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Freure 1. Cireadian excretion cycles of magnesium and calcium (upper panel) and chloride (middle panel).
Creatinine excretion and urine flow (lower two panels) show no regular rhythm.

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REFERENCES

- Buscarno, V. M. and Batzr, R.: Concerning the

Genesis of Sleep; Magnesium and Vitamin B-6, Boll.
Soc. Ital. Biol. Sper. 28: 113-114, 1952,

Brrscor, A. M. and Ragan, C.: Diurnal Variations
in Calcium and Magnesium Excretion in Man.
Metabolism 15: 1002-1010, 1966.

Cannutners, B. M., Copp, D. H. and McIntosu,
H. W.: Diurnal Variation in Urinary Excretion
of Calcium and Phosphate and Its Relation to Blood
Levels. J. Lab. Clin. Med. 63: 959-968, 1964,

Cortovs, E., TrarHam, ‘H. V. and Bowman, R. L.:
An Instrument and Method for Automatic, Rapid,
Accurate and Sensitive Titration of Chloride in
Biological Samples. J. Lab. Clin. Med. 51; 461-468,
1958,

Doz, R. P., Vennes, J. A. and Fux, E. B.: Di-
urnal Variation of 17-Hydroxycorticosteroids, So-
dium, Potassium, Magnesium and Creatinine in
Normal Subjects and in Cases of Treated Adrenal

Insufficiency and Cushing's Syndromée. J. Clin,
Endocr, Met, 20: 258-265, 1960.
. Heaton, F. W. and Hopexinson, A.: External

Factors Affecting Diurnal Variation in Electrolyte
Excretion with Particular Reference to Calcium and
Magnesium. Clin. Chim. Acta. 8: 246-254, 1963.

7.

8.

10.

11.

12.

13,

14.

Huu, J. B.: Automated Fluorometric Method for
Determination of Serum Calcium. Clin. Chem, 11:
122-130, 1965.

Hux, J. B.: An Automated Fluorometric Method
for the Determination of Serum Magnesium. Ann.
N.Y. Acad. Sci. 102: 108-117, 1962.

. Mitts, J. N.: Human Circadian Rhythms, Physiol.

Rev, 46: 128-171, 1966.

Min, H. K., Jones, J. B., and Frinx, BE. B.: Cire
cadian Variations in Renal Excretion of Magnesium,
Calcium, Phosphorus, Sodium and Potassium During
Frequent Feeding and Fasting. Fed. Proc. 25:
917-921, 1966,

Pino, S., Benorrt, J. and Garpyna, H.: An Auto-
mated Method for Urinary Creatinine Which Does
Not Require A Dialyzer Module. Clin, Chem. 11:
664-666, 1965.

Rusino, A. and Bazar, R.: Diurnal Variations in
Magnesium in Relation to the Sleep-wake Cycle.
Acta Neurologica. 6: 618-633, 1951.

Sranpuny, S. W. and THomson, A. E.: Diurnal
Variations in Blectrolyte Excretion. Clin. Sei. 10:
267-298, 1951.

Wesson, L. G., Jr.: Electrolyte Excretion in Re-
lation to Diurnal Cycles of Renal Function. Medi-
cine 43; 547-592, 1964,


Tate 2. Physical Characteristics of Experimental and
Control Subjects Used in Activity Study

Experimental Control
Number 10 10
Age (years) 23 (0.9)* 25 (1.0)
Height (cm) 1771 (2.1) 175.1 (1.9)
Weight (kg) 75.0 (2.6) 76.6 (4.2)

*Values in parentheses are the S.E.

utes rest (recumbent) , 60 minutes activity (tread-
mill walk at 3.560.02 (SE) mph; 4° inclina-
tion), 120 minutes rest (recumbent). To insure
an adequate urine flow over the course of the
test, distilled water was given to experimental
subjects in the following amounts: 3 ml/kg
body weight at the start of the first rest period;
6 ml/kg immediately prior to the activity period;
1.5 ml/kg at the end of the activity period and
every 380 minutes thereafter for 2 hours. One-
hour urine samples were collected covering the
initial 60-minute rest period and the 60-minute
activity period; four 30-minute samples were
collected over the 2-hour recovery period. Ve-
nous blood samples were taken at the end of the
initial rest period, at the end of the activity
period, and at the end of the 2-hour recovery
period.

