ARTICLE
Auteur(s) : Forrest H Nielsen, David
B Milne1, Sandra Gallagher, LuAnn
Johnson, Bonita Hoverson
US Department of Agriculture, Agricultural Research
Service2, Grand Forks, ND, 58202-9034, USA
Magnesium deficiency has been implicated in hypertensive vascular
disease, atherogenesis, ischemic heart disease, arrhythmias,
stroke, osteoporosis, diabetes, migraine headaches, hyperalgesia,
neurological disturbances, and cognitive performance deficits
[1-7]. Experiments involving humans consuming plausible
low-magnesium diets (<160 mg or 6.58 mmol Mg/d) have not been
particularly successful in providing definitive evidence that
magnesium deficiency is a primary factor for a significant number
of any of these disorders. Past experimental approaches have been
focused on determining whether magnesium deprivation could result
in pathophysiological conditions by changing the actions of
hormones and/or enzymes dependent on or influenced by the presence
of magnesium. These approaches have not shown that any enzyme or
hormone action is consistently changed by consuming a low-
magnesium diet similar to one that may occur in the general
population. This is exemplified by the studies of Fatemi et al. [8]
and Nielsen [9]. As a result, a recommendation of the Food and
Nutrition Board [10] in the US was “Investigations are needed to
assess the inter-relationships between dietary magnesium intakes,
indicators of magnesium status, and possible health outcomes that
may be affected by inadequate magnesium intakes, such as
hypertension, hyperlipidemia, atherosclerotic vascular disease,
altered bone turnover, and osteoporosis.” This recommendation is
still valid because many US women regularly consume low amounts of
magnesium. Estimated daily magnesium intakes of 5% of women are
4.65 mmol or 113 mg (aged 19-30 years), 5.55 mmol or
135 mg (aged 31-50 years), 5.68 or 138 mg (aged 51-70
years), and 5.22 mmol or 126 mg (aged 71+ years) [11].Little
attention has been given to the possibility that magnesium
deprivation caused by low dietary intake may be involved in some of
these disorders through an impairment of the regulation of cellular
ionic balance. Hypomagnesemic disorders increase intracellular
calcium [12]. Animal experiments have shown that an early response
to magnesium deprivation is an increased production of oxidative
and inflammatory neuropeptides and cytokines [13]. The suggested
basis for this increase is an excitotoxicity initiated by the loss
of the magnesium-blockade of the N-methyl-D-aspartate receptor and
an increase in intracellular calcium [13], which can induce the
release of neuropetides such as substance P, calcitonin
gene-related peptide and tumor necrosis factor-α. Increased release
of inflammatory neuropeptides may lead to a variety of
pathophysiological responses including bone loss and cardiovascular
disease [14, 15].Increased calcium retention or calcium balance may
be an indication of an alteration in intracellular ionic balance.
Limited animal data suggest that magnesium deficiency does not have
to be severe to result in increased calcium retention. Mature
female rats fed 50% of the dietary requirement for magnesium for
4-10 wk exhibited increased calcium absorption and balance [16]. In
another study, rats exhibited a significantly increased calcium
absorption and balance within a week after being fed a severely
magnesium-deficient diet (about 5% of requirement) [17]. Calcium
deficiency has been found to be protective against the inflammatory
response induced by magnesium deficiency in rats [18]. Two human
studies have suggested that a subclinical dietary magnesium
deficiency (≈ 115 mg or 4.75 mmol/d) compared to an
adequate intake (≈ 330 mg or 13.6 mmol/d) increased
calcium balance [9, 19]. The present experiment was performed to
establish whether naturally occurring inadequate intakes of
magnesium increase calcium retention and alter the excretion of
other minerals (sodium, potassium and phosphorus) involved in
cellular ionic balance, and these changes are associated with a
non-positive magnesium balance and decreased erythrocyte membrane
magnesium concentration.
Subjects and methods
Subjects
Fourteen post-menopausal women were recruited for the study after
they had been informed in detail both verbally and in writing about
the nature of the research and associated risks, and after medical,
psychological, and nutritional evaluation had established that they
were healthy and emotionally suited for the project. Fourteen were
recruited based on the finding of significant changes in calcium,
phosphorus and potassium excretion in a previous study [9] and an
expected attrition of about 20%. One subject was dismissed from the
study during the first week because she could not tolerate the
amount of food required to be eaten. Another subject was recruited
to restore the 14-bed metabolic unit to full occupancy; the
replacement subject had only an 11 days adaptation period. Two
subjects left the study early for personal reasons. One subject who
contracted an illness the last two weeks of magnesium
supplementation that markedly affected many variables examined was
not included in the data analysis. The eleven Caucasian women who
completed the study as designed were not on hormone replacement
therapy, did not smoke, and had a mean ± SD age of 62.9 ± 7.3
(range of 49 to 71 years with five over the age of 65 years). The
women ranged in height from 143.3 to 168.9 cm, in weight from
54.3 to 85.6 kg and in body mass index of 20.4 to 30.3 (mean of
26.2 ± 3.1). Before entry into the study, a physical examination in
a local clinic that included a Pap smear test, lung x-ray,
tuberculosis test, and electrocardiogram (EKG), and laboratory
tests to assess liver, kidney, and thyroid functions established
that the women had no underlying disease. The women were not taking
any medications including those to control hypertension and
cholesterol. The subjects resided for the entire study in the
metabolic research unit of the Grand Forks Human Nutrition Research
Center that provided a common environment for strict control of
food consumption, weight, exercise, and data collection. Subjects
consumed only food and beverages provided by the dietary staff and
were chaperoned on all outings from the metabolic unit to ensure
compliance with the study protocol.
