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ABSTRACT

AN EVALUATION OF THE SEPARATION OF PROTEIN-BOUND
AND FREE SERUM MAGNESIUM BY THERMAL COAGULATION

By

Anna Mae Spencer

A thermal precipitation method for the separation of free from
protein-bound magnesium is described. It is a relatively simple,
reproducible and rapid procedure. It yields estimates of free mag-
nesium compatible to those obtained with slower, more cumbersome ultra-

filtration methods. The validity of the thermal precipitation tech-

 

‘ b l
nique was evaluated by showing that a plot of Mg Alb against Mg++

follows predictions based on the law of mass action.

AN EVALUATION OF THE SEPARATION OF PROTEIN-BOUND

AND FREE SERUM MAGNESIUM BY THERMAL COAGULATION

By

Anna Mae Spencer

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Pathology

1972

For My Mother
and

in Memory of My Father

ii

 

 

LJ

'5';

I"!
“D

b"
(I)

ACKNOWLEDGMENTS

My.appreciation and gratitude are expressed to my major professor,
Dr. A. E. Lewis, of the Department of Pathology. He provided unfailing
support and guidance and the necessary encouragement to make this
manuscript possible.

My sincere appreciation goes to Dr. C. C. Morrill, Chairman of
the Department of Pathology, for his administrative assistance and
guidance throughout my graduate studies. I am grateful to all the other
members of the Department of Pathology, who provided the necessary
instruction and support whenever requested.»

I am especially grateful to Dr. R. B. Foy, Technical Director of
the Clinical Laboratories at Edward W. Sparrow Hospital, for his instruc-
tion, suggestions and understanding throughout the course of my research.
His assistance concerning the biochemical methods and preparation of
this manuscript is deeply appreciated.

I wish to express thanks to Dr. W. E. Maldonado, Director of Labora-
tories at Edward W. Sparrow Hospital, for allowing me the opportunity
to pursue my graduate work and to make available the necessary technical
instrumentation for completion of my research.

As members of my committee, I would like to extend my appreciation
to Dr. R. T. Houlihan, Department of Osteopathic Medicine, and Dr. M.
Jones, Department of Pathology in Human Medicine, for their helpful

comments and suggestions.

iii

To my fellow employees in the Laboratory at Sparrow Hospital, I
am truly indebted for their assistance in collection and donation of
blood samples. Also, for supplying morale and technical assistance
when needed.

Finally, I am deeply grateful to my family and friends for their
encouragement, support, and understanding throughout the course of

this work.

iv

TABLE OF CONTENTS

INTRODUCTION. . . . . . . . . . . . . . . . . . .
REVIEW OF LITERATURE. . . . . . . . . . . .‘. . .
Distribution, Absorption, and Excretion of
Serum Forms of Magnesium . . . . . . . . .
Functions of Magnesium . . . . . . .'. . .
Abnormalities of Magnesium Metabolism. . .
Methods for Magnesium Determinations . . .
MATERIALS AND METHODS . . . . . . . . .'. . . .-.
Thermal Precipitation Method . . . . . . .
pH . . . . . . . . . . . . . . . . . . . .
Total Protein. . . . . . . . . . .'. . . .
Albumin. . . . . . . . . . . . . .'. . . .
Ultracentrifugation Method . . . . . . . .
Additions Curve. . . . . . . . . . . . . .
Absorbance Curve Linearity . . . . . . . .
Check for Magnesium Contamination. . . . .
Pooled Sera Specimens. . . . . . . . . . .
Atomic Absorption Spectrosc0py . . .‘. . .
RESULTS . . . . . . . . . . . . . . . . . . . . .
Results of the Pooled Sera Studies . . . .

The Effect of Increasing the (H+). . . . .

DISCUSSION 0 O O O O O O 0 O I O I b O O O O O O O >

S WY 0 O O O O O I O O O O O O O O O O O O O O

Page

10
13
13
l4
l4
l4
14
15
l6
l6
l6
l7
18
24
31*
47

53

Page
REFERENCES. 0 o o o o o o o o o o o o o o o o o o o o o op. o o o o 55
APPme o o o o o o o o o o o o o o o o o o o o o o o o 0 V o o o o o 61

VITA. O O O O O O O O O O O O O O O O O O O O I O O O O O O O O O O 65

vi

LIST OF TABLES
Table Page

1 Concentration of total magnesium and non-precipitable
fraction in individual serum samples . . . . . . . . . . . . l9

2 Concentration of total magnesium, non-precipitable and
ultrafiltrable fractions in individual serum samples . . . . 21

3 Summary of methods and comparison of normal values for
serum magnesium concentration. . . . . . . . . . . . . . . . 22

4 Summary of methods and measurements of ultrafiltrate
magnesium concentration in normal.serum. . . . . . . . . . . 23

5 pH, total protein and albumin value on the pooled serum
samples. 0 O O O 0 O 0 O O O O O O O I O O O O O O O O O O O 33

6 Results of No. 5 pooled serum samples used to determine
values for reciprocal plotting of l/Y against l/X-Y for
the line of least squares. . . . . . . . . . . . . . . . . . 34

7 Results of No. 6 pooled serum sample used to determine
values for reciprocal plotting of l/Y against 1/X-Y for
the line of least squares. . . . . . . . . . . . . . . . . . 35

8 Results of No. 7 pooled serum sample used to determine
values for reciprocal plotting of l/Y against l/X-Y for
the line of least squares. . . . . . . . . . . . . . . . . . 36

9 Results of No. 8 pooled serum samples used to determine
values for reciprocal plotting of l/Y against l/X-Y for
the line of least squares. . . . . . . . . . . . . . . . . . 37

10 Results of No. 9 pooled serum samples used to determine
values for reciprocal plotting of l/Y against l/X-Y for
the line of least squares. . . . . . . . . . . . . . . . . . 38

11 Results of No. 10 pooled serum samples used to determine
values for reciprocal plotting of l/Y against l/XeY for
the line of least squares. . . . . . . . . . . . . . . . . . 39

12 Results of No. 11 pooled serum samples used to determine

values for reciprocal plotting of l/Y against l/XeY for
the line of least squares. . . . . . . . . . . . . . . . . . 4O

vii

Table

13

14

Page

Results of No. 12 pooled serum samples used to determine
values for reciprocal plotting of l/Y against l/X-Y for
the line of least squares. . . . . . .7. . . . . . . . . .'. 41

Calculated values from pooled sera data giving the line of
least squares, binding sites for magnesium on each albumin'
molecule, dissociation constant (KMg Prot)’ and pK for each
sample 0 C O 0 O 0 O O O O (1 O O 9 O O O O O I O 0 0 O O 'l O 4 6

viii

Figure

LIST OF FIGURES

Serum magnesium fractions as revealed by ultrafiltra-
tion and thermal precipitation . . . . . . . . . . .

Results of additions curve to test linearity for mag-
nesium absorbance curve and the linearity of serum

dilutions in aqueous solution. . . . . . . . . . . . .

Results of the plotting of an absorbance curve using
magnesium standards up to 4.5 mM/L . . . . . . . . . .

Effect of increasing the number of hydrogen ions on

the slope of the line of least squares when reciprocal

plotting O O 0 O O O 0 e e F. 0 O O O 0 Q .0 O I I O O .

Plotting of the line of least squares for the pooled
serum samples Nos. 5, 6, and 7 . . . . . . . . . . .

Plotting of the line of least squares for the pooled

serum samples Nos. 8, 9, and 10. . . . . . . . . . . .

Plotting of the line of least squares for the pooled

serum samples Nos. 11 and 12 . . . . . . . . . . . . .

Plotting of the average line of least squares for the
pooled serum samples Nos, 5, 6, and 7; 8, 9 and 10;
l]- arld 122‘ O 0 r O O 0 h 0 0 0 0 O h o 0 O 0 O 0 O 0

ix

Page

25

26

27

32

42

43

44

45

INTRODUCTION

The purpose of this investigation is to describe a simple,
accurate and reproducible thermo-precipitation method for the separa-
tion of ionized magnesium from protein-bound magnesium. The results
of this method were compared to an ultrafiltrate technique which utilized
centrifugation as a means of separation. All the magnesium fractions
were measured by atomic absorption spectroscopy. The thermo-precipitation
method was evaluated by reciprocal plotting to see if it followed the
law of mass action.

Magnesium is the fourth most abundant cation in the body and second
in concentration intracellularly. About one-half of the total body
magnesium is contained in bone. The remainder is almost equally dis-
tributed between muscle and non-adipose soft tissue. The human erythro-
cyte has only one—fourth of the magnesium concentration of other cells
but three times the magnesium concentration of plasma. Normal dietary
intake, serum protein concentration, thyroid and renal function are
important factors in the regulation of magnesium concentrations in
intracellular tissues and extracellular fluids. However, the factors
that regulate ultrafiltrable magnesium are incompletely understood.

Magnesium exists in the plasma in three forms: ionized, complexed
and protein-bound in chemical equilibrium. This equilibrium is main-
tained according to the law of mass action. The ultrafiltrable fraction
consists mainly of the ionized form. This is the physiologically
active form which can be determined by direct measurement in an

1

2
ultrafiltrate. The complexed portion, the remaining small fraction of
the ultrafiltrate, consists of several components (citrate, phosphate,
bicarbonate, etc.), none of which has a known physiological function.
The protein-bound fraction is non—filtrable. Alterations in these
fractions may accompany changes in magnesium metabolism. These may
result from altered intake, absorption or excretion of the ion, and
shifts of the ionized magnesium or movements of fluid between the
extracellular spaces. Any of these changes may give rise to disturb-
ances of electrolyte balance.

Over the last 15 to 20 years, wide discrepancies have been reported
for the distribution of human plasma magnesium fractions. This is, in_
all probability, due to differences both in analytic methods and in
techniques utilized for the isolation of the ultrafiltrate. The most
important advance in the understanding of the biological role of mag-
nesium and other trace metals has been the develOpment of atomic absorp—
tion spectroscopy (AAS). It provides a simple, precise analytical
method, whereby multiple assays of minute amounts of magnesium can be
made both reliably and quickly. This has already led to a better under-

standing of the role of magnesium metabolism in human disease.

REVIEW OF LITERATURE

Distribution, Absorption, and Excretion of Magnesium

 

Magnesium has an atomic weight of approximately 24 and is divalent.
The adult human body contains approximately 2,000 mEq (21 to 28 grams)
of magnesium and is the fourth most abundant cation of the body.21
Bone contains about one—half of the total body magnesium as complexed
salts, Mg (P04)2 and MgCO3, in the lattice tissue.2’21 The remaining
magnesium (predominantly as an intracellular constituent) is almost
equally distributed between muscle and non-adipose soft tissue, such
as liver, kidney, spleen and brain.2 Extracellular magnesium in the
plasma and other body fluids comprises 0.5 gm or less. Of the non-
osseous tissues, liver and striated muscle have the highest concentra-
tions (15-20 mEq per kilogram), about four times the amount found in
erythrocytes (4.4—6.0 mEq per liter).67 The normal serum level-(1.6—
2.1 mEq per liter), determined by atomic absorption spectrosc0py
methods, is roughly one—third of the erythrocyte concentration.67
Under normal circumstances these concentrations are maintained by
sensitive control mechanisms. Disturbances in magnesium metabolism
may occur as a result of alteration in dietary intake, serum proteins,
renal and thyroid function, and transfer of ions intracellularly or
extracellularly. However, there is relatively little knowledge of the
frequency of these shifts and the factors that govern them.4

In volume-distribution studie822’56’76

utilizing the radioactive
isotOpe Mg 28, the exchangeable magnesium in 24 hours was much less than

3

4

the total amount in the body. Indications are that a large quantity
of magnesium is apparently "fixed" and not readily available to the
metabolic pool.4’22’56

Most of the dietary magnesium (20—40 mEq per day) in a normal diet
is obtained from green vegetables high in chlorophyll.55 Spices, nuts,
whole grains and legumes also have high magnesium concentrations.55
Approximately one—third (10 mEq per day or 0.30-0.35 mEq per kilogram
of body weight per day) of ingested magnesium must be absorbed to main—
tain a positive balance in normal individuals.31 Studies by Ross and
Aikawa indicate that magnesium absorption occurs in the small intestine
with no evidence of active transport of this ion.50 In magnesium
absorption peak levels are seen in,2 to 8 hours24 with maximum absorp-
tion at 4.0 mEq per liter concentration.l Both dietary phosphate and
calcium levels influence the amount absorbed, possibly as a result of

competition for a common absorption pathway 17.24

Excess magnesium
salts ingested are not absorbed, and the volume of water retained in
the intestinal lumen maintaining osmotic equilibrium accounts for their
cathartic action.

