Accurate Preparation of Solutions
of Hygroscopic Salts: Mg2 for
High-Density Lipoprotein
Cholesterol Assay
v.C
To the Editor:
A popular method for high-density lipoprotein (HDL) cholesterol involves
precipitation
of low- and very-lowdensity lipoproteins from plasma or
serum by means of a magnesium chloride-phosphotungstic
acid solution (1).
The commercially available form of
magnesium chloride is the hexahydrate,
which is quite hygroscopic.
Thus the
concentration of a magnesium chloride
solution prepared by weighing the calculated amount of the commercial salt
and dissolving it in the desired volume
of solution may be significantly less than
that calculated, depending on the degree
of hydration
of the salt. Because of the
critical nature of the HDL-cholesterol
test, it would seem imperative that the
concentrations of the components of the
precipitant solutionsbe accurate and
reproducible.
A common procedure used in preparing solutions
of hygroscopic
salts of
definite concentration is to make a more
concentrated
solution and measure its
concentration,then dilute to the desired
concentration. However, solutions of
magnesium chloride more concentrated
than 1 mob/L are cloudy and viscous,
and are not adaptable to the above
technique.
I describe
here a simple
method
for
preparing definite concentrations of
magnesium chloride solutions from the
commercial salt in any degree of hydration. We use a 1.0 mol/L solution for
our method, and the procedure that
followsis designed
to obtain
with Mg2 concentration
and 1.03 mob/L.
a solution
between 0.97
Add 25.0 g of MgCl2.6H20 to 100 mL
of de-ionized water and stiruntil dissolved. Dilute 1.00 mL of this solution to
250 mL with de-ionized
water. Measure
the Mg concentration
of the diluted
solution in duplicate (we use the “MG”
test on the DuPont aca). If the average
Mg concentration is between 94 and 100
mg/L, use the original solution as is. If it
is less than 94 mg/L, use equation 1 to
calculate
the weight of extra salt to be
added to the original solution.
(g)
=
C
-
(96 (v
-
100)/v)
(1)
where C = the Mg concentration of the
diluted solution and v = the volume of
the original solution.
(2)
volume (mL) =
v
The equations can be used in preparing solutions of other concentrations
or other reagents, if cast in the general
forms:
--
weight (g)
=
C
-
g1.(Cf-C)
(C1#{149}
(v v)/v)
-
volume (mL)
where gj
the initial
of substance added,
=
=
v.C
C1
-
v
g
V2
v-vt
-=------orv2=(v-v1).-
gi
(3)
The following equations are derived
of molefL:
(4)
% purity/Mr
(5)
v + V2
(Mr is the relative molecular massformerly called “molecular weight”)
Dividing equation 5 by equation 4:
(gi + g)
g’/v
+ v2)
in high-density
lipoproteins
-
-C1C
separated
Chem.
three different methods. Clin.
882-884 (1977).
William
gj
gj . % purity/Mr
(6)
I. Lopes-Virella, M. F., Stone, P., Ellis, S.,
and Colwell, J. A., Cholesterol determination
are indepen-
with gj:
(gj + g)/(v
-
Reference
Cl ions is assumed to be negligible).
Becausethe amount of water associated
with g is proportional to that associated
=
-
(2a)
-
=
g
= v + (v
v1) .
(C1. gj/v
gj
The above equation is then solved for
g to obtain equation la.
Derivation
of equation
2a: The
number of moles of salt is constant before and after dilution, because the
number of moles is proportional to the
product of volume times concentration:
C.(g1+g)
This equation is then solved for volume (mL) to obtain equation 2a.
dent of the concentration units used. In
practice, the Cf’s in equation la are decreased by the quantity
(C1.volume
taken for analysis)/v and the Cf in
equation 2a is increased by the same
amount to correct for the error incurred
in removing an aliquot of the original
solution for analysis.
Derivation
of equation
la. Let: gj =
grams of salt necessary
to obtain the
initial concentration, C; g = additional
grams of salt necessary to obtain the
final concentration, Cf; v = volume of
solution obtained when gj grams of salt
are added to the initial volume v of
water; and v2 = volume of water associated with the amount of salt g. Then v
= the volume of water associated with
the amount of salt g1 (the volume change
due to the dissolution of the Mg2 and
C
equa-
(la)
number of grams
C1 = the desired
as above. The equations
from the definition
and substituting
v2
v.C(v+mL).C1
final concentration, v = the volume of
water initially added to make the original solution, and the other symbols are
g
Solving for
tion 3:
-
and
Cf
25(96-C)
weight
If the average Mg2+ concentration of
the diluted solution exceeds 100 mg/L,
use equation 2 to calculate the volume of
water to be added to the original solution.