The participants gave their written informed consent to
participate in the experimental protocol that was approved by the
Institutional Review Board of the University of North Dakota and
the Human Studies Review Committee of the United States Department
of Agriculture, and followed the guidelines of the Department of
Health and Human Services and the Helsinki Doctrine regarding the
use of human subjects.
Experimental protocol
The diet, based on ordinary Western foods, is shown in table 1. A 3 days rotating menu cycle was used to
give some variety but assured that variation in nutrient intake was
not consequential. To create the magnesium-low diet, foods rich in
magnesium, including whole grains, green vegetables, and milk, were
limited. Commonly consumed fruit-flavored drinks instead of milk
were provided as beverages during most meals. An analysis of the
diet formulated before the women entered the study showed that it
supplied approximately 3.21 mmol (78 mg) magnesium per 8.4 MJ
(2,000 kcal). Variation in energy intake to maintain body weight
and some variation in the magnesium content of the foods used
resulted in a mean intake of 4.40 mmol (107 mg) and range of
3.33 to 5.96 mmol (81 to 145 mg) magnesium/d. The mean intake
was slightly below the fifth percentile intake (5.27 mmol or
128 mg/d) of all women aged over 19 years according to NHANES
2001-2002 [11]. The diet energy distribution was 10% protein, 35%
fat and 55% carbohydrate. The diet was based on the 1989 US
Recommended Dietary Allowances (RDA) [20] because the experiment
was performed before the issuance of the current US Dietary
Reference Intakes. To assure adequacy, supplements were used for
nutrients present in low or unknown quantities in the diet. These
supplements were (per day) 960 mg (24.55 mmol) potassium as
potassium gluconate, 20 mg (0.36 mmol) iron as ferrous
gluconate, 8 mg (0.12 mmol) zinc as zinc sulfate, 2 mg
(31.5 μmol) copper as cupric sulfate, 2 mg (36.4 μmol)
manganese as manganese sulfate, 1 mg (92.5 μmol) boron as a
boron-amino acid complex, 0.4 mg (2.39 μmol) vitamin
B6, and 10 μg (26 nmol) of vitamin D3.
Calcium was not supplemented to the diet because the mean intake of
19.08 mmol (778 mg)/d was considered adequate based on the
1989 RDA of 800 mg/d [20]. Although formulating a diet low in
magnesium resulted in a relatively low protein intake of 53 g/8.4
MJ (2000 kcal), the protein intake of all women was near or above
the US RDA of 46 g/d. Dietary iron was provided in excess of the US
RDA to mitigate the decline in iron status as a result of
phlebotomy during the experiment. All food was weighed
proportionally with a 1% rounding error during preparation in the
metabolic kitchen and was completely consumed by the subjects with
the aid of spatulas and rinse bottles. Deionized water was consumed
ad libitum. The initial energy requirement for each subject was
determined by using the Harris and Benedict equation [21] and
adding 50% to compensate for normal daily activities. Individually
prescribed exercise was performed three times weekly to maintain
body composition and physical work capacity. Energy intake was
adjusted in 0.84 MJ (200 kcal) increments during the course of the
experiment to maintain body weight (measured daily) within ± 2% of
admission weight.
Upon arrival in the metabolic unit, the subjects were
equilibrated for 18 d with the basal diet supplemented with 9.05
mmol (220 mg) magnesium/d. The equilibration period for one
subject that replaced a dismissed subject was reduced to 11 d.
After equilibration, a double-blind crossover experimental design
was used for feeding the experimental diets. Seven subjects were
fed the basal diet with a lactose placebo similar in appearance to
the magnesium supplement while the other seven subjects continued
with the basal diet supplemented with 9.05 mmol (220 mg)
magnesium/d for 72 d, then each group switched to the other’s diet,
which they consumed for 72 d. Magnesium was supplemented as
magnesium gluconate. Because of variation in energy intakes to
maintain body weight, the supplementation resulted in magnesium
intakes ranging from 12.92 to 14.11 mmol (314 to 343 mg)/d.
The intakes were near the current US RDA for magnesium of 13.17
mmol (320 mg)/d [10]. The supplementation assured that all
subjects had intakes that were more than 50% the estimated average
requirement of 2.36 mg (0.097 mmol)/kg body weight/d [22]. At
the end of the experiment, each subject was given a 90-d supply of
magnesium gluconate capsules with instructions to consume the
number that would supplement the usual diet with 8.23 mmol
(200 mg) magnesium/d.