Magnesium absorbed or released from cells is predominantly excreted
through the kidneys. The amount required to maintain positive balance
is 10 mEq per day. Glomeruli allow the unbound form to filter through
but 94 to 96% is reabsorbed in the proximal portion of the distal
tubule.4 The mechanisms controlling renal excretion are at least
partly independent of those controlling excretion of calcium and potas-
sium ions.4 Under conditions of magnesium deprivation, renal conserva-
tion becomes prominent, limiting urinary losses to less than 1 mEq per

17'

day. ‘ The rate of magnesium excretion apparently follows a diurnal

pattern, but this may reflect responses to the ingestion of carbohydrates

5
or relative metabolic acidosis causing a diminished net tubular
reabsorption.33 Diminution of net tubular reabsorption of magnesium

is also seen following the consumption of alcohol.46

Serum Forms of Magnesium

 

Magnesium, like calcium, in human serum exists in three forms:
diffusible (ionized and complexed) and non—diffusible in chemical
equilibrium. The diffusible forms consist predominantly of ionic_
magnesium (55% of total), the physiologically active state. A small
fraction (10-15% of the total) exists mainly as soluble but undissoci-
ated complexes with citrate, phosphate, and bicarbonate, may play

a role in bone formation.l3’71

The non-diffusible fraction, magnesium
proteinate (32% of total),5 is reversibly bound to the serum proteins.71
Solutions containing proteins and the divalent cations of mag-

nesium and calcium do not appear to follow the laws of electrolyte
distribution (Donnan equilibrium) with respect to ion transport or
semipermeable membrane systems. This effect may be explained by the
formation of a complex cation proteinate.l3 Copeland and Sundermanl3
suggested that the magnesium proteinate in the serum acts as a dissoci-
ated salt according to the law of mass action. Prasad, Flink and
Zinneman45 found that normally albumin and alpha-2 globulin bind mag-
nesium, whereas calcium binds to albumin and beta-globulins. In multiple
myeloma, magnesium binds to the alpha-l globulins and in the hyper-
proteinemias to the beta-globulins. Approximately 0.012 mEq of magnesium
binds to 1.0 gram of albumin, while 0.008 mEq of magnesium binds to

1.0 gram of alpha-2 globulin.l3’45

There is a positive correlation
between concentration of non—ultrafiltrable magnesium and concentration

of total proteins.45

1.;
b0
7-.-
ma

 

nu

~l

6

Magnesium binding is a reversible process dependent on plasma pH.l4
A decrease causes the release of magnesium from the protein-binding
sites and allows the association of hydrogen ions to take place, result-
ing in an increase in the ionic fraction. 0n the other hand, an increase
in pH causes more binding sites to become available for magnesium and

the process is reversed. At a constant pH, magnesium follows the law

of mass action, the ionic magnesium and protein-bound (magnesium
proteinate) have a constant ratio as indicated by the McLean-Hasting

equation.13’36

MgProt Z Mg++ + Prot=

(MgH) (Pret') a
(MgProt)

 

K

Thus, any increase in ionic Mg++ will cause an increase in protein-
bound magnesium to re-establish a constantratio, and vice versa.

Magnesium Shares some important interrelationships with the other
major biologic cations.74 Ions of monovalent potassium and divalent
magnesium are generally present in high concentrations within cells,
whereas the intracellular ion concentrations of monovalent sodium and
divalent calcium are low. These ratios are inverted in extracellular
fluids. The intracellular rations log (K+)/(Na+) and log (Mg++)/(Ca++)
are directly related. In general, the higher the metabolic activity of
a cell, the greater the ratios (Mg++)/(Ca++) and (K+)/(Na+ .67’74
There is a reciprocal relationship between serum Mg++ and Ca

Another striking relationship exists between the intracellular
concentration of magnesium and phosphorus. When the content of intra-
cellular magnesium decreases in the inactive tissue, such as skin and

erythrocytes with a corresponding increase in the very active tissues,

such as liver, brain and striated muscle, there is a concomitant

7

increase in the cellular content of phosphorus.67’74

These relationships
are in keeping with the major requirement of magnesium activity and

emphasize its biologic importance.

Functions of Magnesium

 

Magnesium ions serve as activators for a number of important intra-
cellular enzyme systems engaged in hydrolysis and transfer of phosphate
groups67 such as alkaline phosphatase (from erythrocytes and bone),
prostatic acid phosphatase, hexokinase, creatine kinase and enolase.
Systems concerned with reactions involving adenosine triphosphate (ATP)
include glucose utilization, fat, protein, nucleic acid and coenzyme
synthesis, acetate and formate activation.67

Magnesium is required as a cofactor for intracellular mitochondrial
oxidative phosphorylation.43 The highly organized macro-molecular
structures of DNA, RNA and ribosomes are stabilized by the presence of
this ion. Maximum stabilization of DNA to thermal disruption occurs
when a 1:1 stoichiometric relationship is reached between equivalents
of magnesium ion and DNA phosphate residues.l6 Magnesium is essential
for the structural integrity of ribosomal components, and the subunit
aggregation or dissociation is critically dependent on this
concentration.4

Variation of magnesium concentration is further involved in protein
synthesis by contributing to the binding of messenger RNA to the 708
ribosome9 and in the interaction of S-RNA with a site on a 508 subunit
of the 708 ribosome.lo DNA synthesis and degradation require the magnesium
ion. The amino acid activating systems are dependent upon this divalent
ion in which a specific amino acyl S-RNA synthetase forms a complex with
its amino acid in the presence of ATP, with well-defined Mg++zATP

ratios.67

 

 

 

r-v

'1

Ir;

J.
6-4

U

[‘1

8
Magnesium and its interrelationships with the other biologic cations
helps to maintain electrolyte balance.67
Magnesium participates in bone formation with the deposition of its

salts, Mg (P0 and Mg C0 , in the lattice tissue underneath the surface
3 3

£32

bound ion layer.2’2'l

Magnesium aids in maintenance of cardiac rhythm and has a function
in the regulation of blood pressure and vasodilationfl’67
Magnesium and calcium have complex interdependent influences on

the regulation of neuromuscular excitability. Depletion of either ion
leads to increased neuronal excitability and enhanced neuromuscular
transmission by decreasing end-plate potential and by blocking the
release of acetylcholine.67 Studies by Fatt and Katz,19 utilizing an
intracellular electrode with frog muscle, observed that on reducing the
+1- . H- '
Ca and raising the Mg concentrations in Ringer 5 solution, the neuro-
muscular junction could be blocked and the end-plate potentials result-
ing from nervous stimulation could be reduced. Boyd and Martin6’7
demonstrated the same effect on mammalian muscle. The main action of
Mg++ is to reduce the amount of acetylcholine liberated by the nerve
impulse. The action of Ca++ is not confined to antagonizing the effects
++ ,, -H-
of excess Mg for increasing the concentration of Ca in normal
Ringer’s solution actually increased the size of end-plate potential
in response to a nerve stimulus. Presumably it increases the amount of
, -H- -H- .
acetylcholine liberated at the junction. Ca -Mg antagonism can be
explained by these ions competing for some site or carrier molecule in
the nerve endings.7
In the contractile process of muscle, the degree to which the,

enzyme ATPase is affected by the Mg++ and Ca++ concentration will depend

on the degree to which it is complexed with free myosin or with

9
actomyosin. Significant in muscular liberation of necessary energy are
the observations that Ca++ stimulates the ATPase activities of both
proteins, Mg++ inhibits myosin ATPase but stimulates actomyosin ATPase
activity. At pH 7.0, the phosphate portions of ATP are completely
dissociated so that the molecule bears 4 negative charges and may bind
15

divalent cations such as MngTP or CaZATP.

Abnormalities of Magnesium Metabolism

 

In magnesium deficiency states, measurement of serum levels is the
quickest, simplest and most effective initial approach, followed by
measurement of erythrocyte magnesium or urinary excretion. Most of the
symptomatic magnesium deficiencies have serum concentrations less than
1 mEq per liter with 25% or more cellular depletion.35 It leads to
neuromuscular dysfunction manifested by hyperexcitability and is some—
times accompanied by tetany, convulsions, vertigo, irritability, depres-
sion, psychotic behavior, tremors, muscular weakness and cardiac
arrhythmias. All these disturbances are reversed by magnesium

therapyfl’67

A human tetany syndrome with hypomagnesemia can occur
in the presence of normal values for other serum electrolytes and acid-
base balance.67 Symptomatic magnesium deficiencies apparently are not
the result of dietary inadequacy, but are associated with malabsorption
syndromes, acute pancreatitis, chronic alcoholism and delirium tremens,
chronic renal failure with defective renal tubular reabsorption, hyper—
parathyroidism and hyperaldosteronism.67

The manifestations of hypermagnesemia in man were first noted as
a result of pharmacologic studies on the properties of this ion as a

23,42

potential anticonvulsant and anesthetic. In symptomatic hypermag-

nesemia, the toxic responses associated with impairment of neuromuscular

10

transmission start to occur at serum concentrations in excess of 4 mEq
per liter. Approaching 10 mEq per liter there is loss of deep tendon

reflexes, peripheral vasodilation with hypotension, and altered cardiac
conduction with complete heart block or respiratory paralysis at levels

47 69
’ Increased serum values are associ-

near or above 15 mEq per liter.
ated with the acute ingestion of toxic doses of magnesium sulfate

(Epsom salts and cathartics) or its use in treatment of eclampsia,
severe diabetic acidosis, Addison's disease, but most frequently occurs
with chronic renal insufficiency where inadequate glomerular filtration,
results in retention.“67 Supposedly, effective treatment depends on
antagonizing the sedating effect of magnesium with the calcium ion4

or by hemodialysis.4O

Methods for Magnesium Determinations

 

Serum magnesium determinations should be performed on non-hemolyzed
blood samples drawn in the fasting state. Samples are stable for
several days if the serum is separated from the erythrocytes and stored
at 4° C. The methods utilized most frequently to determine total
magnesium are:

(l) Precipitation as magnesium ammonium phosphate.

(2).Titan yellow dye in alkaline solution.

(3) Complexometric titrations with ethylenediaminetetraacetic
acid (EDTA) and eriochrome black T.

(4) Complexing fluorometric methods.

(5) Flame emission.

(6) Atomic absorption spectroscopy.