Laboratory
St. Anthony’s
Medical
10010 Kennerly
Rd.
St. Louis, MO 63128
by
23,
A. Joern
Center
Effect of Cyanide on Radioassay
for Serum Cobalamin
To the Editor:
Cooper and Whitehead (1) recently
presented results of a study demonstrating that the serum cobalamin (vitamin B12) concentration in 10-15% of
patients with pernicious
anemia
may
appear to be within the normal range
(>200 ng/L)
when measured by ra-
dioassay, whereas no patient had a
serum concentration exceeding 100 ng/L
when measured microbiobogically by use
of Euglena gracilis. In the same issue
with this report, studies by Kolhouse et
al. (2) elegantly demonstrated that these
high serum cobalamin values obtained
by radioassay were attributable to unidentified and biologically inactive cobalamin analogs that compete with radioisotopic cyanocobalamin for binding
sites on the R binder. This results in a
spuriously high serum cobalamin concentration, even though the true serum
cobalamin concentration is low. This
was not observed
when intrinsic factor
(IF) was used as the binding determinant in the radioassay, because these
inactive analogs do not bind to this
macromolecule.
Our laboratory has been using a ra-
CLINICALCHEMISTRY,Vol. 25, No. 4,
1979
639
Table 1. Intrinsic Factor and R Binder Used In Radloassay of Cobalamin in
Five Sera Extracted in the Presence of Zero, Low, and High Cyanide
Concentration
serum cobalamln. naiL
Apparent
No cyanIde
R
10 sg sodium cyanIde
IF
R
27
102
0
56
51
56
95
39
45
138
Mean ± SD 32.4 ± 20 89.4 ± 3.4
p < 0.01
We describe the measurement
cyanIde
IF
Analyzer
To the Editor:
100 sg potassIum
IF
Measurement of Total Iron-Binding
Capacity with a Centrifugal
R
67
119
103
251
49
33
115
33
67
120
112
238
51
226
87
0
49
132
95
385
43.2 ± 28 101.4 ± 34 78.8 ± 35 243 ± 96
p<0.01
p<0.01
of total
iron-binding
capacity (TIBC) in serum
with a centrifugal
analyzer. This involves saturation
of the iron binding
sites with ferric ammonium
sulfate in
tris(hydroxymethyl)aminomethane
at
pH 8.5, followed by a direct analysis for
excess
triazine
using
2,4,6-tripyridyl-s(TPTZ). The physiological iron
iron
concentration in serum is measured
separately. From these two analyses, the
total iron-binding
lated.
dioassay with R binder for about 10
years (3), and although we have ob-
30% of circulating cobalamin (6). The
analog resulting
from this extraction
served higher values for serum cobalamm in patients with pernicious anemia
than reported
for microbiological
assay
(4), we cannot recall seeing such a patient with a florid megalblastic
anemia
whose serum cobalamin concentration
exceeded200 ng/L. This suggested to us
procedure would appear to be more
that the unidentifiable cobalamin analog may not be a natural component of
serum but rather an artifact of the radioassayprocedure. One specific step in
the method that could result in cobalamm alteration is the heat extraction and
ommend
deproteination
of serum in the presence
with R binder than the [57Co] cyanocobalamin
tracer, because a spuriously high serum cobalamin concentration is measured when the pre-extraction serum cobalamin concentration
reactive
is low. In view of these findings, we recthat the concentration of cyanide be low (<2.5 mg/L of extraction
mixture),
or even omitted
the binding
dioassay.
Our
preference
is to use R
binder rather than IF, because of its
greater stability and higher affinity for
cobalamin.
It is clear that with or without cyanide
in the extraction mixture, the apparent
cobalamin concentration when R binder
ficiency becausecurrent
is used significantly
higher
than
the
results obtained when using IF. However, when the cyanide in the extraction
mixture was increased from 0.2 tmol to
1.53 4mol, the cobalamin concentration
obtained with R binder was within the
normal range for four of the five sera.
Higher values were also obtained with
high cyanide and IF as the binder, but
all were still within the diagnostically
low range.