Table 1 Food composition of the 3-d rotation diet.
|
Meal
|
Day 1
|
Day 2
|
Day 3
|
|
Breakfast
|
Orange drink, Tanga
|
Cranapple juice
|
Orange drink, Tanga
|
|
Cornflakes w/non-dairy creamer
|
Apple cinnamon coffeecake
|
Waffles w/margarine
|
|
Sugar
|
|
Maple syrup
|
|
Biscuit w/butter & honey
|
|
|
|
Lunch
|
White bread w/turkey breast
|
Drink mix, strawberry w/sugar
|
Drink mix, raspberry w/sugar
|
|
Miracle Whipa dressing
|
Ham broccoli casserole
|
Pizza, Canadian bacon
|
|
Vegetable soup
|
Lettuce w/French dressing
|
Jelloa, strawberry banana
|
|
Cottage cheese, 2%
|
Cottage cheese, 2%
|
Pears
|
|
Jelloa, cherry
|
Sherbet, orange
|
Whipped topping, Cool Whipa
|
|
Dinner
|
Drink mix, grape w/sugar
|
Carbonated soda, 7-Upb
|
Drink mix, strawberry w/sugar
|
|
Chicken rice casserole
|
Crispy chicken w/potatoes
|
Beef stew
|
|
Lettuce w/French dressing
|
White dinner roll w/butter
|
Biscuit w/butter & grape jelly
|
|
Cheddar cheese
|
Cheddar cheese
|
Cheddar cheese
|
|
Apple crisp
|
Jelloa, orange
|
Lemon pie
|
|
Mandarin oranges
|
|
|
Snack
|
Crackers, Ritzc
|
Drink mix, cherry w/sugar
|
Angel food cake
|
|
Cheddar cheese
|
Sugar cookies
|
|
Vanilla frosting
|
aKraft Foods, Inc., Rye Brook, NY, and Glenview, IL.
bDr Pepper/Seven Up, Inc. Plano, TX.
cRitz, Nabisco, East Hanover, NJ.
Physical measurements
Because potentially harmful electrocardiographic changes have been
found in people deficient in magnesium [12, 23-26], a 20-hr EKG
using a Holter Recorder was performed on each volunteer every two
weeks during the study. Tapes obtained were machine (Model 363,
Accuplus, Del Mar Avionics, Irvine, CA) scanned for signs of
abnormal rhythm by trained nurses under the direction of a
physician who confirmed the findings of the nurses. If the EKG
showed a significant increase (four times baseline obtained from
the initial two Holter EKGs) in ventricular premature discharges,
the appearance of AV heart blocks, or the appearance of atrial
flutter and fibrillation, the change was confirmed by immediately
performing another Holter EKG. Confirmation of one of these changes
while consuming the low magnesium diet prompted the supplementation
with 9.05 mmol (220 mg) magnesium/d.
Because magnesium deficiency has been correlated with
hypertension [27], blood pressures were measured daily during the
study. Upon waking-up in the morning, each subject would notify by
intercom the on-duty nurse trained in blood pressure measurement.
The nurse then went to the room of each woman and determined their
blood pressure by using a mercury sphygmomanometer while the
subject was lying in bed. If the subject had to use the toilet upon
waking, she was required to return to her bed and recline for 20
minutes before her blood pressure was determined.
At the time the Holter EKGs were performed, standard
neuromuscular magnesium deficiency tests, checking for Trousseau’s
and Chvostek’s signs (reflexes) were done. The test for Trousseau’s
sign involved inflating a sphygmomanometer cuff to 10 mm above
systolic blood pressure and holding this pressure for two minutes.
Carpal spasm with relaxation 5 to 10 s after deflation was
considered positive. The test for Chvostek’s sign involved tapping
the facial nerve just anterior to the ear lobe and just below the
zygomatic arch (or between the arch and the corner of the mouth). A
positive response ranges from a simple twitching of the corner of
the mouth to a twitching of all facial muscles on the stimulated
side.
Sample collection
For balance determinations, duplicate diets of 8.4 MJ (2 000
kcal) were prepared daily for analysis and blended in a plastic
container with stainless steel blades. Urine and feces were
collected in plastic containers and bags, respectively, to avoid
mineral contamination. Venous blood, collected in plastic syringes
from antecubital veins that had been distended by the temporary use
of a tourniquet after the subjects had fasted for 10 hr overnight,
was obtained each wk for routine health assessment, and magnesium
and calcium determinations. Additional blood was obtained during wk
4, 7, 9, and 10 of each dietary period for the determination of
other experimental variables. Total blood collected was limited to
≤ 250 mL/mo.