The determination of magnesium by precipitation as magnesium ammonium
phosphate dates back to 1877. Calcium is first removed by precipitation

as oxalate and the magnesium in the supernate then precipitated as a

ll

double salt, magnesium ammonium phosphate. The magnesium is first pre-
cipitated in slightly acid solution as MgHPO4 and then converted to
M'gNH4P04 by addition of NH4OH. The phosphate in the precipitate is
determined colorimetrically by the molybdenum blue method utilizing
hydroquinone or amino-naphthol-sulfonic acid as the reducing agent.72

A direct magnesium method depends on the formation of a red lake
with the dye, titan yellow (Clayton yellow, Thiazole yellow), in an
alkaline solution. The dye is specifically absorbed on the surface of
colloidal particles of magnesium hydroxide upon alkalinization with
sodium hydroxide. The resulting dye lake color is intensified and
stabilized with the aid of polyvinyl alcohol. The color complex is
read spectrophotometrically at 540 nm.54

An indirect measurement of magnesium is obtained by the disodium
ethylenediaminetetraacetate (EDTA) titration of deproteinized calcium-
free extracts of plasma or whole blood. The alkaline earth metal indi-
cator Eriochrome black T, in ammonium hydroxide, is used with the visual
end point as the change from royal blue to aquamarine in white fluorescent
light.70 Also, photometric methods can be used that are based on the
fact that the color intensity with eriochrome black T is proportional
to the concentration of magnesium present.11

In the complexing-fluorometric methods, magnesium ions and 8-hydroxy-
5 quinoline sulfonic acid forms a chelate. The metal complex can be
read fluorometrically when excited at 380 to 410 nm.54

The flame emission photometric determination of magnesium has led
to difficulty due to interference from high concentrations of sodium.
Preliminary separation of the magnesium with 8-hydroxyquinoline or as
magnesium ammonium phosphate plus the use of a photomultiplier and
acetylene-air flame has achieved sufficient sensitivity by reducing the

interfering sodium ions.20

12
Magnesium measurement by atomic absorption spectrosc0py depends
on the amount of light absorbed at the wavelength (285.2 nm) of the
resonance line by the unexcited atoms of the element. The sample is
sprayed into a flame to provide a reproducible and clearly defined cloud
of atoms. A hollow cathode lamp that emits the line spectrum of mag-

nesium is used as the energy source.52’61’75

Ionic magnesium determinations require the separation of diffusible

from the non-diffusible (magnesium proteinate) by either ultrafiltra-

44,63

tion or ultracentrifugation.8 Ultrafiltration techniques utilize

cellophane membranes of specific pore size with either hydrostatic

pressure32 or centrifugal force.“’63

MATERIALS AND METHODS

Twenty milliliters of venous blood was collected by Vacutainer
technique into #4710 blood tubes that contained no anticoagulant
(less than 9 pg magnesium content). The blood was then allowed to
clot for at least one hour, after which it was centrifuged, serum
removed from the cells, stoppered and serum recentrifuged.

The first study was designed to measure the total magnesium and
the thermal precipitation fractions in individual serum samples. The
thermal precipitation method requires 4 ml. of serum. The remaining
portion of the serum (about 6 ml.) was utilized for the measurement of

total magnesium, pH, total protein and albumin.

Thermal Precipitation Method

 

Preparation of the coagulum's supernatant consisted of heating 4
m1. of serum in a 10 ml._glass-stoppered Pyrex centrifuge tube (140 x
17 mm.) at 100° C. (:_3° C) in boiling water bath for five minutes.73
The coagulum formed was then mashed with a stirring rod until the solid
mass was broken into fine pieces. The tube was centrifuged at 3500
rpm for 10 minutes yielding 110 to 1.5 ml. of clear supernatant used for
non—precipitable magnesium studies.

The supernatant magnesium value was considered the non-precipitable
or free magnesium. This value subtracted from the total serum concen-
tration equals the value for the thermoprecipitable or protein-bound

magnesium.

l3

14

[3:

All pH's were measured on the Radiometer-blood micro system EMS-3.

Total Protein

 

Total protein was measured with a standard AutoAnalyzer N-Method
using a modified biuret reaction.18 Copper in alkaline solution forms
a purple complex with the peptide linkages of amino acids in,a protein.
The protein stream is mixed with a biuret reagent and the developed

color is measured at 550 nm in a 15 mm. flow well.

Albumin

An adaptation of the bromocresol green procedure for the auto-
analyzer was utilized for albumin measurement. The albumin stream is
mixed with bromcresol green (BCG) and the developing color due to

albumin-dye binding is measured at 600 nm.27

Ultracentrifugation Method

 

The second study compared the magnesium concentrations in the non-
precipitable fraction of the thermal precipitation technique and the
ultrafiltrate fraction in an ultracentrifugation method. A modifica-
tion of Prasad's44 ultracentrifugation method was utilized to obtain
the ultrafiltrate. A double layer of narrow-meshed white gauze, 15 x
15 cm., was suspended inside a 40 ml. Kimax glass-steppered centrifuge
tube (130 mm. x 27 mm.). The gauze was secured around the outside of
the tube by means of two small rubber bands. Any excess of gauze was
trimmed and then a piece of plastic adhesive tape, 1 inch wide, was
placed around the tube so that the gauze and the rubber bands were
firmly adherent to the glass tube. A piece of ce110phane casing (dialy-

sis tubing, Union Carbide, Food Products Division), 15 cm. long, was

 

15
moistened in distilled water for ten minutes. The outside of the casing
was wiped with a piece of gauze and then Opened by simply.blowing into
the lumen. One end was double—folded lengthwise and secured with a
figure-8 knot. This was then placed inside the tube supported on the
gauze as a U-tube. The casing was opened by blowing into the outer
lumen. Immediately afterward, 5 ml. of serum were delivered inside the
casing as quickly as possible, making sure not to touch the pipette
to the casing. The outer end of the casing was securely knotted and
attached to the outside of the tube by tape and the tube was stoppered
with a Size 3 or 4 rubber stepper. The stopper helps to keep the C02 N

tension inside the tube constant and also gives extra support to the

 

gauze to prevent it from being drawn to the bottom while centrifuging.
The tube was then centrifuged in an International IEC Model K centri-
fuge for 75 minutes at 2000 rpm. This yields about 1 to 1.5 ml. of

protein-free ultrafiltrate.

Additions Curve

 

Equal portions (0.25 ml.) of each intermediate magnesium standard
were added to 0.25 ml. aliquots of a Control Serum (XPT-56) and two
separate individual serum samples. Mixed specimens were diluted 1:25
with deionized water. Undiluted Control Serum (XPT-56) and the two
individual serum samples were diluted 1:50. Intermediate magnesium
standards were diluted 1:50 with deionized water to give the working
standards: 0.83 mM/liter, 1.25 mM/liter, and 1.65 mM/liter, in determin-
ing the absorbance curve. Total magnesium.was determined on each addi-
tion specimen and plotted against percentage absorbance. The lineari-
ties of these curves were compared to the absorbance curve of the pure

standards.

 

C)

r)

’L’l

16

Absorbance Curve Linearity

 

To determine linearity, an absorbance curve was prepared using
dilutions up to 4.5 mM/liter of magnesium. The absorbance of the

standards was plotted on the ordinate and concentrations on the abscissa.

Check for Magnesium Contamination

 

The glassware used was all acid washed. All glassware and non-
glass materials used for analysis, such as Vacutainer tubes, dialysis
tubing, gauze, disposable plastic vials, deionized water, etc., were
checked for amount of magnesium contamination. All demonstrated negli-

gible magnesium concentration.

Pooled Sera Specimens

 

Approximately 90 ml. of fresh pooled sera (less than 4 hours from
drawing) was recentrifuged twice and kept stoppered. The total protein,
albumin and pH were measured. The pH was adjusted to 7.35 to 7.45

in three pooled sera samples by "high-gas" bubbling (12% CO and 88%

2
oxygen). In the remaining samples, the pH was adjusted by addition
of either 0.006 ml. or 0.012 ml. of 2N HCl per ml. of serum to maintain

pH of supernatant to 8.1 :_0.10 or 7.6 :_0.15 after CO loss resulting

2
from heating in thermal precipitation procedure.34
Five-milliliter aliquots of pooled sera were pipetted into 10 ml.
glass-stoppered centrifuge tubes. To all but the first sample, 0.1 ml.
of various concentrations of magnesium chloride were added to the appro-
priate tube. The first sample was a representative sample of pooled
sera. Magnesium standards were prepared from a stock magnesium chloride

solution (100 mM/liter magnesium: 20.32 grams MgCl '6H20/500 ml.

2
deionized H20).

17
One-milliliter aliquots were removed from each of the addition
mixtures for total magnesium assay. The remaining 4 ml. of each mixture
was utilized in the thermal precipitation method. All serum and super-
natant samples were diluted 1:50 with deionized H

20 and assayed for

magnesium content by atomic absorption spectrosc0py.

Atomic Absorption Spectroscopy

 

All serum and ultrafiltrable and non-precipitable magnesium samples
were assayed on the Perkin-Elmer Model 305 Atomic Absorption Spectro-
photometer (Perkin-Elmer, Norwalk, Connecticut). The energy source
of the 285.2 nm was supplied with an operating current of 15 milliamps.
The burner was supplied with acetylene fuel at 8 to 9 pounds per square
inch and air at 30 pounds per square inch. Sample values were.recorded
on the Perkin-Elmer Recorder #165. Sensitivity is about 0.007 ug./ml.
magnesium for 1% absorption under standard conditions. The working
range for magnesium is linear up to concentrations of approximately
0.5 Lg./ml. in aqueous solutions.

Unknown samples to be assayed by atomic absorption spectro-
scopy were diluted 1:50 with double-deionized water and concentra-
tions determined by the following method:

The absorbance of the standards was plotted on the ordin-

ate and concentrations on the abscissa. The concentra-

tions of the unknowns were determined from their absorbance
which was calculated from their percent absorption.

RESULTS

The results of individual serum concentration of total magnesium
and the fractions obtained by thermal precipitation are given in Table l.
The 50 specimens analyzed were reported in mM per liter with the percent
nonprecipitable magnesium being calculated. Total serum proteins were
measured in all samples. Albumin concentration was determined in about
one-half of the specimens. The mean (E), standard deviation (S.D.)
and the range of all results were calculated and recorded. Serum pH
ranged from 7.35 to 7.55, while the pH of the supernatant after heating
was 8.8 to 9.0.

Table 2 contains the comparative concentration in individual serum
samples of total magnesium, nonprecipitable fraction by the thermal
precipitation method and the ultrafiltrable fraction by the ultracentri-
fugation method of Prasad. The percent free magnesium was calculated
and compared in the thermal precipitation and the ultrafiltration
methods. The mean (R), standard deviation (S.D.) and range were calcu-
lated and recorded. Serum pH ranged from 7.38 to 7.48; nonprecipitable
fraction, pH from 8.8 to 9.0; and ultrafiltrate, pH ranged from 7.60
to 7.80.

Summary of the reported methods and comparison of normal values
for serum magnesium concentrations are given in Table 3, along with the
results of this study.

Table 4 lists a summary of the reported methods and mean values of the
measurements of total and ultrafiltrable magnesium concentration in

18

19

Table 1. Concentration of total magnesium and non-precipitable fraction
in individual serum samples

 

 

 

 

 

Magnesium in mMgper liter % Non-
Non- Protein- Proteins in gm/dl. precipitable
No. Total precipitable bound Total Albumin Mg.
1 0.90 0.72 0.18 7.60 79.5
2 0.94 0.69 0.25 6.80 73.0
3 0.87 0.67 0.20 7.40 76.5
4 0.89 0.67 0.22 7.00 75.0
5 0.84 0.62 0.22 8.10 73.5
6 0.85 0.55 0.30 7.50 64.0
7 0.86 0.61 0.25 7.20 70.5
8 0.95 0.65 0.30 7.70 68.0
9 0.89 0.65 0.24 7.60 73.0
10 0.92 0.64 0.28 6.80 69.5
11 0.88 0.62- 0.26 7.40 71.0
12 0.84 0.63 0.21 7.00 74.5
13 0.79 0.54 0.25 8.10 67.5
14 0.84 0.53 0.31 7.50 64.0
15 0.84 0.60 0.24 7.20 71.0
16 0.92 0.63 0.29 7.70 69.0
17 0.84 0.56 0.28 7.00 67.0
18 0.96 0.63 0.33 8.10 65.0
19 0.88 0.63 0.25 7.00 71.0
20 0.91 0.61 0.30 6.90 66.5
21 0.821 0.58 0.24- 6.80 71.0
22 0.83 0.59 0.24 7.70 71.0
23 1.08 0.73 0.35 8.30 3.75 67.5
24 0.87 0.55 0.32 6.00 63.5
25 0.89 0.58 0.31 5.70 3.70 65.0
26 0.78 0.53 0.25 5.60 3.25 68.0
27 0.93 0.62 0.31 5.95 3.50 66.5
28 0.87 0.63 0.26 4.80 2.60 72.0
29 0.84~ 0.58' 0.26 6.00 69.0
30 0.88 0.57 0.31 8.00 64.5

20

Table 1 (cont'd.)

