Study design and objectives
This study was conducted in Germany as a single-center, randomized, open, 2-period, 2-supplementation, 2-sequence, single-dose, cross-over study to compare the bioavailability of two different Mg compounds: Mg citrate and Mg oxide. Both Mg compounds were provided as capsules (please refer to the methods’ subsection study products below for more information).
The bioavailability of magnesium was assessed by measuring the renally eliminated Mg quantity during the interval of 24 h after Mg administration (Ae 0-24h) as primary endpoint. Additionally, and on an exploratory basis, the magnesium concentrations in a subset of leukocytes (i.e., monocytes and lymphocytes) as well as in erythrocytes and the magnesium concentration in blood serum were measured (please refer to the methods’ subsection sample collection and analyses below for details regarding the analytical methods).
All target parameters of the study were objective variables that could not be influenced by intention of the study subjects. Due to the cross-over design, subjects served as their own control for the parameters under investigation. The integration of a placebo group and the blinding of products administered were not considered necessary.
Study subjects and eligibility criteria
A total of about 20 healthy male subjects of Caucasian origin aged between 18 and 45 years (inclusive) were to be included in this study. Only male subjects were eligible because the female hormone estrogen affects Mg distribution and therefore Mg excretion [20].
Subjects were only included if they had a body mass index (BMI) within the range of 18.0 to 29.0, a normal blood pressure (systolic blood pressure ≥95 ≤ 140 mmHg and diastolic blood pressure ≥55 ≤ 90 mmHg), a resting pulse of ≥45 and ≤95 bpm, a normal digestion (i.e., no current obstipation or diarrhea), a normal renal function (serum-creatinine <1.2 mg/dL), an ECG recording without clinically significant abnormalities and if they reported no febrile or infectious illness for at least 7 days prior to the Screening Visit.
Illnesses or use of medication with influence on renal function (e.g., diabetes, diuretics), conditions which might interfere with the absorption of the study products (e.g., cholecystectomy, bowel resection) or any gastrointestinal complaints within 7 days prior to the Screening Visit were considered criteria for exclusion. Additionally, subjects presenting with symptoms of Mg deficiency (e.g., muscle cramps or fasciculations) as determined by physical examination and anamnesis or any active physical disease (acute or chronic) were excluded. Moreover, clinical chemical, hematological or any other laboratory parameters (i.e., urinalysis and serology) clinically relevant outside the normal range as judged by the investigator were considered criteria for exclusion.
Those with any history of chronic or recurrent metabolic, renal, hepatic, pulmonary, gastrointestinal, neurological, endocrinological, immunological, psychiatric or cardiovascular disease and bleeding tendency were also excluded. Moreover, a history of alcohol or drug abuse, chronic gastritis, peptic ulcers, drug hypersensitivity, asthma, urticaria or other severe allergic diathesis as well as acute symptoms of hay fever were considered criteria for exclusion.
Also excluded were current smokers, subjects reporting a history of smoking within the last 3 months or those who consumed >35 g of ethanol regularly per day, respectively >245 g ethanol regularly per week or more than 5 cups of coffee (or equivalent) per day. Moreover, consumption of alcohol or xanthine-containing food or beverages (e.g., coffee or black tea) as well as grapefruit juice was not allowed within 48 h prior to first administration of magnesium. Alcohol consumption was determined by questioning and regular alcohol breath tests.
Study flow and procedures
The study consisted of a Screening Visit, an In-house Phase and an End-of-Study Visit. Subjects were screened for eligibility within 14 to 2 days prior to the In-house Phase. Figure 1 presents an overview of the study schedule.
The In-house Phase included two profile days (P1 and P2). P1 was preceded by an initial magnesium saturation phase of 5 days, followed by one stop-of-saturation day (S1). P1 and P2 were separated by an intermediate magnesium saturation phase of 2 days, followed by another stop-of-saturation day (S2). The End-of-Study Visit was performed on the day after last study product administration. The stop-of-saturation days were integrated in the study schedule to avoid distortion of renal magnesium excretion on profile days by the high magnesium load administered during the saturation phases.
During the magnesium saturation phases, five single doses of 100 mg magnesium were to be taken at approximately 08.00 am, 10.00 am, 01.00 pm, 04.00 pm, and 06.00 pm together with 200 mL of water of normal Mg content (approximately 15 mg/L) each. On stop-of-saturation days, subjects did not receive magnesium supplementation.