Laboratory methods
The magnesium, calcium, and phosphorus content of 6 d-composites of
diets and feces were determined throughout the experiment by
inductively coupled argon plasma emission spectroscopy (ICAP)
(Jarrell-Ash Atom Comp 1140, Thermo Elemental, Franklin, MA) after
wet digestion of lyophilized blended samples with nitric and
perchloric acids [28]. Urinary minerals were determined by ICAP of
a diluted aliquot. For diets and feces, concurrent replicate
analysis of a standard reference material NIST 1577a bovine liver
(National Institute of Standards and Technology, Gaithersburg, MD)
yielded means (μg/g) ± SD of 118 ± 8 for calcium, 621 ± 28 for
magnesium, and 10456 ± 266 for phosphorus compared with certified
values (means ± confidence values) of 120 ± 7 for calcium, 600 ± 15
for magnesium, and 11100 ± 400 for phosphorus. For urine,
concurrent replicate analysis of UriChem 1 (Fisher Scientific,
Orangeburg, NY) yielded means (μg/mL) ± SD respectively of 67 ± 2
for calcium, 44.2 ± 2.4 for magnesium, 673 ± 15 for phosphorus, and
2442 ± 163 for potassium compared with certified values of 64 ± 3
for calcium, 43.5 ± 11.7 for magnesium, 650 ± 90 for phosphorus,
and 2600 ± 332 for potassium. Calcium, magnesium and phosphorus
balances were calculated as the differences between intake and
excretion (feces plus urine) for all days of each dietary period.
The balance or retention calculations did not include surface or
phlebotomy losses.
Blood was processed within 90 min to obtain serum or
plasma. The blood was allowed to clot for 20 min before
centrifuging at 2000 RPM for 10 min to obtain serum. Serum
(after dilution with a 0.5% lanthanum oxide in deionized water)
calcium and magnesium concentrations were determined by ICAP.
Concurrent analysis of Sera Chem controls (Fisher Scientific,
Orangeburg, NY) yielded the values (mg/L) of 95.9 ± 6.0 for calcium
and 20.4 ± 1.7 for magnesium compared with the certified values of
99 ± 20 for calcium and 20.9 ± 6.6 for magnesium. The method of
Dodge et al. [29] was used to obtain red blood cell membranes from
10 mL of blood collected using EDTA as the anticoagulant. The
protein content of the membranes was determined by using a
commercially available kit (Biorad Protein Kit #500-006, Standard
#500-0007, Biorad, Hercules, CA). Each membrane sample was diluted
by a factor of 5 with deionized water before analyzing for
magnesium by ICAP.
Commercially available radioimmunoassay kits were used to
determine serum calcitonin, 25-hydroxycholecalciferol, mid-molecule
parathyroid hormone (PTH-MM) and osteocalcin (Incstar Corporation,
Stillwater, MN). Serum alkaline phosphatase activity was determined
by using a standard method of the Cobas Fara Centrifugal Analyzer
(Roche Diagnostic Systems, Sommerville, NJ). Urinary hydroxyproline
excretion was measured by using the method of Podenphant et al.
[30].
Data analysis
Biochemical determinations made during the last 36 days of each
dietary period were used in the statistical analysis. Because fecal
excretion was highly variable, for each diet period, data from
every two consecutive six-day collection periods were combined to
give 12 d excretion data. These data were used to estimate mean
daily excretion values and for balance estimates. The data were
analyzed by repeated measures analysis of variance with a SAS
general linear model program (SAS 8.02, SAS Institute, Cary, NC). A
p ≤ 0.05 was considered significant. Variances in the data are
expressed as a pooled standard deviation, calculated as the square
root of the mean square error from the analysis of variance.
Results
Three subjects showed changes during magnesium deprivation that
resulted in stopping the deprivation and supplementing with 9.05
mmol (220 mg) magnesium/d. All three were in the magnesium
deprivation period that followed the adequate magnesium period. One
subject showed a confirmed significant increase in ventricular
premature discharges 52 d after starting the magnesium deprivation
period; 14 days after magnesium supplementation started, the
ventricular premature discharges returned to baseline. Another
subject showed an increase in blood pressure starting about 51 d
after starting magnesium deprivation. When blood pressures reached
hypertensive levels (about 150/90 mm mercury) 10-14 d later,
magnesium supplementation was started. Blood pressures decreased
until the end of the experiment with the last reading being 126/80.
This finding was similar to that reported for one subject in a
previous dietary magnesium deprivation experiment [26]. A third
subject had a cancerous skin lesion (not detected prior to entering
magnesium deprivation) removed 18 d after starting magnesium
deprivation. The wound did not heal well and antibiotics were
required. Another cancerous skin lesion was removed 52 d after
starting magnesium deprivation that did not heal. Because of the
possibility that magnesium was affecting wound healing, the subject
was supplemented with magnesium 63 d after starting magnesium
deprivation. Upon dismissal from the study 9 d later, both wounds
were healing nicely.
Except for the one subject described above, blood pressure was
not significantly affected during magnesium deprivation. During
magnesium deprivation and supplementation, the mean
systolic/diastolic pressures of the 11 volunteers were
112/69 mm and 112/68 mm of mercury, respectively. No
positive Chvostek’s and Trousseau’s signs were obtained during
magnesium deprivation.