 

 

Magnesium in mM per liter % None
Non- Protein- Proteins in gm/dl. precipitable

 

 

 

 

No. Total precipitable bound Total Albumin Mg.
31 1.54 1.19 0.35 4.30 2.70 77.0
32 0.94 0.64 0.30 5.85 3.85 68.0
33 0.95 0.63 0.32 5.55 3.30 66.0
34 0.95 0.61 0.34 6.20 3.80 64.0
35 0.98 0.68 0.30 5.70 3.30 69.0
36 1.00 0.62 0.38 7.85 4.90 61.5
37 1.00 0.68 0.32 6.20 4.10 68.5
38 1.10 0.66 0.44 7.50 4.10 59.5
39 0.88 0.62 0.27 5.40 3.25 69.5
40 0.85 0.50 0.35 8.30 4.60 59.0
41 0.86 0.55 0.31 6.70 3.10 63.0
42 0.89 0.54 0.35 6.00 4.15 60.5
43 0.76 0.54 0.22 4.30 2.30 70.5
44 0.92 0.58 0.34 7.30 4.00 63.0
45 0.94 0.57 0.37 7.30 3.90 60.5-
46 0.96 0.59 0.37 7.20 4.00 61.5
47 0.81 0.58 0.23 7.20 3.90 72.0
48 0.88 0.56 0.32 7.80 63.5
49 0.86 0.56 0.30 7.70 65.0
50 0.92 0.63 0.29 7.10 3.90 68.5
i 0.91 0.62 0.29 6.90 3.65 67.9
S.D. 0.17 0.10 0.05 1.00 0.62 4.7
Range

0.76- 0.50- 0.18- 4.3— 2.3- 59-

1.54- 1.19 0.44 8.3 4.9 79.5

pH of the serum ranged from 7.35 to 7.55; non-precipitable fraction from
8.8 to 9.0

 

21

 

 

 

 

 

.x; *1I_‘. flfl
'6

 

 

Table 2. Concentration of total magnesium, non-precipitable and ultra-
filtrable fractions in individual serum samples
Magnesium in mM per liter % free magnesium
Non— Ultra- Non- Ultra-
No. Total precipitable filtrable precipitable filtrable
1 0.840 0.560 0.695 67.0 83.0
2 0.880 0.625 0.730 71.0 83.0
3 0.910 0.605 0.710 66.5 78.0
4 0.825 0.585 0.725 71.0 87.0
5 1.080 0.730 0.780 67.5 72.0
6 0.865 0.550 0.620 63.5 72.0
7 0.780 0.530 0.555 68.0 71.0
8 0.925 0.615 0.680 66.5 73.5
9 0.865 0.625 0.665 72.0 77.0
10 0.835 0.575 0.650 69.0 78.0
11 0.875 0.565 0.615 64.5 70.0
12 1.540 1.185 1.195 77.0 77.5
13 0.945 0.625 0.640 66.0 68.0
14 0.980 0.675 0.720 69.0 73.5
15 1.000 0.685 0.750 68.5 75.0
16 0.885 0.615 0.690 69.5 78.0
17 0.845 0.500 0.655 59.0 77.5
18 0.865 0.545 0.575 62.0 67.0
19 0.885 0.535 0.600 60.5 67.5
20 0.760 0.535 0.540 70.0 70.0
i 0.92 0.62 0.69 67.4 74.9
S.D. 0.16 0.14 0.14 4.2 5.5
Range 0.76- 0.50— 0.54- 62-77 67-87
1.54 1.19 1.20

pH of the serum ranged from 7.35 to 7.48; non-precipitable fraction from
8.8 to 9.0; ultrafiltrable fraction ranged from 7.6 to 7.8.

 

22

 

 

 

 

Table 3. Summary of methods and comparison of normal values for serum
magnesium concentration
Mean Magnesium
conc. mEq/L No. of
Author Method Serum Plasma Subjects S.D.
41 .

Smith (1950) Spectrographic - 1.57 103 0.43

Orange (1951)70 Titan yellow 1.87 - 45 -
photometric

Hunter (1958)29 Eriochrome Black 1.67 - 34 -
T and murexide

Wacker (1957)66 Emission flame 2.05 - 14 0.18
spectrOphotometric

Van Fossan (1959)65 Emission flame 1.67 - 73 0.17
spectrophotometric

Schachter (1959)53 Fluorometric - 2.05 33 0.22

Vallee (1960)64 Emission flame 2.00 - 30 0.16
spectrophotometric

Alcock (1960)3 Emission flame - 1.66 76 0.07
spectrOphotometric

Schachter (1961)54 Fluorometric 1.80 - 14 0.26

Montgomery (1961)37 Emission flame 1.70 - 46 -
spectrophotometric

Heaton (1962)26 Atomic absorption - 1.64 176 0.12
spectrophotometric

Wallach (1962)70 EDTA titration - 2.00 77 0.15

Ryan (1967)51 Titan yellow 1.92 - 31 -
Emission flame 1.75 — 31 -
Fluorometric 1.76 - 31 -
Atomic absorption 1.71 - 31 —

Thin (1967)62 Atomic absorption 2.13 - 138 -
spectrophotometric

Jackson (1968)30 Atomic absorption 1.78 — 5100 0.15
spectrophotometric

Present study (1972) Atomic absorption 1.81 — 50 0.17

SpectrOphotometric

 

 

23

 

muse:

 

 

 

 

 

 

4.46 a~.H Hm.a mo .uaa Hmaumas
commum mo hmoomouuommm
mm wm.a Hm.H coaumw:MHuusoomuuHD coaunuomnm oHaou< om AHanv moouw mammoum
ouommoum haoumouuomam Amomav xooomom
6.6m ~N.H ao.H owcaumm m>auamom anfiumuomnm UHaou< ma we can comuuonom
mmuoa>ma mo coaumuu nmAammHV “momma
m.mo 6H.H RN.H uaammuuas smamasoz sous.» cause as. amauo>aam
as mAH.H Hw.H coaumwsmauuamomuuas sesame amuse om osAoomHV swamps
pwom oHcowaom
cofiuosm .mm .88 ona onwnamocfiamlq.m.a %n m mHANmmHv psmaumpcom
no m~.H RN.H m was coaaoaflou om mzmz .uaa mumaamosm as as. uamamaou
mmumeamoaum m.~ Naamsaav
mo mo.H as.H .mamaaoaamo com .62 .uaa mumaamoam 44 uses as. maoo
.wm .80 mm
ca sm.a mm.a .mcmaaoaamu om. .oz .uaa mumaamoam mm .zm mamamav Hammmam
.mm .80 mm a a .Amqmav monfim
mm MN.H mm.a .oapOpomcm .oamsmoaamo om mzwz .umm .msfi:m< mm can mouofi>mq
am .afi.am\mna om «Hummumumsnsuos
om NR.H mo.N .mamaaoaamo coo .oz ..uaa mamaamoaa as mmflaaaav condom
coauoom mm .85 oma mufimaom m oaocfiovouvms Ammmav cosmooz
ma wa.a so.m was coasoaaoo .uaa mumaamosa .wcaama m Na as. auoaoumz
mHnHmsMMHw oumuuaam HouOH oumuuaflmmuuaa annoy muoomnzm uoaua<
N Imuuaa muonumz
n\mma
aofimocwmz
Boson awake: as soaumuusmocoo anamocmms oumuuaammuuas mo musoamuommoa can awesome mo mumaaom .q maan

24
serum. Also, the results of this study are given and compared to
the previous studies.

Figure 1 is a graphic representation of comparative serum magnesium
fractions as revealed by ultrafiltration and thermal precipitation tech-
niques in this study.

The additions curve used in testing the linearity of the magnesium
absorbance curve indicated that between 2.50 and 3.00 mM per liter, the
line started to become non—linear (Figure 2). As the concentrations
increased, the line was less linear. This was demonstrated by comparing
the results of the direct reading for total magnesium of the three sera
agains' the negative intercept on the abscissa of the lines for the three
addition curves. If the absorbance curve was linear at these higher
concentrations, the results would read the same. The addition curve,
also, demonstrates the linearity of the dilution of the specimen in
aqueous solution.

The results of the plotting of an absorbance curve using magnesium
concentrations up to 4.5 mM per liter indicated that there was linearity
up to about 2.05 mM per liter (Figure 3). As the concentration increases,

the line becomes less linear.

Results of the Pooled Sera Studies

 

The pooled sera studies were performed in an attempt to demon-
strate that the binding of magnesium to the serum proteins obeys the law
of mass action when the thermal precipitation method is used. The law

of mass action equation for magnesium proteinate may be written as follows:

Mg Prot 3 Mg++ + Prot=

. 1‘ 5-o\l .‘.;-" .LE

 

25

Total Serum Magnesium

 

 

 

 

 

1.0 mM/L
F “\
’ \
’ \
I/ \
l \
/ \
Non- / \>Heat precipitable
ultrafiltrate\ _ /
257. (Mg ) // 32/.
\ /
\___ ____________ /
/ /
/
l
l \\
’ \
/ 5 \
x, . '1 \\
/ \
Ultra- I \ Non-heat
filtrable I PrECIPitfble
’ (Mg Prot) I 68%
75% \ I
I
X I
I
\ I
\ I
‘ I
\ /
\
\
\
\
\
\
\
\
\
\— -
Figure 1.

and thermal precipitation.

Serum magnesium fractions as revealed by ultrafiltration

 

26

Absorption

OH

ON

on

oq

cm

00

On

ow

om

OCH

7 ‘
l“ 1‘15... E 1.. ill»!

.cowuaaom maooovm aw ucowuaaap assoc

 

mo muwuwoafla can one o>uoo coconuomnm aaamuswma now hufiumonfifl anon on o>uoo mcoaufiuvm mo muasmom .N ouswfim
A\za as anamoawoz mo cowumuucoocoo
mo.H m~.H mm.o o n.01 HI m.H| N: m.~|
IV. _ T _ _ h _ \L -
_ A I _ fl 4 _ .
doom.
as»
\Oé
.rWEJK
2%

 

.Fv—
6

q\za mo.~

iza no;

A\za om.o

 

 

A\za mm.~ omnemx
A\za mm.H moauuou
.128 3.0 3:5

 

m>uoo maoaufivwm
aoum wafivmom

 

abflmumwma Hmuou
weapons uooufin

 

 

100

90

80

70

6O

50

Absorbance

40

30

20

10

using

27

 

- 6.1 d1.
mgm/

<3____ 4.9 mgm/dl.

 

l I J l I

 

 

 

if f

1 2 3 4‘ 5
Concentration of Magnesium in mM/L

Figure 3. Results of the plotting of an absorbance curve
magnesium standards up to 4.5 mM/L.

The equation for the dissociation

as:

28

constant (KMg Prot) may be expressed

K = <Mg++> (Prot‘)
Mg Prot

(Mg Prot)

If we assume that magnesium is quantitatively bound to albumin, i.e.,

(Mg Prot)= (Mg Alb), this dissociation constant equation is simplified

to:

K Mg Prot _

(Alba) is the total negative site

binding and is equal to:

 

- 3422+) (Alb') .
(Mg Alb)

concentration available for magnesium

(Alb‘) = ncAlb) - (Mg Alb);

where (Alb) is the molar concentration and (n) is the average maximum

number of negative sites available for magnesium binding per albumin

molecule. Substituting in the preceding equations:

_(Mg++
K Mg Alb

we now have two unknowns: K' and

values for (K' ) and (n) can

Mg Alb

(Alb)
(Mg Alb)

1 l

) (n(Alb ) - (Mg Alb))

(Mg Alb)

n. By rearranging the equations the

be obtained:

K' 1

=£+ -—-x
n n Mg?!

 

or:

1 1

(Mg Alb) = n(Alb) + Alb )6? >41?”

 

(Mg Alb) ‘ n<Alb> + nuub) <Mg++>

-L‘II '

‘u ‘ "TN-J" 138.4‘..'x
.