These findings confirm the observations of Kolhouse
et al. that some en-
dogenous cobalamin analogs may be
present in serum. In addition, however,
a high cyanide concentration in the extraction evidently may result in the
conversion
of additional serum cobalamm to these analogs. Although their
extraction procedure did not appear to
affect [57Co]cyanocobalamin,
it is known
that excess cyanide at an acidic pH can
alter adenosylcobalamin
(5), and this
analog may constitute
640
between 20 and
CLINICAL CHEMISTRY,
Vol.
25,
No. 4,
is
in the ra-
Whereas in our method 10 sg
cyanide is used in the extraction mixture, Kolhouse et al. use 100 tg
of potassium
cyanide. Accordingly,
we
measured serum cobalamin
from five
patients
with pernicious
anemia, after
extracting
each sample without any cyanide, with 10 tg of sodium cyanide, and
with 100 zg of potassium
cyanide, using
both IF and R binder in the radioassay
(Table 1).
of cyanide.
of sodium
if R binder
determinant
References
1. Cooper, B. A., and Whitehead, V. M., Evidence that some patients with pernicious
anemia are not recognized by radiodilution
assay for cobalamin
in serum. N. Engi. J.
Med. 299, 816-818 (1978).
2. Kolhouse, J. F., Kondo, H., Allen, N. C., et
al., Cobalamin
analogues are present in
human plasma and can mask cobalamin deradioisotope dilution
assays are not specific for true cobalamin. N.
Engi. J. Med. 299, 785-790 (1978).
3. Rothenberg, S. P., A radioassay for serum
B12 using unsaturated transcobalamin
I as
the the B12 binding protein. Blood 31,44-54
(1968).
4. Rothenberg,S. P.,Applicationof competitive ligand binding for the radioassay of
vitamin B12 and folic acid. Metabolism
22,
1075-1082 (1973).
5. Hogenkamp, H. P. C., The Chemistry
of
Cobalamins and Related Compounds: Cobalamin, Biochemistry and Pat hophysiology, B. M. Babior, Ed., John Wiley and Sons,
New York, NY, 1975, pp 21-73.
6. Linnell, J. C., Hussein, H. A.-A., and
Matthews, D. M., A two-dimensional chromatobioautographic method for complete
separation of individual plasmacobalamins.
J. Clin. Pat hol. 23,820-821(1970).
Sheldon
P. Rothenberg
John Lawson
New York Medical College
Metropolitan
Hospital
Medical
Center
New York, NY
1979
capacity
is calcu-
We analyzed for iron in serum with
the Basic AutoAnalyzer
(Technicon
Corp., Tarrytown,
NY 10591) by the
method
of Kaupinen and Gref (1).
Serum iron analysis with the CentrifiChem System 400 was performed
by the
method of Johnson (2), adapted for the
centrifugal
analyzer
from the manual
procedure
of O’Malley et al. (3). In this
assay, ferric ion is reduced to the ferrous
state by ascorbic acid, complexed
with
TPTZ, and measured colorimetrically.
Serum specimens
are run twice in the
centrifugal
analyzer: first to obtain absorbancy
values
without
TPTZ
and
second with TPTZ present, to form the.
colored complex. Serum iron concentration is then calculated
from the absorbancy
difference.
Table 1 shows the
centrifugal
analyzer
settings
for this
analysis.
TIBC in serum was measured
with
the basic AutoAnalyzer
by the semiautomated
method of Giovanniebo et al.
(4), with ascorbic acid as the reducing
agent.
TIBC was measured
with the centrifugal analyzer by the following procedure:
Dilute 0.6-mL of serum to 1.4 mL with
tris(hydroxymethyl)aminomethane
(3.0
mol/L, pH 8.5, and containing
32.5 mg
of ferric ammonium
sulfate.12
H20).
This is equivalent
to 5000 g/L of serum.
Incubate
the mixture for 5 mm at room
temperature
and analyze for serum iron
(Table 1). The final pH of the reaction
mixture after addition of TPTZ reagent
should be 7.3.
Calculations:
TIBC = [5000 + serum
iron (physiological value)] - (serum iron
excess).
were collected from 87
whose serum iron data ranged
from iron deficiency to iron excess.
These sera were analyzed for TIBC by
Blood samples
patients
the two methods described here, and the
results are shown in Figure 1. The TIBC
methods
correlate
well over a wide
clinical range.
The standard
curve for the TIBC
method with the centrifugal analyzer is
linear from 1000 to 5000 isg/L. No interference was observed with icteric