Table 2 shows that magnesium balance
was positive when the diet provided an average of 13.46 mmol (327
mg) magnesium/d. The percent of ingested magnesium absorbed was not
increased during magnesium deprivation as indicated by a higher
percentage of magnesium appearing in the feces. However, urinary
magnesium excretion was significantly decreased when the diet
provided an average of 4.40 mmol (107 mg) magnesium/d. This
homeostatic response was not enough to prevent a non-positive
magnesium balance based on just fecal and urine excretion during
the deficient dietary periods.
Red blood cell membrane magnesium was significantly decreased by
magnesium deprivation (table 3). Serum
magnesium concentration was significantly lower during magnesium
deprivation than supplementation when deprivation occurred first,
but was higher during magnesium deprivation when deprivation
followed supplementation; just the opposite occurred with serum
calcium concentration (table 3). Table 3 also shows that magnesium deprivation
decreased the urinary excretion of potassium, but did not
significantly affect the urinary excretion of hydroxyproline.
Magnesium deprivation significantly affected calcium and
phosphorus metabolism or utilization. During magnesium deprivation
the urinary excretion of calcium was significantly decreased and
the percentage of ingested calcium appearing in the feces tended (p
≤ 0.08) to be decreased (table 4).
This resulted in calcium balance being significantly higher during
magnesium deprivation (+0.82 mmol or +35 mg/d) than supplementation
(-0.02 mmol or -1 mg/d). Magnesium deprivation significantly
decreased the percentage of ingested phosphorus appearing in the
feces but increased the amount of phosphorus excreted in the urine
(table 5). The net result of these
opposing changes was that magnesium deprivation did not
significantly affect phosphorus balance.
Magnesium deprivation had little effect on serum indicators of
calcium metabolism (table 6). Serum
calcitonin was significantly lower during magnesium deprivation
than supplementation when deprivation followed supplementation but
not when magnesium deprivation was first. The opposite was found
with serum osteocalcin. It was lower during magnesium deprivation
than supplementation when deprivation occurred first but not when
deprivation followed supplementation. Magnesium deprivation did not
significantly affect the serum concentrations of PTH-MM,
25-hydroxycholecalciferol, and alkaline phosphatase.
Urinary creatinine (data not presented) was not significantly
affected by magnesium deprivation and thus the PTH-MM results
apparently were not affected by a possible change in renal
function.
Table 2 Mean magnesium balance during each dietary
period.
|
Dietary Treatment
|
Diet Mg, mmol/d
|
Fecal Mg, mmol/d
|
|
|
|
Mg Balance, mmol/d
|
|
Mg Def-first
|
4.12
|
2.30
|
55.9
|
1.93
|
47.2
|
-0.12
|
|
Mg Sup-second
|
13.33
|
6.95
|
52.4
|
3.99
|
30.1
|
+2.35
|
|
Mg Def-second
|
4.65
|
2.59
|
56.6
|
2.35
|
51.5
|
-0.29
|
|
Mg Sup-first
|
13.58
|
6.95
|
51.1
|
4.53
|
33.3
|
+2.10
|
|
Mg Def - All
|
4.40
|
2.47
|
56.2
|
2.14
|
49.3
|
-0.21
|
|
Mg Sup - All
|
13.46
|
6.95
|
51.7
|
4.28
|
31.7
|
+2.22
|
|
Pooled SD
|
|
0.74
|
9.4
|
0.45
|
4.4
|
0.82
|
|
Analysis of Variance – p values
|
|
Diet
|
|
0.0001
|
0.005
|
0.0001
|
0.0001
|
0.0001
|
|
Sequence
|
|
0.76
|
0.95
|
0.25
|
0.46
|
0.36
|
|
Diet x Sequence
|
|
0.22
|
0.53
|
0.57
|
0.48
|
0.91
|
Table 3 Effect of dietary magnesium on erythrocyte
membrane and serum magnesium, plasma calcium, and urinary potassium
and hydroxyproline concentrations.
|
Dietary Treatment
|
- RBC membrane Mg
- (nmol/mg protein)
|
Serum Mg (mmol/L)
|
|
|
Urinary OH-Proline (mmol/d)
|
|
Mg Def-first
|
2.55
|
0.80
|
2.34
|
2.55
|
0.082
|
|
Mg Sup-second
|
2.72
|
0.88
|
2.31
|
2.72
|
0.084
|
|
Mg Def-second
|
2.47
|
0.92
|
2.33
|
2.47
|
0.093
|
|
Mg Sup-first
|
2.67
|
0.88
|
2.35
|
2.67
|
0.090
|
|
Mg Def - All
|
2.51
|
0.86
|
2.34
|
2.51
|
0.087
|
|
Mg Sup - All
|
2.67
|
0.88
|
2.33
|
2.67
|
0.087
|
|
Pooled SD
|
0.021
|
0.02
|
0.02
|
0.021
|
0.013
|
|
Analysis of Variance – p values
|
|
Diet
|
0.05
|
0.04
|
0.50
|
0.05
|
0.75
|
|
Sequence
|
0.75
|
0.03
|
0.58
|
0.75
|
0.55
|
|
Diet x Sequence
|
0.90
|
0.0001
|
0.05
|
0.90
|
0.64
|
Table 4 Mean calcium balance for each dietary period.