 

29

___l____.= ___l__. 1 + K'
(Mg Alb) n(A1b) x Mgff ’

. 1 . 1
If the equation holds, a plot of (Mg Alb) against (Nip?) should yield

a straight line, with intercept of and slope of from which

1 K'
n(A1b) n(A1b)

K' is calculated.
From the Tables 6 through 13, the values for the reciprocal of

protein—bound magnesium, ;%; are substituted for MEIKIb'and non-

precipitable magnesium, i-for fig, then:

1__1 g
§:§'- n(A1b) (E +y :>

K' 1

or. _1_.= —————————-+-—-———-
° x-y y(n(Alb)) n(A1b)

or: (A) + B (the line of least squares).

'~<IF-'

_l_.=
x-y

The line of least squares can be determined for each pooled sera speci-

men, with

1

A n(A1b)°

K
- n(A1b) and B -

Results on the pooled serum samples No. 5 through 12, used to
determine values for reciprocal plotting of %-against ;%§-for the line
of least squares are given individually in Tables 6 through 13. The
values for %-and ;%§- were calculated. The percent free magnesium for
each dilution was also determined. Figure 5 gives the graphic represen-
tation of the line of least squares for the pooled serum samples Nos.

5, 6 and 7. They have supernatant pH values of 8.8 to 9.0. The plot-

ting of the results gave negative intercept values for Figure 6

._;L_.
n(A1b)°

30
represents the graphic illustration of the line of least squares for
the pooled serum samples Nos. 8, 9 and 10. They had supernatant pH
values ranging from 8.10 to 8.15. The plotted results showed positive
1 .
n(A1b) of 0.59 to 0.88. Figure 7 is a graphic

representation of the line of least squares for the pooled serum

intercept values for

samples Nos. 11 and 12. The supernatant pH values ranged from 7.56 to
7.68. The plotted results gave positive intercept values of 0.51 for
both specimens. Figure 8 is a graphic representation of the average
line of least squares for the three groups, according to the pH's of
the supernatants.

The variation in the lines in Figure 8 can be explained by the
assumption that the pH of the supernatant in Numbers 5, 6 and 7 a
portion of the magnesium precipitated as magnesium phosphates during

the heating process. This resulted in altered values for —l—-and l3

X‘Y Y
with a negative intercept for H(%Ib)7 Negative values for 3(1167 makes
it impossible to calculate the number of magnesium binding sites on the
albumin molecule or the dissociation constant (K') for magnesium
albumin. The plotting of the average line of least squares for Numbers
8, 9 and 10 and Numbers 11 and 12 resulted in positive intercepts close
to each other (0.74 and 0.51, respectively). The slopes of Numbers 8,
9 and 10, however, are lower than those of Numbers 11 and 12 (1.8 and
4.1, respectively).

The results of the pH, total protein and albumin values on the

pooled serum samples are given in Table 5. Albumin was also calculated

in mM per liter using a molecular weight of 64,000. (mM per liter of

1
Mol. Wt. of Albumin

 

albumin = grams per liter of albumin x x 1000).

31
Table 14 is a summary of the calculated values from the pooled sera
data giving the line of least squares, the binding sites for magnesium
on each albumin molecule and the dissociation constant (K') for each

sample.

The Effect of Increasing the (H+)

 

In Figure 8, to explain the increase in the slope of the line as

the supernatant pH is decreased, let us assume the law of mass action

++— =
applies. Then: Mg + Pr 2 MgPr

2H+ + Pr: .1: H2Pr,

and the equations for the dissociation constants are:

 

 

(My?) (Pr‘) = K

(MgPr) 1

and, <H+)2 <Pr‘) K
(HzPr) 2'

If (n) is the average maximum number of binding sites available for

albumin and (Alb) is the molar concentration for albumin, then:

n(A1b) = (MgPr) + (Pr=) + (HzPr).

 

 

+ 2 =
(
Rearranging K2 to read: (HzPr) = ‘H )K (Pr ) ,
2
= / 01.32
Substituting: n(A1b) = (MgPr) + (Pr ) x K: + _R——— .
2
= -(
Rearranging: (Pr ) = n(A1b) (H;?§Pr)
1'*'7z“‘
2

Substituting in the dissociation constant Kl:

= (Mg++) (Pr=> = (Mg++> x n(Alb) - (MgPr)
l (MgPr) (MgPr) l + (HI)2 °
K
2

 

K

32

 

Rearranging: THEE—7 x (n(A1b) — (MgPr»= K1 1 + K2
. n(A1b)_ (H+ )
Then“ (MgPr) W(} 1' 22:>x (Mg++) ’
. _Ala__ = (H+ )2 ._; 1 1
And finally. (MgPr) 1 + K2 x n x (Mg++) +—

In this formula, the effect of increasing the number of H+ or
lowering the pH would increase the slope of the line. The intercept
(1/n) would remain the same. Thus, the (A) value in the line of least

squares would also be directly related to the increase in (H+).

effect of increasing

\

(MgPr)

 

sli—I
”a

 

1
(MgTI)

Figure 4. Effect of increasing the number of hydrogen.ions on
the slope of the line of least squares when reciprocal plotting.

33

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N

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.asumm voaooa
mo .Ha om ou aum ZN .Ha qw.o mo coaufipum can nouwm new Boson :mwum mo ma I OH pom m .m .02

oo NNHV :wcaannan mmwuan: umuwm ssumm mo me I A 6cm 0 .m .02

 

 

 

 

 

 

 

 

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mom.o m.q 0.x mH.m Hw.o om.m m
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moo.o m.m N.m O¢.w wm.~ m
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asumm
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moaaamM Eamon woaooa can so msam> awasaam new samuoum anuOu .ma .m oHAMH

34

 

 

 

 

 

 

Table 6. Results of No. 5 pooled serum samples used to determine values
for reciprocal plotting of l/Y against 1/X-Y for the line of
least squares

Magnesium Concentration
X Y
Total Noneprecipitable Protein-bound %

Tube 1_ 1 free
No. mM/L mM/L Y mM/L X-Y Mg.
U 0.87 0.59 1.70 0.28 3.57 68
A 1.04 0.66 1.52. 0.38 2.64 63.5
B 1.26 0.78 1.28 0.48 2.08 62
C 1.42 0.92 1.09 0.50 2.00 65
D 1.67 1.03 0.97 0.64 1.56 62
E 1.83 1.13 0.89 0.70 1.46 62
F 1.97 J 25 0.80 0.72 1.39 63
G 2.19 1.35 0.74 0.84 1.19 67
H 2.42 1.47 0.68 0.95 1.05 61
I 2.47 1.58 0.63 0.89 1.12 64
J 2.63 1.71 0.59 0.92 1.08 65
K 2.98 1.87 0.54 1.11 0.90 63
L 3.19 2.01 0.50 1.18 0.85 63
M 3.42 2.38 0.42. 1.10 0.91 68
N 3.90 2.60 0.39 1.30 0.77 67
0 ---- 2.84 0.35 ---- ---- --

 

35

Table 7. Results of No. 6 pooled serum sample used to determine values
for reciprocal plotting of l/Y against l/X-Y for the line of
least squares

 

 

Magnesium Concentration

 

 

 

X Y X-Y
Iggal_ Noneprecipitable Protein-bound %
Tube 1. _g;_ free
No . mM/L mM/ L Y mM/L X—Y Mg .
U 0.83 0.57 1.75 0.26 3.85 68
A 1.06 0.67 1.49 0.37 2.70 65
B 1.24 0.80 1.25 0.44 2.28 65
C 1.40 0.92, 1.09. 0.48 2.08 66
D 1.56 1.03 0.97 0.53 1.89 66
E 1.74 1.19 0.84 0.55 1.82 68
F 1.93 1.31 0.76 0.62 1.61 68
C 2.11 1.42 0.71 0.69 1.45 67
H 2.34 1.52 0.67 0.82 1.22 65
I 2.53 ---- ---- ---- ---- --
J 2.69 1.77 0.57 0.92 1.09 66
K 3.05 1.99 0.50 1.06 0.94 66
L. 3.43 2.28 0.43 1.15, 0.87 67
M 3.77 2.50 0.40 1.27 0.79 66
N 4.15 2.67 0.37, 1.48 0.68 64
0 ---- 3.12 0.32 ---e ---- --

 

36

 

 

 

 

 

 

Table 8. Results of No. 7 pooled serum sample used to determine values
for reciprocal plotting of l/Y against l/X-Y for the line of
least squares

Magnesium Concentration
X
Total Noneprecipitable Protein-bound %
Tube 1_ 1 free
No . mM/L mM/ L Y mM/L X-Y Mg.
U 0.79 0.55 1.82 0.24 4.17 70
A 0.98 0.68 1.47 0.30 3.34 69
B 1.15 0.84 1.19 0.31 3.23 73
C 1.43 0.98 1.02 0.45 2.22 69
D 1.54 1.11 0.90 0.43 2.33 72
E 1.84 1.29 0.78 0.55 1.82. 70
F 2.06 1.43 0.70 0.63 1.59 69
G* 2.28 1.56 0.64_ 0.72 1.39 68
H 2.56 1.74 0.58 0.82 1.22 68
I 2.71 1.86 0.54 0.85 1.18 69
J 3.05 2.00 0.50 1.05 0.95 66
K 3.42 2.30 0.44 1.12 0.89 67
L 3.82 2.62 0.38 1.20 0.83 69
M 4.15 3.05 0.33 1.10 0.91 73
N —--— 3.19 0.31 ---- ---- --
0 ---- 3.53 0.28 ---- ---- --

 

37

Table 9. Results of No. 8 pooled serum samples used to determine values.
for reciprocal plotting of 1/Y against 1/X-Y for the line of
least squares

 

 

Magnesium‘Concentration

 

 

 

 

X Y X-Y
Total Nonfprecipitable Protein-bound 2
Tube 1_ _1__ free
No. mM/L mM/L Y mM/L X-Y Mg.
U 0.83 0.53 1.89 0.30 3.33 64
A 0.95 0.68 1.47 0.27 3.70 72'
B 1.18 0.85 1.17 0.33 3.03 72
C 1.34 0.95 1.05 0.39 2.56 71
D 1.54 1.14 0.88 0.40 2.50 74
E 1.70 1.24 7 0.81_ 0.54 1.85 68
F 1.86 1.36 0.73 0.50 2.00 73
G 2.05 1.63 0.61 ' 0.42 2.38- 79
H 2.36 1.78 0.56 0.58 1.72 , 75
I 2.48 1.94 0.52 0.54 1.85 78-
J 2.73 2.07 0.49 0.66 1.52. 76
K 2.95 2.45 0.41 0.50 2.00 83
L 3.40 2.73 0.37 0.67 1.49- 80
M 3.68 2.90 0.35 0.78 1.28 79
N 4.15 3.15 0.32 1.00 1.00 76
O 4.48 3.40 0.29 1.08 0.93 76

 

38

Table 10. Results of No. 9 pooled serum samples used to determine values
for reciprocal plotting of l/Y against 1/X-Y for the line of
least squares

 

 

Magnesium Concentration

 

 

 

X Y X-Y
Total. Noneprecipitable Protein-bound %
Tube 1_ _1;_ free
No . mM/ L mM/ L Y mM/L X-Y Mg .
U 0.86 0.59 1.69 0.27 3.70 69
A 1.02 0.71 1.41 0.31 3.22 70
B 1.22 0.88 1.13 0.34 2.94 72
C 1.44 1.05 0.95 0.39 2.56 73
D 1.66 1.24 0.81 0.42 2.38 75
E 1.82 1.38 0.73 0.44 2.28 71‘
F 2.05 1.58 0.63 0.47 2.12 77
G 2.38 1.77 0.57 0.61 1.64 74
H 2.70 1.96 0.51 0.74 1.35 73
I 2.95 2.20 0.46 0.75 1.33 75
J 3.23 2.45 0.41 0.78 1.28 76
K 3.56 2.74 0.38 0.82 1.22 77
L 4.04 3.20 0.31 0.84 1.19 79
M 4.32 3.44» 0.29 0.88 1.14 80
N ---- 3.80 0.26 ---- ---- --
0 -—-— 4.15 0.24 ---- -—-- --

 

39

 

 

 

 

 

 

Table 11. Results of No. 10 pooled serum samples used to determine
values for reciprocal plotting of 1/Y against l/X-Y for the
line of least squares

