatively short time frame (12–14 days), but a higher concentration of Mg²⁺ cream, a larger number of subjects given the serum Mg²⁺ variance, and perhaps a longer study is required to make any real conclusion.

Although participants were told to refrain from exercise for 24 hours before blood and urinary collection, 4 participants were undertaking regular high intensity training, the effects of which may affect Mg²⁺ parameters that can last longer than 24 hours [18]. When the data from these four subjects were removed from the statistical analysis a significant effect of the Mg²⁺ cream on serum Mg²⁺ concentrations could be seen.(p = 0.02). Additionally, the %rise in serum Mg²⁺ in the non-athletes (+22.7%) was much larger than that shown in all subjects, i.e. both athletes and non-athletes (+8.54%). This is too small a sample size to reach a firm conclusion but this increase in percentage change in the non-athlete intervention group may be due to an additional uptake of Mg^2+.from the cream which may have been utilised during exercise or for replenishment of Mg²⁺ stores rather than being transferred to serum in the athletic participants. This is an area of interest for further study.

Further, this analysis of the serum Mg data for the non-athletes in the intervention group showed a mean increase from 0.75 to 0.92 mmol/l which may have clinical relevance in particular with relation to cardiovascular disease. In a meta-analysis by Del Gobbo et al (2013)[20], it was found that a rise of 0.2 mmol/L circulating Mg was associated with a 30% lower risk of cardiovascular disease and fatal ischemic heart disease. In addition, Lutsey et al.(2014) [21] found that after 20+ yrs follow up serum Mg showed a linear inverse association with the risk of incident heart failure. Relative to those in the highest category of serum Mg, those in the lowest category were at 2.58 times greater risk of incident heart failure after demographic adjustments. In these quintiles, the lowest serum Mg quintile was 0.7 mmol/L, the second was 0.75, the third was 0.8, the 4th was 0.85 and the highest was 0.9 mmol/L, i.e. each quintile was 0.05 mmol/l higher than the next lowest quintile (these results were converted from mEq/L by dividing by 2 to attain mmol/L). Our results show a mean rise of 0.05 mmol/L serum Mg²⁺ with daily application of transdermal Mg²⁺ for only two weeks, and when considering this Lutsey[21] study and the Del Gobbo[20] results, our finding suggests a possibly significant favourable impact of transdermal Mg on risk of heart failure that needs full study.

Transdermal Mg effect on urinary Mg²⁺

Previous study of humans has suggested that 24-hr urinary Mg²⁺ excretion cannot be used as a measure of changes in whole body Mg²⁺ status [17].

Upon analysis for all subjects, as well as for only non-athletes, use of the transdermal Mg²⁺ cream showed no significant rise in urinary Mg²⁺, a measurement that reflects short term intestinal absorption of Mg²⁺. However, subjects in the Mg²⁺ group showed slight rises in urinary Mg²⁺ (+9 to 11%) while those in placebo group showed a substantial decrease in urinary Mg²⁺ (-32%). Possibly the decreased urinary Mg²⁺ excretion in the placebo cream group represents more active physiological Mg²⁺ retention processes that are not apparent in the Mg cream group ([15,16]. It has been suggested that 24-hour urine excretion of Mg²⁺ may be a better indicator of tissue status than the serum Mg²⁺ concentration, but it is highly variable and it is questionable whether it can be used to reliably assess a given individual’s Mg²⁺ status.

The trans-dermal cream contained 56 mg of Mg²⁺ administered daily. This is at the lower end of creams sold commercially. The recommended dose of the few commercially available creams range between 70mg/d to 400mg/d per day, therefore results of this study may represent an “underdose” of transdermal Mg²⁺.