|
Dietary Treatment
|
|
Fecal Ca (mmol/d)
|
|
|
|
Ca Balance (mmol/d)
|
|
Mg Def-first
|
18.01
|
14.12
|
78.0
|
3.02
|
17.4
|
+0.87
|
|
Mg Sup-second
|
18.66
|
15.37
|
82.1
|
3.37
|
18.8
|
-0.07
|
|
Mg Def-second
|
20.03
|
14.32
|
71.0
|
4.97
|
25.0
|
+0.77
|
|
Mg Sup-first
|
19.61
|
14.22
|
72.6
|
5.34
|
27.2
|
+0.05
|
|
Mg Def – All
|
19.01
|
14.22
|
74.5
|
3.99
|
21.2
|
+0.82
|
|
Mg Sup – All
|
19.14
|
14.80
|
77.3
|
4.37
|
23.0
|
-0.02
|
|
Pooled SD
|
|
2.00
|
9.6
|
0.32
|
1.9
|
1.87
|
|
Analysis of Variance – p values
|
|
Diet
|
|
0.09
|
0.08
|
0.0001
|
0.0001
|
0.009
|
|
Sequence
|
|
0.76
|
0.11
|
0.04
|
0.12
|
0.98
|
|
Diet x Sequence
|
|
0.05
|
0.45
|
0.77
|
0.26
|
0.70
|
Table 5 Mean phosphorus balance for each dietary
period.
|
Dietary Treatment
|
Diet
|
Fecal
|
Fecal
|
Urine
|
Urine
|
Balance
|
|
(mmol/d)
|
(mmol/d)
|
(% intake)
|
(mmol/d)
|
(% intake)
|
(mmol/d)
|
|
Mg Def-first
|
33.61
|
10.36
|
30.2
|
23.89
|
72.1
|
-0.65
|
|
Mg Sup-second
|
33.81
|
11.30
|
33.0
|
23.35
|
70.1
|
-0.84
|
|
Mg Def-second
|
36.29
|
10.11
|
27.7
|
26.93
|
74.8
|
-0.71
|
|
Mg Sup-first
|
36.62
|
11.49
|
31.5
|
26.28
|
71.8
|
-1.16
|
|
Mg Def - All
|
34.97
|
10.24
|
28.9
|
25.41
|
73.4
|
-0.68
|
|
Mg Sup - All
|
35.23
|
11.40
|
32.3
|
24.83
|
71.0
|
-1.00
|
|
Pooled SD
|
|
1.61
|
4.3
|
1.58
|
4.8
|
2.10
|
|
Analysis of Variance – p values
|
|
Diet
|
|
0.0001
|
0.0001
|
0.03
|
0.003
|
0.38
|
|
Sequence
|
|
0.98
|
0.69
|
0.14
|
0.71
|
0.82
|
|
Diet x Sequence
|
|
0.40
|
0.48
|
0.85
|
0.50
|
0.73
|
Table 6 Effect of dietary magnesium on serum indicators
of calcium metabolism.
|
Dietary Treatment
|
Calcitonin
|
PTH-MM
|
25-OH-Vit D
|
Osteocalcin
|
AP
|
|
(ng/L)
|
(pmol/L)
|
(nmol/L)
|
(μg/L)
|
(U/L)
|
|
Mg Def-first
|
54
|
61
|
82
|
0.81
|
108
|
|
Mg Sup-second
|
49
|
58
|
79
|
1.69
|
106
|
|
Mg Def-second
|
28
|
53
|
86
|
1.16
|
77
|
|
Mg Sup-first
|
48
|
50
|
89
|
0.91
|
77
|
|
Mg Def - All
|
42
|
57
|
84
|
0.97
|
94
|
|
Mg Sup - All
|
49
|
55
|
84
|
1.33
|
93
|
|
Pooled SD
|
9
|
6
|
4
|
0.56
|
4
|
|
Analysis of Variance – p values
|
|
Diet
|
0.09
|
0.39
|
0.96
|
0.22
|
0.48
|
|
Sequence
|
0.26
|
0.14
|
0.29
|
0.12
|
0.07
|
|
Diet x Sequence
|
0.009
|
0.97
|
0.09
|
0.04
|
0.69
|
Discussion
Previous studies have indicated that magnesium balance can be used
as a method for determining whether subjects are being depleted of
magnesium when fed a magnesium-restricted diet [9, 26]. A highly
positive magnesium balance upon feeding a magnesium adequate diet
after magnesium deprivation confirms the presence of a low or
deficient magnesium status. Thus, the magnesium balance findings in
the present study indicate that subjects were becoming magnesium
deficient when fed the diet providing about 4.40 mmol (107 mg)
magnesium/d. The positive magnesium balance exhibited by subjects
fed the magnesium supplement before the placebo suggests that these
subjects had a low magnesium status when they entered the study.