Magnesium Concentration
X Y X—Y
Total Non—precipitable Protein-bound 2
Tube 1_ 1 free.
No. mM/L mM/L Y mM/L X—Y Mg.
U 0.84 0.58 1.72. 0.26 3.85 69
A 1.00 0.69 1.45 0.31 3.23 69
B 1.23 0.84 1.19 0.39 2.56 68
C 1.45 1.08 0.93 0.37 2.70 74
D 1.65 1.26 0.80 0.39 2.56 76
E 1.89 1.42 0.71 0.47 2.12 75
F 2.13 1.58 0.63 0.55 1.82 74
G 2.37 1.76 0.57 0.61 1.64 74
H 2.46 1.95 0.51- 0.51- 1.96 79
I 2.62 2.08 0.48 0.54 1.85 80
J 2.90 2.18 0.46 0.72. 1.39 75
K 3.30 2.58 0.39 0.72 1.39 78
L 3.80 3.06 0.33 0.74. 1.35 80
M 4.18 3.40 0.29 0.78 1.28 81
N ---- 3.70 0.27 ---- -—-- --
0 ---- 4.10 0.24 —--— —--- --

 

40

 

 

 

 

 

 

Table 12. Results of No. 11 pooled serum samples used to determine
values for reciprocal plotting of 1/Y against 1/X-Y for the
line of least squares

Magnesium Concentration
X Y XrY
Total Non-precipitable Protein-bound Z

Tube 1_ 1 free
No. mM/L mM/L Y mM/L X-Y- Mg.
U 0.82 0.64 1.56 0.18 5.55 82
A 0.98 0.82 1.22 0.16 6.25 84
B 1.20 1.01 0.99 0.19 5.26 84
C 1.39 1.14~ 0.88 0.25 4.00 82'
D 1.58 1.35 0.74 0.23 4.35 85
E 1.85 1.56 0.64 0.29 3.45 84
F 2.04 1.73 0.58 0.31 3.23 85
G 2.33 1.91 0.53 0.42 2.38 82
H 2.53 2.09 0.48 0.42- 2.38. 83
I 2.73 2.27 0.46 0.46 2.17 83
J 2.90 2.42 0.41 0.48 2.08 83
K 3.26 2.75 0.36 0.51 1.96 84
L 3.82 3.19 0.31 0.63 1.58 84
M 4.30 3.60 0.28 0.70 1.43 84
N ---- 4.00 0.25 ---- ---- --
0 ---- 4.35 0.23 ---- ---- --

 

I)...

41

 

 

 

 

 

 

Table 13. Results of No. 12 pooled serum samples used to determine
values for reciprocal plotting of 1/Y against l/X—Y for the
line of least squares

Magnesium Concentration
X Y X-Y
Total Non—precipitable Protein-bound Z
Tube 1_ 1 free
No. mM/L mM/L Y mM/L X—Y Mg.
U 0.82 0.66 1.52 0.16 6.25 81
A 1.03 0.85 1.18 0.18 5.55 82
B 1.24 1.03 0.97 0.21 4.75 83
C 1.40 1.16 0.86 0.24. 4.17 83
D 1.65 1.38 0.73 0.27 3.70 84
E 1.89 1.58 0.63 0.31 3.23 84
F 2.10 1.76 0.57 0.34 2.94 84
G 2.42 2.05 0.49 0.37 2.70 85
H 2.64 2.23 0.45 0.41 2.44 84
I 2.87 2.42 0.41 0.45 2.22 84
J 3.20 2.72- 0.37 0.48 2.08 85
K 3.58 3.03 0.33 0.55 1.82 85
L 3.97 3.35 0.30 0.62 1.61 84
M 4.32 3.64 0.27 0.68 1.47 84
N ---- 3.91 0.26 ---- ---- -—
0 ---- 4.30 0.23 ---- —--- --

 

42

 

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43

 

 

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46

 

 

 

Table 14. Calculated values from pooled sera data giving the line of
least squares, binding sites for magnesium on each albumin
molecule, dissociation constant (KM ) and pK for each

I g Prot 1
sample
Magnesium Dissociation
No. Line of Sites on. Constant
Pooled Serum Least Squares Albumin Molecule KMg Prot pK
5 Y = 1.90X - 0.ll_ ---— --—-
6 Y = 2.04X - 0.06 ---- ----
7 Y = 2.40X - 0.08 ---- ----
8 Y = 1.6OX + 0.88 1.60 1.83 x 10‘3 2.73
9 Y = 1.95x + 0.59 2.40 3.30 x 10"3 2.48
10 Y = 1.80X + 0.75 2.03 2.40 x 10'3 2.62
11 Y = 4.07x + 0.51 2.99 7.98 x 10‘3 2.09
12 Y = 4.11x + 0.51 2.99 8.05 x 10‘3 2.09

 

DISCUSSION

Newer analytic methods, current interest of a number of investi-
gators, and recent evidence suggest that disturbances of magnesium in-
the body fluids are of increasing clinical interest. The study of
magnesium metabolism has been hampered by difficulty in attaining
accurate analytic methods for biological fluids. The older methods
of precipitation as magnesium ammonium phosphate or as magnesium
quinolate are cumbersome and usually involve removal of some magnesium
in the preliminary separation from calcium. Complexing with the titan
yellow dye or complexo-metric titration with ethylenediaminetetraacetic
acid (EDTA) led to inaccurate, unreproducible and time-consuming methods.
Emission flame spectrOphotometric techniques require isolation of the
magnesium resonance line (285.2 nm) from the sodium line at 285.3 nm
resulting in interference. Background interference due to other con-
stituents in biological materials is also a problem when using the
emission techniques because of high flame temperature necessary to
excite the magnesium atoms.-

In 1955, Walsh61 suggested that the principle of atomic absorption
spectroscopy has significant advantages over emission methods. The
technique has been investigated by several workers including Willis,75
Dawson and Heaton,26 and Stewart et al.61 They have.emphasized that
atomic.absorption spectrosc0py may be the most sensitive and
reliable method for the determination of magnesium. The flame tempera-
ture is lower than in the emission studies because the absorption of

47

48
the apprOpriate radiation by the magnesium atoms requires them to be in
the ground state. Thermal energy is used to evaporate the solvent and
to convert the magnesium in the solution to the atomic state.‘ Atoms in
the ground state will absorb apprOpriate radiation from a source outside
the flame, and the amount of absorption is quantitatively related to
the concentration of atoms in the flame. This in turn is mainly.
dependent on the efficiency of the atomizer. Also, the monochromator
slit width is not critical because the magnesium hollow cathode lamp
does not emit any other lines near 285.2 nm. This study utilized a
1:50 dilution of serum with redistilled water. The high dilution was
used to minimize protein interference. It was found to be a sensitive
and reliable technique which can be performed rapidly on small samples.

There is some variation in reported normal serum total magnesium
concentration depending on the analytic method used (Table 3). Mean
normal values for atomic absorption spectroscopy have been reported
ranging from 0.80 to 1.10 mM per liter. The values of the 50 individuals
in this study were comparable to most reported values (X) 0.91 mM per
liter and (S.D.) 0.17 mM per liter.

Over the last twelve years, many methods have been employed to pro-
duce protein-free ultrafiltrates of serum under various conditions.48
Table 4 shows the variability in the percentage of ultrafiltrable frac-
tions of magnesium in normal serum by various workers using these
methods. A simple, reproducible thermal precipitation technique is
described which quickly produces an.adequate volume of supernatant.

It compares favorably to most methods of ultrafiltration where ionic
magnesium is measured. Results of the thermal precipitation super-
natant non—precipitable magnesium, X 67.4% is very comparable to the

ultracentrifugation method by Prasad for ionic magnesium, X 64%.

49

However, the comparative results in this study showed higher values for
ultracentrifugation, X 74.9%. This may be partly due to difficulty in-
adapting to the technique, especially in the first five specimens where
the percent ionic magnesium shows exceptionally high values, up to 87%.

Upon pH measurement, it was found that a serum pH of about 7.4 was
altered when using the thermal precipitation technique to a pH of approxi-
mately 8.8 to 9.0 for the supernatant. This is probably largely due to
the removal of CO2 when the serum is heated. The ultracentrifugation

method resulted in ultrafiltrate pH values of about 7.6 to 7.8, a com-

paratively smaller loss of CO resulting from the handling of the

2
Specimen. The pH of the ultrafiltrate is comparable to the results in
Prasad's study,45 in which the pH ranged from 7.9 to 8.0. It is

probable that the wide variation in both mean values and ranges of
percentage ultrafiltrate magnesium is a complex function of the type

of method employed, type of membrane used, and temperature of the
ultrafiltration and the degree of pH control.

Magnesium is mainly an intracellular ion. The level of ultra-
filtrable magnesium in serum, which can be regarded as representing the
extracellular fluid concentration, appears to remain constant in normal
individuals within reasonable limits. The mechanism which governs this
equilibrium between the intra- and extracellular magnesium concentra-
tion is not well understood. In the serum, the law of mass action
determines the magnesium-protein relationship,45 and as such, the pro-
tein affects the percentage of ultrafiltrable magnesium. Serum proteins
and thyroid and renal functions are partially responsible for maintenance
of magnesium level in the serum and ultrafiltrate. However, apparently

in addition to the above-mentioned factors, some other unknown factor(s)

must be operating to maintain the level of ultrafiltrable magnesium

50
within a narrow range. Increased levels of ultrafiltrable or non-
precipitable magnesium was always associatedeith increased total
magnesium (Table 1).

The linearity of magnesium absorbance by atomic absorption spec-
troscopy technique was shown to be linear up to about 2.5 mM per
liter when using an additions curve. Also, the additions curve showed
that a 1:50 dilution of the serum Specimen in water was linear,
demonstrating that interfering substances were negligible. A more
precise measurement for linearity is absorbance curves using the
magnesium standards. They illustrated linearity up to about 2.05 mM
per liter and as the magnesium concentration increased the curve
became proportionately less linear.

The major fractions of magnesium in serum are (1) ionized, or
magnesium associated with other ions in a dissociated form and (2) a
portion held in solution by the serum proteins, or the protein-bound
fraction. McLean and Hastings36 suggested that magnesium and calcium
both react with the serum proteins in a very similar way. They expressed
the relationship by the empirical mass-law formation of:

(MgH) (Prof) ..
(Mg Prot) '

 

K

One would anticipate, therefore, that the ratio between bound magnesium
and total serum magnesium should remain relatively constant despite
marked changes in total serum magnesium. It also would hold that any
procedure for the measurement of total and ionic magnesium should abide
by the law of mass action. As described by Moore,38 an explanation of
the formulas is given in this study under the results. A series of
pooled serum studies was performed to test the reliability of the thermal

precipitation technique. Reciprocal plotting on the first series of

51

l . 1 .
pooled serum of Mg Alb against fig;;-y1e1ded a straight line. However,
the intercept ETX%Ey-resulted in a negative value from which the dissoci-

ation constant (K) or negative binding sites (n) on the albumin molecule
could not be calculated. The cause of the negative intercept values
was thought to be the heating process in which the precipitation of
some ionic magnesium complexed with phosphates at this alkaline pH.

To demonstrate that binding of magnesium to serum proteins is
pH dependent, a series of pooled sera with lower pH's was studied. To
correct for the high alkaline supernatant, 2N HCl was added to the
pooled serum lowering the pH to about 6.8 to 6.9. The supernatant pH

was 8.1. Lowering the pH resulted in a straight line with positive

__1___
n(A1b) ’

the number of negative binding sites could be calculated. The dissoci-

intercept values for from which the dissociation constant and
ation constant for magnesium proteinate ranged from pK 2.48 to 2.73
(average 2.61), a value higher than that found by Prasad45 (pK 1.93)
or reported by Copeland and Sunderman13 (pK 1.77). The pH of the ultra-
filtrate by Prasad was 7.9 to 8.0 with the pH of the supernatant being
adjusted to about 8.1. The maximum number of negative binding sites
for magnesium on the albumin molecule averages to about 2.0 at the
supernatant pH of 8.1.