Control subjects followed the same schedule
for urine and blood sampling outlined above,
but rested in a recumbent position over the 4-
hour test period. Distilled water was given to
control subjects at a dose of 3 ml/kg/hour for
the first 2 hours and 1.5 ml/kg/30 minutes over
the remaining 2 hours. Urine volumes over the
designated periods were recorded and a portion
of each urine sample reserved for analysis.
Magnesium and calcium levels in both urine and
plasma were measured by techniques indicated
earlier. Urine was also analyzed for creatinine.
The excretion of magnesium and calcium was
expressed per unit excretion of creatinine.

Results
Figure 1 presents the pattern of excretion of
magnesium and calcium, chloride, and creatinine
over the 120-hour test period. The variation in
urine flow over this time is shown in the lower
panel. A clear circadian periodicity in excretion
of both magnesium and calcium was observed.
Peak excretion levels for magnesium were ob-
served between the hours 0200-0600 and were
reproducible from day to day. Trough levels

for magnesium excretion occurred between the
hours 1000 and 1400 and were also consistent.
Peak excretion levels for calcium were only
slightly less regular than those for magnesium.
During days 1, 3, and 4, calcium peaks coincided
with magnesium peaks (0200-0600); on days 2
and 5, calcium peaks occurred between 2200 and
0200. Trough levels for calcium excretion al-
ways occurred between 1000 and 1400.

The pattern of chloride excretion was also
circadian, but out of phase with the magnesium/
calcium cycles. Peak levels for chloride excre-
tion occurred each day between 0600 and 1000.
Minimum excretion levels of chloride were ob-
served between 2200 and 0600. A secondary
peak in chloride excretion was noted on days
2-5 occurring between the hours 1800-2200. No
regular pattern was observed in either creatinine
excretion or in urine flow.

The influence of activity on the urinary ex-
cretion of magnesium and calcium is shown in
Figure 2. Exercise induced a marked decrease
in the excretion of both ions to about 45% of
beginning excretion levels. During the period
of rest following exercise the excretion of mag-
nesium and calcium tended to return to the
original level; however, after two hours of rest,
the excretion of both ions was still only 60%
of the original level. By comparison, only small
changes were detected in the calcium and mag-
nesium excretion of control (resting) subjects
over the 4-hour period.

Creatinine excretion fluctuated slightly over
the 4-hour test in both groups. The changes
observed, however, were similar for both groups
and were not statistically significant. Urine
flow gradually increased in the control group
as a result of water loading. In the exercise
group urine flow decreased, as was expected,
during the exercise period and during the first
30-minute post-exercise rest period. Beyond that
time urine flow increased rapidly to the same
levels occurring in control subjects.

Plasma levels of magnesium and calcium for
both experimental and control groups are given
in Table 3. No significant difference between
groups was detected in either ion at the times
sampled.

Discussion
Under the conditions described in this paper
we have observed (1) a reproducible circadian


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URINE FLOW ml/min CREATININE EXCRETION
mi

TIME — HOURS

Fiours 2, Reduced excretion of magnesium and calcium
during and following moderate exercise (upper
panel). Creatinine excretion and urine flow are
shown in the lower panels.

cycle in the urinary excretion of magnesium and
calcium, and (2) a marked decrease in the uri-
nary excretion of magnesium and calcium during
and following a period of moderate activity.
Although other (shorter term) studies have
suggested a circadian rhythm in the excretion
of calcium and magnesium**” the time at which

excretion is maximal has varied widely among
the several reports. This variability has pre-
cluded any statement linking the excretion of
these ions with other exogenous rhythms. The
reproducibility of our own data leads us to sug-
gest that the excretion of both ions is greatest
during the sleeping hours and decreases during
the waking hours. Since the sleep-wake cycle
also represents an activity cycle it is a reasonable
assumption that the circadian periodicity in
magnesium and calcium excretion simply reflects
the exogenous activity pattern. The demonstra-
tion of a marked decrease in magnesium and
calcium excretion after exercise supports this
view.