The finding of decreased red blood cell membrane magnesium
concentrations during the magnesium deprivation periods provided
confirmation that the low magnesium diet was inducing a deficient
magnesium status. Red blood cell membrane magnesium has been found
to be an indicator of decreased magnesium status in humans fed a
magnesium-restricted diet [26]. As indicated in a previous study
[26], serum magnesium concentration was not useful as an indicator
of magnesium status.
The present calcium metabolism findings are consistent with
those obtained in previous studies using different experimental
protocols [9, 19]. The findings from three different studies
indicate that decreased urinary excretion and increased retention
of calcium are signs of mild to moderate magnesium deprivation in
humans. The magnesium deficiency in the present experiment
apparently was only mild to moderate because it did not change
serum parathyroid hormone or decrease serum calcium concentrations.
Severely magnesium-deficient subjects exhibit hypocalcemia [8] and
increased urinary calcium excretion [31, 32]. These changes
apparently are caused by decreased secretion of, and end-organ
resistance to, parathyroid hormone [8, 33], which does not occur in
the early stages of severe magnesium deficiency or with mild
magnesium deprivation.
Decreased urinary excretion and increased retention of calcium
are signs of magnesium deficiency in animals and they have been
associated with soft tissue calcium accumulation [16, 34-37].
Although soft tissue calcium retention occurs, calcium is lost from
bone in severely magnesium-deficient animals. Mice and rats fed
severely magnesium-deficient diets (10% or less of the requirement)
exhibit bone loss apparently caused by increased bone resorption
and inadequate bone formation during remodeling [14, 38-40]. In a
study more relevant to the present study, mature female rats fed
50% of their magnesium requirement, which would be comparable to
6.58 mmol (160 mg) magnesium/d for humans (50% of the RDA for
magnesium), for seven months exhibited decreased humeral bone
mineral density and content in trabecular bone [41]. Rude et al.
[42] found that rats fed 25% of their magnesium requirement (25% of
the RDA for humans is 3.28 mmol or 80 mg/d) for 2, 4 and 6 months
exhibited decreased bone volume and trabecular thickness. Katsumata
et al. [43] found that moderate magnesium deficiency (50% of the
requirement) increased indicators of bone resorption in rats. Thus,
the magnesium deficiency-induced calcium retention in the present
study most likely did not increase the amount of calcium as bone
mineral and did not increase extracellular calcium, but instead
increased soft tissue (including bone marrow) calcium
concentrations. This suggestion is supported by another human study
that found sublingual cellular calcium concentration was increased
by magnesium deprivation [44].
Intracellular calcium may have increased because of decreased
magnesium regulation of its entry into the cell. The decreased
urinary excretion of potassium found in the present study and in a
previous experiment [9] may be another indication that the
magnesium deprivation was affecting the intracellular content of
ions. Magnesium activates the Na+,K+-ATPase
pump that has a major role in regulating Na+ and
K+ transport across the cell membrane [12]. In addition,
one of the first responses to a moderate magnesium deficiency may
be a reduction of the magnesium-blockade of the
N-methyl-D-aspartate receptor [13] and/or a change in the
regulation of calcium influx into cells [45]. This blockade results
in increased intracellular calcium that may increase the transport
of potassium into cells, which would result in less potassium
available for excretion in urine.
Other findings indicated that magnesium deprivation affected
calcium balance or retention by affecting calcium at the soft
tissue or cellular level, and not by affecting its absorption from
the gastrointestinal tract or regulation at the kidney level. These
findings are that the moderate magnesium deficiency did not affect
the circulating amounts of alkaline phosphatase, parathyroid
hormone (involved in regulating calcium excretion), and
25-hydroxycholecalciferol (precursor of the active form of vitamin
D that stimulates calcium absorption). Moderately
magnesium-deficient rats showed similar responses. Two months after
being fed only 25% of their magnesium requirement, rats exhibited
no change in circulating parathyroid hormone concentration and
alkaline phosphatase activity [42]. The finding that serum
calcitonin was significantly decreased by magnesium deprivation
when it followed supplementation without any apparent effect on
plasma calcium concentration may be also reflecting that magnesium
deprivation affected calcium balance and retention by increasing
soft tissue or cellular calcium.
The calcitonin decrease may have been caused by a change in the
activation of voltage-dependent calcium channels that are involved
in calcitonin excretion [46].