Two more pooled serum samples with the pH of the sera lowered to
about 6.4 by the addition of 2N HCl were analyzed. The supernatant pH
values were about 7.6. Again the reciprocal plotting resulted in a

straight line with a positive intercept value of from which the

_1__
n(A1b)
dissociation constant and number of negative binding sites for albumin

could be calculated. The dissociation constant for magnesium proteinate

was about pK 2.09. The number of negative binding sites for magnesium

on the albumin molecule was 2.99 at the supernatant pH of 7.6.

52

Because the serum proteins are part of the buffer systems of the
blood, changes in the hydrogen ion concentration that permit competi-
tion between hydrogen and other cations for their binding sites results
in alterations in the proportion of the ionic fraction of magnesium.
This would result in the apparent change by the dissociation constant
for magnesium proteinate according to the law of mass action (Figure 4).
The effect of lowering the pH is that magnesium is released from the
albumin binding sites to allow the association of hydrogen ions to
take place. This released magnesium is detected in the serum as an
increase in the ionic fraction. 0n the other hand, with a rise in .

pH more binding sites become available for magnesium and the process

 

is reversed. In Figure 4, the effect of increasing the hydrogen ions
or lowering the pH would be to increase the lepe of the line of least
squares. Therefore, the (A) value in the equation for the line of
least squares would also be directly related to the increase in

hydrogen ions.

SUMMARY

A simple, reproducible, and relatively rapid thermal precipitation
method is described for the separation of free from protein-bound
magnesium. It yielded a supernatant containing a non-precipitable
magnesium fraction that was comparable to that reported from ultra-
filtrate methods for ionic magnesium. Measurement of total serum
magnesium and the non—precipitable fraction--3 was determined by atomic
absorption spectroscoPy, utilizing a 1:50 aqueous dilution. Mean
value for the total magnesium was 0.91 mM per liter with a standard
deviation of 0.17 mM per liter. Non-precipitable fraction determinations
resulted in a mean value of 0.62 mM per liter, a standard deviation of
0.10 mM per liter and a percentage of 67.9.

The thermal precipitation technique was compared to an ultracentri-
fugation method as an ultrafiltrate measurement of ionic magnesium.

The reliability of the thermal precipitation technique was evaluated
in accordance to whether reciprocal plotting of fiéigig-against ii;;

8 8
follows the law of mass action. The reciprocal plotting yielded a
straight line when determining the line of least squares. Precipita—
tion of magnesium phosphates at the highly alkaline pH of the super-
natant resulted in negative intercept values. Lowering the pH with the
addition of 2N HCl resulted in positive intercept values, from which
the dissociation constant for magnesium proteinate was determined: pK
2.61 at supernatant pH of 8.1; and pK 2.09 at supernatant pH of 7.6.
The maximum number of negative binding sites for magnesium on the

53

54
albumin molecule was calculated as 2.0 at the supernatant pH of 8.1,
and 2.99 at the supernatant pH of 7.6.
The effect of increasing the hydrogen ions or lowering the pH in
the pooled serum studies was an increase in the slope or a direct
relationship to the (A) value in the formula for the line of least

squares.

REFERENCES

 

10.

ll.

12.

13.

REFERENCES

Aikawa, J. K.: Gastrointestinal absorption of magnesium28 in
rabbits. Proc. Soc. Exper. Biol. and Med., 100, (1959): 293-295.

Aikawa, J. K.: The Role of'Magnesium in Biologic Processes.
Springfield, Illinois, Thomas, 1963.

Alcock, N., Maclntyre, I., and Raddle, 1.: The determination of
magnesium in biological fluids and tissues by flame spectropho-
tometry. J. Clin. Path., 13, (1960): 506.

Barker, E. S.: Physiologic and clinical aspects of magnesium
metabolism. J. Chron. Dis., 11(3), (March, 1960): 278-291.

Bissell, G. W.: Magnesium partition in hyperthroidism.with
Special reference to effect of thiouracil. Am. J. Med. Sci., 210,
(1945): 195.

Boyd, 1. A., and Martin, A. R.: Spontaneous subthreshold activity
at mammalian neuromuscular junctions. J. Physiol., 132, (1956):
61-73.

Boyd, I. A., and Martin, A. R.: The end-plate potential in
mammalian muscle.. J. Physiol., 132, (1956): 74-91.

Breen, Morra, and Smith, Freeman: The use of centrifugal force in
separating plasma calcium into a protein-bound and protein-free
fractions. Clin. Chem. Acta, 6, (1961): 181-187.

Brenner, 3., Jacob, F., and Meselson, M.: Unstable intermediate
carrying information from genes to ribosomes for protein synthe-
sis. Nature (Lcndon), 190, (1961): 576-581.

Cannon, M., Krug, R., and Gilbert, W.: Binding of s-RNA to
Escherichia coli ribosomes. J. Molec. Biol., 7, (1963): 360-378.

Connerty, H., and Briggs, A. R.: Spectrophotometric determination
of serum magnesium by Eriochrome Black T. Amer. J. Clin. Path.,
50, (1968): 499.

Cope, C. L., and Wolf, B.: The ultrafiltrable magnesium in hypo—
thryroidism. Biochem. J., 36, (1942): 413.

COpeland, B. E., and Sunderman, F. W.: The magnesium-binding
property of the serum proteins. J. Biol. Chem., 197, (1952): 331.

55

14.

15.

l6.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

56

Danesh, M. B., Walker, H. M., and Mathers, N. P.: The relation
of post—dialysis plasma calcium and magnesium to dialysate levels
and to changes in blood pH. New Eng. J. Med., 282(14), (1970):

Davison, H.: Textbook of’GeneraZ Physiology. Third Edition,
Little, Brown and Co., Boston, (1964): 966—987.

Dove, W. F., and Davidson, N.: Cation effects on denaturation
on DNA. J. Molec. Biol., 5, (1962): 467-478.

Dunn, M. J., and Walser, M.: Magnesium depletion in normal man.
Metabolism, 15, (1966): 884—895.

Failing, J. F., Buckley, M. W., and Zak, B.: Automatic determina-
tion of serum proteins. Am. J. Clin. Path., 33, (1960): 83-88.

Fatt, P., and Katz, B.: Spontaneous subthreshold activity at
motor nerve endings. J. Physiol., 117, (1952): 109-128.

Fawcett, J. K.,and Wynn, V.: The determination of magnesium in
biological materials by flame photometry. J. Clin. Path., 14,
(1961): 403.

Forbes, R. M., Mitchell, H. H., and Cooper, A. R.: Further studies
on the gross composition and mineral elements of the adult human
body. J. Biol. Chem., 223, (1956): 969.

Glicksman, A. 3., Schwartz, M. K., Bane, H., Roberts, K. E., and
Randall, H. T.: Physiologic distribution and excretion of Mg28
in dogs and man. Clin. Res., 4, (1956): 14.

Goodman, L. 8., and Gilman, A., edited: The Pharmacological Basis
of Therapeutics. Third Edition, New York, Macmillan, 1968.

Graham, L. A., Caesar, J. J., and Burger, A. S. V.: Gastroin-
testinal absorption and excretion of magnesium in man. Metabolism,
9, (1960): 646-659.

Hansen, J. L., and Freier, E. F.: The measurement of serum mag-
nesium by atomic absorption spectrophotometry. Am. J. Med. Tech.,
33(3), (1967): 1-9.

Heaton, F. W., Pyrah, L. N., Beresford, C. C., Bryson, R. W., and
Martin, D. F.: Hypomagnesemia in chronic alcoholism. Lancet, 2,
(1962): 802.

Hernandez, 0., Murray, L., and Doumas, B.: Automated determination
of serum albumin with bromcresol green. Clin. Chem., 13, (1967):
701.

HOpkinS, T. R., Howard, J. E., and Eisenberg, H.: Ultrafiltration
studies on calcium and phosphorus in human serum. Bull. Johns
Hopkins Hosp., 91, (1952): l.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

57

Hunter, G.: Calcium and magnesium content of normal human blood
serum. Nature, 182, (1958): 263.

Jackson, C. E., et aZ.: Routine serum magnesium analysis. Correla-
tion with clinical state in 5100 patients. Ann. Intern. Med., 69,
(1968): 743-748.

Jones, J. E., Manalo, R., and Flink, E. B.: Magnesium requirements
in adults. Am. J. Clin. Nutr., 20, (1967): 632-635.

Lavietes, P. H.: Anaerobic ultrafiltration. J. Biol. Chem., 120,
(1937): 267.

Lennon, E. J., and Piering, W. F.: A comparison of the effects of
glucose ingestion and NH4CL acidosis on urinary calcium and mag-
nesium excretion in man. J. Clin. Invest., 49, (1970): 1458-1465.

Loken, H. F., Havel, R. J., Gordan, G. S., and Whittington, S. L.:
Ultracentrifugal analysis of protein-bound and free calcium in
human serum. J. Biol. Chem., 235(12), (1960): 3654-3658.

Maclntyre, 1., Hanna, S., Booth, C. C., and Read, A. B.: Intra-
cellular magnesium deficiency in man. Clin. Sci., 20, (1961):
297—305.

McLean, F. C., and Hastings, A. B.: The state of calcium in fluids
of the body. J. Biol. Chem., 108, (1935): 285.

Montgomery, R. D.: The estimation of magnesium in small biological
samples by flame spectrophotometry. J. Clin. Path., 14, (1961):
400.

Moore, E. W.: Studies with ion-exchange electrodes in biological
fluids: Some applications in biomedical research and clinical
medicine. In symposium on ion-selective electrodes. R. Durst,
editor, National Bureau of Standards, Gaitherburg, Maryland,
(1970): 245-249.

Novelli, G. D.: Amino acid activation for protein synthesis.
Ann. Rev. Biochem., 36, (1967): 449-484.

Odgen, D. A., and Holmes, J. H.: Changes in total and ultrafil-
trable plasma calcium and magnesium during hemodialysis. Trans.
Amer. Soc. Artif. Intern. Org., 12, (1969): 200.

Orange, M., and Rhein, H. G.: Microestimation of magnesium in
body fluids. J. Biol. Chem., 189, (1951): 379.

Peck, C. A., and Meltzer, S. J.: Anesthesia in human beings by
intravenous injections of magnesium sulfate. J.A.M.A., 67, (1916):
1131-1133.

Potter, V. R.: Assay of animal tissues for respiratory enzymes.
VI. Further Studies on oxidative phosphorylation. J. Biol.
Chem., 169, (1947): l7-37.

. r-E

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

58

Prasad, A. S., and Flink, B.: Effect of carbon dioxide on concen-
tration of calcium in an ultrafiltrate of serum obtained by centri-
fugation. J. Appl. Physiol., 10, (1957): 103.’

Prasad, A. S., Flink, E. B., and Zinneman, H. H.: The base binding
property of theserum proteins with respect to magnesium. J. Lab.
and Clin. Med., 54(3), (1959): 357-364.

Prasad, A. S., Flink, E. B., and McCollister, R.: Ultrafiltration
studies on serum magnesium in normal and diseased states. J. Lab.
and Clin. Med., 58(4), (1961): 531-541.

Randall, R. E., Cohen, M. D., Spray, C. C., and Rossmeisel, E. C.:
Hypermagnesemia in renal failure: Etiology and toxic manifestations.
Ann. Intern. Med., 61, (1964): 73.

Robertson, W. G., and Peacock, M.: New techniques for the separa-
tion and measurement of the calcium fractions of normal human.
serum. Clin. Chim. Acta, 20, (1968): 315-326.

Rodgers, A.: Magnesium ions and structure of E. coli ribosomal
ribonucleic acid. Biochem. J., 100, (1966): 102-109.

Ross, D. B.: In vitro studies on the transport of magnesium across
the intestinal wall of the rat. J. Physiol., 160, (1962): 417-428.

Ryan, M. P., and Hingerty, D.: Comparison of four methods for
determining serum magnesium. J. Clin. Path., 21, (1968): 220.

Savory, J., Wiggins, J. W., and Heigtess, M. G.:. Measurements of
calcium and magnesium in serum and urine by atomic absorption
spectrophotometry. Am. J. Clin. Path., 51(6), (1969): 720-727.