In the morning of each profile day at about 08.00 am to 09.00 am, the subjects received a single dose of 300 mg Mg in the form of Mg citrate or Mg oxide after an overnight fast of at least 8 h. Immediately after intake, the subjects were to drink 200 mL of water of normal Mg content. Water was allowed until 1 h prior to administration.
During the In-house Phase, all subjects received a balanced, mixed diet containing approximately 300 to 400 mg magnesium per day. The diet was composed by a dietitian according to standard nutrient tables. On each profile day, standardized meals (i.e., identical meals) were provided. On each profile day, starting 2 h after administration, non-carbonated water of normal Mg content at room temperature was served.
On study days with urine sampling, subjects were requested to follow a standardized drinking schedule. Subjects were to drink 200 mL of water of normal Mg content every 2 h. An additional portion of 200 mL water was to be drunk together with lunch. On the other study days, subjects were free to consume water of normal Mg content in common amounts. Subjects were asked to drink approximately 2 l a day.
Study products
The test product was Magnesium Verla® purKaps capsules, containing the organic magnesium compound magnesium citrate, marketed by Verla-Pharm Arzneimittel GmbH & Co. KG, Germany. The product is also marketed under the trade name Xenofit® Magnesiumcitrat pure in Germany. Each capsule provides a dose of 150 mg elemental magnesium.
The reference product was Biolectra® Magnesium 300 mg Kapseln capsules, containing the inorganic magnesium compound magnesium oxide, manufactured by HERMES Arzneimittel GmbH, Germany. Each capsule provides a dose of 300 mg elemental magnesium.
For the saturation phases, capsules containing magnesium citrate (100 mg elemental magnesium per capsule) were used.
All study products were supplied by Verla-Pharm Arzneimittel GmbH & Co. KG, Germany.
Sample collection and analyses
Urine
Urine was collected over 24 h in nine fractions (0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16 and 16-24 h) on stop-of-saturation days and on profile days.
Directly after the first micturition, urine samples were acidified with 200 μL highly purified concentrated HNO3. After each micturition, samples were mixed and stored at 2-8 °C. The volume of each fraction was recorded and two aliquots per fraction (5 mL each) were filled in sample shipment tubes. Samples were stored under continuous temperature control below -20 °C and were shipped frozen on dry ice to the bioanalytical site (Helmholtz Zentrum Muenchen, Neuherberg, Germany).
Urine samples were analyzed for total Mg content by inductively coupled plasma optical emission spectrometry (ICP-OES), using a “Spectro Ciros Vision” system (Spectro-Ametek, Kleve, Germany). ICP-OES has been established for more than 35 years for trace element analysis in biological matrices. This technique determines elements, such as magnesium, as total element content, independently of their physico-chemical state (oxidation state or chemical binding form) [21, 22].
Instrumental parameters: Sample introduction was performed by the instrument’s peristaltic pump at 1.0 mL/min and a Meinhard nebulizer which was fitted into a cyclone spray chamber. The measured spectral element line was: Mg 279.079 nm.
The radio frequency (RF) power was set to 1000 W; the plasma gas was 15 L Ar/min and the nebulizer gas was 600 mL Ar/min. Analytical quality control for Mg determination was performed by analysis of blanks and certified control standards (PE# N0691745, Perkin Elmer Pure) after every ten samples. Accuracy of measurements was determined according to IUPAC guidelines [23] by analysis of an adequate certified standard reference material, BCR® 304, from the Joint Research Centre, Institute for Reference Materials and Measurements (IRMM) of the European Union. The found value was 1.85 ± 0.01 mmol/L and the certified value is 1.85 ± 0.03 mmol/L, resulting in an accuracy of 100%. The serial precision (N = 10) was 1.9% and the day-to-day precision (N = 10) was 2.3%.
Monocytes and lymphocytes
On profile days, blood samples (15 mL) were collected at predose, 9 and 24 h post dose in heparinized plasma tubes. The anticoagulated blood was diluted with 7.5 mL phosphate buffered saline (PBS), mixed and carefully poured into Leucosep® tubes (Greiner Bio-One, No. 227288). Samples were centrifuged for 15 min at 1000 × g at room temperature. After centrifugation, the interphase containing monocytes and lymphocytes was harvested and washed twice with PBS. In the end, the cells were pelleted by centrifugation and the remaining liquid supernatant was removed. Cell pellets were stored under continuous temperature control below −20 °C and shipped frozen on dry ice to the bioanalytical site (Helmholtz Zentrum Muenchen, Neuherberg, Germany).