The mechanism behind the change in excretion
with activity remains obscure. In our experi-
ments the excretion of both ions was not simply
related to urine flow. During the post-exercise
recovery period excretion of calcium and mag-
nesium was only 60% of control at the same
time that urine flow was nearly 200% of control.
Nor did changes in blood levels of the ions ac-
count for the decreased excretion after exercise.
The decrease therefore, must have been the re-
sult of a decrease in renal blood flow, or an
increase in tubular reabsorption of magnesium
and calcium, or both.

In contrast to the excretion pattern of the
divalent ions, chloride ion excretion increases
and peaks during waking (active) hours and
decreases to its lowest levels during the sleeping
(inactive) hours (Figure 1). The circadian
pattern of chloride excretion observed in the
present study is consistent, with respect to peak-
ing times, with several other studies of mono-
valent ion excretion.**® The fact that chloride
(along with sodium and potassium) excretion
is lowest during the sleeping (inactive) hours
at the same time that magnesium and calcium
excretion is highest suggests several possibilities
for the role of the kidney. Assuming no active
changes in the renal handling of the ions in
question, the above findings might be the reflec-
tion of a circadian cycle in the filtered load of
those ions due to rhythmically fluctuating plasma
concentrations. This idea has not been generally
substantiated*** although some workers have re-
ported day-night differences in plasma mag-
nesium levels“? Two other possibilities are
(1) the rhythmic response of the kidney to other
regularly fluctuating blood levels of “renal regu-


AM 68-4

CONTRIBUTION OF ACTIVITY TO THE CIRCADIAN
RHYTHM IN EXCRETION OF MAGNESIUM
AND CALCIUM

Vincent Fiorica, Ph.D.
Mary Jo Burr, M.S.
Russell Moses, B.S.

Approved by Released by
J. Roserr Diuiz, M.D. P. V. Siecen, M.D.
Curer, Civ, AEROMEDICAL Feperat Air Surgeon
InsTITUTE
March 1968

Department of Transportation

FEDERAL AVIATION ADMINISTRATION
Office of Aviation Medicine



Taste 8. Plasma Concentrations of Magnesium and Calcium

in Resting and Active

Subjects.
‘Experimental Subjects (10)
After 60 min. After 60 min. After 120 min, rest
rest exercise following exercise
Magnesium 1.72 (0.04)* 1.67 (0.05) 1,67 (0.20)
MEQ/1
Calcium 6.36 (0.09) 6.57 (0.07) 6.48 (0.10)
MEQ/1

Control Subjects (10)

After 60 min.

After 120 min.

rest rest After 240 min, rest
Magnesium 172 (0.03) T72 (0.03) Li (0.03)
MEQ/1
Calcium 6.47 (0.09) 6.57 (0.11) 6.58 (0.09)
MEQ/1

*Values in parentheses are the S.E.

Neither magnesium nor calcium plasma Jevels were significantly different between experimental and control

group at times sampled.

lators” [aldosterone, “glomerulokininTM*], and
(2) an excretion/reabsorption regulating system
operating under the influence of an intrinsic
“renal clock.” ‘The involvement of the above
“mechanisms” as factors responsible for the
circadian excretion cycles is largely speculative,
and reflects the lack of data appropriate to the
question.

The results of this study suggest the general
conclusion that the circadian periodicity ob-
served in the excretion of magnesium and cal-
cium is primarily a reflection of the circadian

activity cycle. Data are not yet available, either
from the present study or from other studies
concerned with the problem, to answer the ques-
tion whether the excretion of magnesium and
calcium follows an endogenous circadian cycle.

Appropriate experiments to test this point are
necessarily difficult to accomplish’, and, in addi-
tion to controlling diet and activity factors, must
exclude all exogenous time cues. Further at-
tempts to distinguish an endogenous excretory
pattern for magnesium and calcium are presently
under consideration.