Schachter, D.: The fluorometric estimation of magnesium in serum
and urine. J. Lab. and Clin. Med., 54, (1959): 763.

Schachter, D.: The fluorometric estimation of magnesium with 8-
hydroxy, 5—quinoline sulfonate. J. Lab. and Clin. Med., 58, (1961):
495.

Schroeder, H. A., et aZ.: Essential metals in man:~ Magnesium.
J. Chron. Dis., 21, (1969): 815-841.

Silver, L., Robertson, J. S., and Dahl, L. K.: 'Magnesium turnover
in humans studied with Mg28. J. Clin. Invest., 39, (1960): 420-425.

Silverman, S. H., and Garner, L. ‘L: Ultrafiltration studies on
serum magnesium. New Eng. J. Med., 250, (1954): 938.

Smith, R. G., Craig, P., Bird, E. J., Boyle, A. J., Iseri, L. T.,
Jacobson, S. D., and Myers, G. B.: Spectrochemical values for

sodium, potassium, iron, magnesium and calcium in normal human
plasma. Am. J. Clin. Path., 20, (1950): 263.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

59

Soffer, L. J., Cohn, C., Grossman, E. B., Jacobs, M., and Sobotka,
H.: Magnesium partition studies in Grave's disease and in clinical
and experimental hypothyroidism. J. Clin. Invest., 20, (1941):
429.

Spare, P. D.: A study of the Titan Yellow dye lake methods for
estimation of serum magnesium. Am. J. Clin. Path., 37, (1962):
232.

Stewart, W. K., Hutchinson, F., and Fleming, L. W.: The estimation
of magnesium in serum and urine by atomic absorption spectrophotometry.
J. Lab. and Clin. Med., 61(5), (1963): 858.

Thin, C. G., and Thomson, P. A.: Estimation of calcium and mag-
nesium in serum and urine by atomic absorption spectrOphotometry.
J. Clin. Path., 20, (1967): 280.

Toribara, T. Y., Trerpka, R. A., and Dewey, P. A.: The ultra— f
filtrable calcium of human serum. 1. Ultrafiltration methods h
and normal values. J. Clin. Invest., 36, (1957): 738.

 

Vallee, B. L., Wacker, W. E. C., and Ulmer, D. D.: The magnesium
deficiency tetany syndrome in man. New Eng. J. Med., 262, (1960):
155.

Van Fossan, D. D., Baird, E. E., and Tekell, G. S.: A simplified
flame spectrophotometric method for estimation of magnesium in
serum. Am. J. Clin. Path., 31, (1959): 368.

Wacker, W. E. C., Iida, C., and Suwa, K.: Accuracy of determina-
tion of serum magnesium by flame emission and atomic absorption
spectrophotometry. Nature (London), 202, (1964): 659-661.

Wacker, W. E. C., and Parisi, A. F.: Magnesium metabolism. New
Eng. J. Med., 278(12), (1968): 658-663.

Wacker, W. E. C., and Vallee, B. L.: A study of magnesium metabol-
ism in acute renal failure employing a multichannel flame Spectrometer.
New Eng. J. Med., 257, (1957): 1254.

Wacker, W. E. C., and Vallee, B. L.: Magnesium metabolism. New
Eng. J. Med., 259, (1958): 431-438; 475-482..

Wallach, S., Cahill, L. N., Rigan, F. H., and Jones, H. L.: Plasma
and erythrocyte magnesium in health and disease. J. Lab. and Clin.
Med., 59, (1962): 195.

Walser, M.: Ion association. VI. Interactions between calcium,
magnesium, inorganic phosphate, citrate and protein in normal human.
plasma. J. Clin. Invest., 40, (1961): 723-730.

Watchorn, E., and McCance, R. A.: Inorganic constituents of CSF-II.
The ultrafiltration of calcium and magnesium from human sera.
Biochem. J., 26, (1932): 5’.

6O

73. White, Fred J.: Some properties of calcium-containing thermopre-
cipitated fractions of human blood serum. Master's thesis, Michigan
State University, 1971.

74. Williams, R. J. P., and Wacker, W. E. C.: Cation balance in bio—
logical systems. J.A.M.A., 201, (1967): 18-22.

75. Willis, J. B.: The determination of metals in blood serums by

atomic absorption spectroscopy. 11. Magnesium. Spectrochem.
Acta, 16, (1960): 273.

76. Zumoff, B., Berstein, E. H., Imarisio, J. J., and Hellman, L.:
Radioactive magnesium (Mg28) metabolism in man. Clin. Res., 6,
(1958): 280.

General References

 

Davidsohn, 1., and Henry, J. B.: Clinical Diagnosis by Laboratory
Methods. Fourteenth Edition, Philadelphia, W. B. Saunders Co.,
1969.

Kahn, H.: Principles and Practices of’Atomic Absorption. Advances in
Chemistry Series, Number 73, American Chemical Society, (1968):
183-229.

Lewis, A. E.: Biostatistics. New York, Reinhold Publishing Corp.,
1966.

Slavin, W.: Atomic Absorption Spectroscopy. Wiley, 1968.

Tietz, N. W.: Fundamentals of’Clinical Chemistry. Philadelphia, W. B.
Saunders Co., (1970): 651-654.

APPENDIX

APPENDIX.

Stock Magnesium Standard for Absorbance Curve

1000 ppm magnesium. Salt is magnesium acetate [Mg (CHZCOOH)2] with
matrix of dilute acetic acid (Harleco, Philadelphia, Pa.). Equivalent
to 1000 mg./ml.; 100 m1./d1.; 82 mEq./L. or 41 mM/L. Intermediate

Standards are made up from the Stock magnesium standard by the following

 

 

 

dilutions:
Concentration in Working Standards
Stock Mg. Std. Deionized H29_ mEg./L. Egyg:_
0.2 ml. dilute to 10 ml. 1.65 0.82
0.3 m1. dilute to 10 ml. 2.50 1.25
0.4 m1. dilute to 10 ml. 3.30 1.65
0.5 m1. dilute to 10 ml. 4.10 2.05

Working standards are prepared by diluting each of the above intermediate
standards 1:50 in deionized H20. Working standards were prepared up to
4.5 mM/L. in the absorbance curves utilized for reciprocal plotting where

magnesium chloride was added to aliquots of pooled sera.

Total Protein Method for Auto—Analyzer

 

A. STOCK BIURET AR-59-60

 

Chemical Composition

 

1. Sodium Potassium Tartrate (KNaC4H4O6°4H20) 45 gm.
2. COpper Sulfate (Cu804°5H20) 15 gm.
3. Potassium Iodide 5 gm.
4. 0.2 N_Sodium Hydroxide, q.s. 1000 ml.

61

62

Preparation_

 

1. To 45 gm. of sodium potassium tartrate in a one liter
volumetric flask, add 400 m1. of 0.2 N_sodium hydroxide.

2. Dissolve the tartrate and while stirring add 15 gm. of
c0pper sulfate. Continue stirring until the copper
sulfate is dissolved.

3. Add 5 gm. of potassium iodide. Dissolve and dilute to
1 liter with 0.2 N sodium hydroxide.

4. Filter and store in polyethylene bottle.

B. ALKALINE IODIDE_ AR-127-62

 

Chemical Composition

1. Potassium Iodide 5 gm.
2. Sodium Hydroxide 8 gm.
3. Distilled Water, q.s. 1000 m1.
Preparation

 

1. Add 8 gm. sodium hydroxide to approximately 800 ml. dis-
tilled water in a one liter volumetric flask. Stir until
dissolved.

2. Add 5 gm. potassium iodide and stir until dissolved.

3. Dilute to volume with distilled water, filter and store
in a one liter polyethylene bottle.

C. WORKING BIURET

 

Chemical Composition

1. Stock Biuret AR-S9-60 200 ml.
2. Alkaline Iodide, AR-127-62, q.s. 1000 m1.
Preparation

 

1. Place approximately 500 ml. of alkaline iodide in a one
liter volumetric flask.

2. Add 200 m1. stock biuret and mix.

3. Dilute to volume with alkaline iodide and mix.

Albumin Method for Auto-Analyzer

A. DISTILLED WATER WITH BRIJ-35

Wetting Agents

 

1. Add 0.25 ml. Brij-35fL. and mix.

63

B. STOCK BROMCRESOL GREEN (6 x 10’4 M)

 

Chemical Composition

 

l. Bromcresol Green 419 mg.
2. 0.1 N NaOH 10 m1.
3. Distilled Water, q.s. 1000 ml.
Preparation

 

1. Dissolve the BCG dye in the 10 m1. of NaOH and dilute to
1000 ml. with distilled water.
2. Filter. Store in refrigerator.

C. STOCK CITRATE BUFFER (0.5 M, pH 4.0)

 

Chemical Composition

 

l. Citric Acid Monohydrate 210 gm.
2. Distilled Water 1000 ml.
3. 10% NaOH

4. Distilled Water, q.s. 2000 m1.
Preparation

 

1. Dissolve the citric acid in 2 liter volumetric flask with
approximately one liter of water.

2. Adjust the solution to pH 4.0 with 10% NaOH. (Takes
approximately 600 ml. of NaOH.)

3. With distilled water, q.s. to 2 liters.

NOTE: Store in refrigerator.

D. WORKING BCG DYE

 

Chemical Composition

 

1. Stock BCG 100 ml.
2. Stock Citrate Buffer 100 ml.
Preparation

 

1. Add the BCG dye to one liter volumetric flask and slowly
add the Brij-35 and stock citrate buffer down the side.
2. 0.5. with distilled water (add slowly).

Acid-Washed Glassware

 

Wash glassware in hot, soapy water and then rinse well in tap water.

Soak glassware in 10% nitric acid for at least one hour. Rinse in running

64
tape water for not less than 20 minutes, then rinse each article twice
with the running tap water. Immerse in deionized water and rinse
three times. Drain and rinse each article twice with running double-
deionized water directly from the bottle. Dry in air oven. Follow the'

same procedure for glass pipettes.

Mg Cl Standards for Addition to Pooled Sera

2

 

Intermediate magnesium chloride standards were prepared from a stock

solution containing approximately 100 mM/L. magnesium (20.32 gm. MgClz'

6H 0 in 500 ml. deionized H20) as follows:

 

 

 

2
Intermediate Concentration, 100 mM/L. Deionized
Mg . S tandard Mg. mM/ L . M _Ii20
A 5 0.1 ml. 1.9 m1.
B 10 0.2 1.8
C 15 0.3 1.7
D 20 0.4 1.6
E 25 0.5 1.5
F 30 0.6 1.4
G 35 0.7 1.3
H 40 0.8 1.2
I 45 0.9 1.1
J 50 1.0 1.0
K 60 1.2 0.8
L 70 1.4 0.6
M 80 1.6 0.4
N 90 1.8 0.2
O 100 2.0 0.0

0.1 ml. of individual intermediate magnesium standards is added to 5 m1.
of pooled sera. One milliliter of this mixture is removed from each
specimen for total magnesium analysis. Remaining 4 m1. is used to obtain
the supernatant sample by the thermal precipitation method. All sera

and supernatant specimens are diluted 1:50 with deionized H20 and assayed

on the atomic absorption spectrosc0py.

VITA

The author was born in Almont, Michigan, on September 13, 1932.
She graduated from Almont High School in June, 1950. Enrolling at
Michigan State University in September, 1950, she received a B.S.
degree in Medical Technology in June, 1954. After an internship from
October, 1953, to October, 1954, at Grace Hospital, Detroit, Michigan,
she was registered as a Medical Technologist with the American Society
of Clinical Pathologists in 1954.

After completion of her training, she worked at St. Lawrence
Hospital, Lansing, Michigan, until June, 1955. McVing to California,
she worked at Ross-Loos Medical Group in Inglewood, California, from
July, 1955, to October, 1956. Since October, 1956, she has been
employed at Edward W. Sparrow Hospital, Lansing, Michigan, as Section
Chief in Hematology and Special Hematology. In January, 1969, She
enrolled in a program of graduate study in Clinical Laboratory Science

in the Department of Pathology.

65

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