Unfortunately, commercial kits for indicators of bone loss
better than hydroxyproline, including N-telopeptide and
deoxypyridinoline, were not available at the time the present study
was performed. Thus, only urinary hydroxyproline excretion was
determined and the results suggest that the magnesium deprivation
was not sufficient to cause a measurable change in bone loss. It
also is possible that dietary magnesium did not affect this urinary
marker of bone resorption because the magnesium treatments were
only 72 days in length. It is recognized that bone turnover markers
take at least 3 months to reach a steady state following the
introduction of a treatment. However, the hydroxyproline results
were similar to deoxypyridinoline findings obtained from moderately
magnesium-deficient rats. Rats fed 25% of their magnesium
requirement for 2, 4 and 6 months exhibited no change in the bone
turnover marker of urinary pyridinoline excretion [42].
The finding that osteocalcin was decreased during magnesium
deprivation that occurred before supplementation but not when
deprivation occurred after supplementation is supportive of the
suggestions that the magnesium deprivation was mild to moderate,
and suggests that the magnesium deficiency after magnesium
supplementation was milder, or did not have time to develop as well
as when the deficiency occurred before supplementation. Carpenter
et al. [47] reported that decreased circulating osteocalcin
occurred after two days in rats after being placed on a severely
magnesium-deficient (5% of the requirement) diet. After being fed
the magnesium-deficient diet for eight days, the rats still did not
show any change in circulating parathyroid hormone or 1,
25-hydroxyvitamin D concentrations. After 12 days, the
magnesium-deficient rats finally exhibited a slight increase in
circulating parathyroid hormone.
The changes in phosphorus metabolism were the same as those
found in a previous study with a different experimental design [9].
The findings were also consistent with those reported by other
investigators who found a substantial decrease in phosphorus
absorption when dietary magnesium was increased [32, 48], but
phosphorus balance apparently was not affected [32]. The apparent
increase in phosphorus absorption (indicated by decreased fecal
phosphorus content) combined with the increased urinary excretion
of phosphorus during magnesium deficiency may be reflecting a
change in high-energy phosphate metabolism. This suggestion is
supported by reports that magnesium deficiency increased the energy
needs of post-menopausal women [49] and magnesium supplementation
of athletes improved energy utilization [50].
The findings in the present study suggest that the moderate
magnesium deficiency which can result from consuming a diet low in
magnesium may have pathological consequences through altering the
intracellular concentrations of signaling or regulatory ions.
Increased intracellular calcium induces the release inflammatory
neuropeptides and causes oxidative stress [13]. In animal models,
magnesium deficiency-induced increases in neuropeptides such as
substance P and tumor necrosis factor-α have been associated with
increased bone resorption or bone loss [14, 40, 42]. This may be
the basis for epidemiologic studies linking a low magnesium intake
to osteoporosis and bone loss [6]. For example, the Framingham
Osteoporosis Study found that magnesium intake was positively
associated with the maintenance of bone mineral density in elderly
women [51].
Also, changing the intracellular concentrations of calcium and
potassium may result in heart arrhythmia. For example, Feyertag et
al. [52] found that a magnesium supplement of 15 mmol magnesium
citrate compared to a placebo for 3 weeks given to patients after
myocardial infarction decreased ventricular extrasystoles. Urinary
potassium excretion also increased in the magnesium-supplemented
patients. An electrolyte imbalance may have been the cause of heart
arrhythmia found in one subject in the present study, and in
several other subjects experimentally deprived of magnesium [9,
26].
Conclusion
A moderate magnesium deficiency was induced in post-menopausal
women when fed a magnesium-restricted diet (about 4.40 mmol or
107 mg/d) under experimental conditions for 72 d. A
non-positive magnesium balance during magnesium deprivation and a
highly positive magnesium balance with magnesium supplementation,
and decreased red blood cell membrane magnesium concentration,
support the conclusion that the subjects had decreased magnesium
status. The moderate magnesium deprivation resulted in increased
calcium retention which is consistent with an increased soft tissue
calcium accumulation and intracellular calcium. The moderate
magnesium deficiency also altered phosphorus metabolism and urinary
potassium excretion. The changes in distribution of calcium and
potassium may lead to pathophysiological conditions including heart
arrhythmias and bone loss. The study shows that an intake of 4.40
mmol (107 mg) magnesium/d is inadequate for postmenopausal
women. Because 5% of all women aged over 19 years in the United
States consume just slightly more than this amount (128 mg/d)
[11], magnesium deficiency may be a significant factor compromising
cardiovascular and bone health.
Acknowledgments
The authors express gratitude to the members of the Grand Forks
Human Nutrition Research Center clinical staff whose special
talents and skills made this study possible: James Penland and
staff (psychological monitoring), Henry Lukaski and staff (exercise
prescriptions and oversight), Leslie Klevay (cardiac monitoring),
Betty Vetter and nursing staff (metabolic unit care), Donna Neese
(protocol processing and scheduling), dietary staff (meal
preparation), analytical chemistry staff (mineral analysis),
clinical chemistry staff (biochemistry analyses), and Christine
Bogenreif (manuscript processing).
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2 The US Department of Agriculture,
Agricultural Research Service, Northern Plains Area, is an equal
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are available without discrimination. Mention of a trademark or
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the US Department of Agriculture and does not imply its approval to
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