Cell pellets were lysed in suprapure HNO3 and analyzed for total Mg content by ICP-OES analogously to the urine samples.
Erythrocytes
Blood samples for determination of total Mg concentration in erythrocytes (2.7 mL for hematocrit determination and 1.2 mL for Mg measurement in whole blood) were taken on profile days at predose, 9 and 24 h post dose. The blood samples were collected in closed potassium EDTA tubes. Blood samples for Mg measurement were stored and shipped at 2-8 °C. Samples for hematocrit determination were stored and shipped at room temperature. The bioanalytical analyses were performed by the contract laboratory Medizinisches Versorgungszentrum Labor Muenchen Zentrum, Munich, Germany.
Hematocrit (Hct) was measured with a particle counter and calculated by the number of erythrocytes and their mean cellular volume. It represents the proportion of erythrocytes in whole blood (in Vol %).
Magnesium concentration in serum (MGA) was measured directly by a clinical chemistry method. Magnesium concentration in whole blood (MGV) was measured directly by flame atomic absorption spectrometry. The intra-assay precision ranged from 1.4 to 3.7% and the inter-assay precision ranged from 3.2 to 5.2%. The limit of detection was 0.03 mmol/L and the lower limit of quantification was 0.07 mmol/L.
The magnesium concentration in erythrocytes (MGER) was calculated as follows:
$$ MGER=\left(MGV-\left(MGA*\left(1-\left(Hct*0.01\right)\right)\right)\right)/\ \left(Hct*\ 0.01\right)\left[\mathrm{in}\ \mathrm{mmol}/\mathrm{L}\right] $$
(1)
Serum
On profile days, blood samples (2.6 mL) were collected at predose, 60 min, 2, 3, 4, 5, 6, 9 and 24 h post dose in closed serum tubes.
Immediately after taking, the samples were incubated at room temperature for at least 20 min but no longer than 60 min for clotting. To ensure comparability, the preferred incubation time was 30 min. Thereafter, samples were centrifuged at room temperature and 1700 × g for 10 min. The complete resulting serum supernatant was then transferred into polypropylene tubes and shipped to the bioanalytical site (Medizinisches Versorgungszentrum Labor Muenchen Zentrum, Munich, Germany).
Serum samples were analyzed for Mg2+ content by a spectrophotometric method. The intra-assay precision ranged from 0.49 to 3.7% and the inter-assay precision ranged from 0.30 to 3.33%. The lower limit of quantification was 0.29 mmol/L.
Statistics
Sample size calculation
Considering an analysis of variance (ANOVA) for a difference of means in a 2 × 2 cross-over, a sample size of N = 18 was determined to reach 80% power for the primary endpoint. The calculation was performed using nQuery Advisor + nTerim 2.0® software with the following assumptions: mean difference = 5%, standard deviation (SD) of difference = 8%, power ≥80%.
In order to allow for withdrawals, at least 20 subjects were to be randomized.
Randomization
On Profile Day 1, subjects were randomly assigned to 1 of the 2 following sequences of study product administration:
The randomization code for assigning random numbers to sequence groups was created using SAS® 9.4 software.
Statistical analyses
At first, the renally excreted Mg quantity during the interval of 24 h after Mg administration (Ae 0-24h) was calculated from the nine urine fractions taken and was analyzed by descriptive statistics for each group.
In order to compare the bioavailability of the test and reference product, an ANOVA was performed. Based on the result of a Kolmogorov-Smirnov data normality test, raw data or log-transformed (natural logarithm) values were used for the ANOVA. The ANOVA was performed at the 5% level [P < 0.05], two-sided, using SAS® 9.4 software. Effects considered in the ANOVA model were: study product, sequence, period and subject within sequence.
Statistical significance at a level of 5% was given if the 95% confidence interval (CI) of the adjusted mean difference resulting from the ANOVA did not include zero, respectively if the adjusted mean ratio resulting from the ANOVA with log-transformed data (log-ANOVA) did not include 1.
The statistical analyses of the Mg concentrations in a subset of leukocytes, in erythrocytes and in serum were carried out analogously to the analysis of the primary endpoint.