ACKNOWLEDGMENT

The authors express their appreciation to Paul Maeder and Edwin Wat-
kins for technical assistance, and to Miss Elinore Galerston for help with
data reduction. The work of Mr. W. V. Flores and Miss Betty Gatliff for
the research data drawings is also appreciated.

Qualified requesters may obtain Aviation Medical Reports from Defense Documentation
Center. The general public may purchase from Clearinghouse for Federal Scientific and
Technical Information, U.S. Dept. of Commerce, Springfield, Va. 22151


CONTRIBUTION OF ACTIVITY TO THE CIRCADIAN RHYTHM
IN EXCRETION OF MAGNESIUM AND CALCIUM

Vincent Fiorica, Ph.D., Mary Jo Burr, MS., and Russell Moses, B.S.
Physiology Laboratory, Civil Aeromedical Institute,
Federal Aviation Administration, Oklahoma City, Oklahoma

In man, the urinary excretion of the divalent ~

cations, magnesium and calcium, appears to con-
form to a general circadian periodicity.” How-
ever, there is little agreement among the reports
available on the time of day during which ex-
cretion levels are maximal and minimal. Al-
though the influence of diet on the excretion
levels of calcium and magnesium seems to be
well recognized, the effect of activity in generat-
ing or moderating the periodicity in excretion
has been given less attention.

Of the studies concerned with the “diurnal
variation” in excretion of magnesium and cal-
cium, several do not permit an accurate evalua-
tion of periodicity because of the use of too long
a collection interval? or the examination of only
a single 24-hour cycle.**"* It is the purpose of
this report to examine the reproducibility of the
circadian pattern in magnesium and calcium
excretion and to consider the role of physical
activity in this pattern.

Methods

The Circadian Pattern of Magnesium and
Calcium Excretion: Bight men, 16-17 years of
age, were given a standardized, balanced dietary
regimen for 144 hours (Table 1). Each meal
consisted of 8 ounces of Metrecal liquid, five
Metrecal wafers and 8 ounces of distilled water.

‘Tastz 1. Physical Characteristics of Subjects Used in
Circadian Excretion Studies.

Number 8

Height (cm) 177.5 (2.2)*
Initial weight (kg) 63.9 (2.4)
Final weight (kg) 63.7 (2.8)

*Values in parentheses are the S.B.

Meals were taken at 4-hour intervals over the
course of the study. This regimen provided
2,100 kcal per day or a daily mean for the group
of 32.9 kcal/kg body weight. Magnesium in the
diet amounted to 521 MEQ/day (daily mean
for the group, 0.82 MEQ/kg body weight) ;
calcium amounted to 277.2 MEQ/day (daily
mean for the group, 4.34 MEQ/kg body weight).
Activity of the subjects was not controlled by
design, but was uniform over the period of the
study. School activities occupied most of the
daytime hours; evenings were devoted to studies.
Subjects slept between 2200 and 0600 hours.

During the first day of the study subjects fol-
lowed the dietary routine, but collected no
samples. Urine sample collections were started
at 0600 on the second day of the study and con-
tinued at 4-hour intervals for 120 hours. Each
4-hour sample volume was recorded and a por-
tion of urine reserved for analysis. Samples
were analyzed for magnesium®, calcium’, and
creatinine." Chloride was measured by the
method of Cotlove‘ Because the creatinine ex-
cretion did not vary with a regular pattern in
this study, the excretion of magnesium, calcium
and chloride was expressed per unit excretion
of creatinine.

The Effect of Activity on the Excretion of
Magnesium and Calcium: Twenty male subjects
(20-29 years of age) were arbitrarily assigned
to an experimental group or to a control group.
Table 2 gives the physical characteristics of each
group. One control and one experimental sub-
ject were studied simultaneously. All tests were
conducted in the morning and all subjects were
post-absorptive at the start of the tests. The
format for experimental subjects was: 60 min-