Note: Descriptions are shown in the official language in which they were submitted.
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BE~IRINGWERKE AKTIENGESELLSCHAFT 87/B 020 - Ma 636
Dr. Ha/Bn
The use of polyvinylpyrrolidone (PVP) to r~duce the
turbidity of sera, sera containing PVP, and a process
for the preparation thereof
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The invention relates to the use o~ polyvinylpyrrolidone
to diminish a turb;dity in a dried and redissolved serum,
in particular in a dried control serum for wh;ch, after
redissolution, turbidity is to be prevented~ In this
sense, poly~inylpyrrolidone is also suitable for control
sera for lipid determinations, especially for those with
an elevated lipid content.
Control sera are to be understood to be sera which are of
human or animal origin and have a composition which is,
where appropriate, changed but like that of serum, and
which sera contain serum constituents, for example proteins,
enzymes, substrates which can be determined enzymatically,
and electrolytes, in known concentration, and are suitable
for checking methods of determination of these serum cons-
tituents. Standard sera are also to be understood to be
control sera within the meaning of the invention.
Processes for the preparation of control sera of these
types, including the adjustment of individual constitu-
ents to desired contents, have been disclosed.
In order to ensure the stability of labile components
such as, for example, enzymes or lipoproteins, it is
possible for control sera to be freeze-dried and s~ored
at low tempera~ure. Undesired side effects of freeze-
drying are turbidities ~hich occur after reconstitution
of the control sera owing to a change in the solubility
behavior of, in par~icular, the lipoproteins. These
turb;dities often interfere with spectrophotometric
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methods, so that an additional sample blank is necessary.
Turbidity gives rise to particular problems in measure-
ments in the region of 340 nm in ~hich, in particular,
those enzyme activity determinations based on NADH/NAD
measurement are carried out. The extinction of NADH,
which is intrinsically high, is further raised by the
turbidity so that it is often necessary to measure in a
range in which precise measurements are impossible. The
results become less accurate and depend greatly on the
quality of the photometer.
Methods for avoiding turbidities have already been d;s-
closed~ German Patent 3,107,060 describes the addition
of organic substances which are not sugar-like, such as
methanol, alanine, triethylene glycol, valine, acetate,
~S lactate or sodium 2-hydroxymethylbutyrate. An addition
of this type may, in the case of alanine and methylbuty-
rate, result in interferences with enzyme react;ons.
Add;tion of methanol is generally a risk to health. If
sodium acetate is used, the control serum c3n no longer
2û be used as a universal control serum for electrolyte
determinat;ons. Addition of ammonium compounds inter-
feres with determinations of urea. Other substances may
cause general interferences with assays.
Another method for avoiding turbid;t;es has already been
descr;bed in German Offenlegungsschrift 3,329,952 by
addition of proline and Na deoxycholate. However, ~his
addition has the disadvantage that it results in arti-
facts on protein fractionation by electrophoresis, and
it ren~ers the deproteinization with $richloroacetic acid more diffi
30 cult, for exampLe in the determination of creatinine.
Hence, the invention has the object of preparing a uni-
versal control s@rum which has a reduced tendency t~
turbidity after reconstitution from a dried form and
which dses not have the described disadvantages of the
known agents.
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It has been found, surprisingly, that reconstituted con-
trol sera which have less turbidity and are more homo-
geneous can be prepared if polyvinylpyrrolidone (PVP) is
added to the control serum before drying. This not only
makes the serum overall more homogeneous but also
improves the precision of determinations of the concen-
tration and activity of parameters contained in the con-
trol serum, i.e. the finding again of the declared
required values is facilitated for the user of quality
control sera.
Hence the invention relates to the use of polyvinyl-
pyrrolidone (PVP) for diminishing a turbidity and improv-
ing the homogeneity in a dried and reconstituted serum.
PVP is added in a suitable concentration to such sera~
espec;ally control sera.
It has also been found that a turbidity of the control
serum to which Lipids such as cholesterol and triglycer-
ides are added is likewise reduced with polyvinyLpyrroli~
done as further additive, and the homogeneity of the
reconstituted serum is improved.
Thus the invention also relates to a human- or animal-
based control serum in dry form and containing polyvinyl-
pyrrolidone, and to a process for the preparation of such
sera.
The effective concentration of PVP with a molecular weight
of 10,000 - 750,000 is between 1 and 2n y/l~ Preferably
2-6 g/l, and particularly preferably 3 g/l, with a mole-
cular weight of 25,000 - 30,000 are used~ The process
according to the inven~ion is suitable for the preparation
o~ control sera for clinical chemistryO i.e. of products
which are intended for use for the quality control of
clinically interesting serum parameters such as the
enzyme, substrate, (ipid, metabolite, hormone or elec-
trolyte content. It is also suitable for the preparation
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of a control serum containing measured amounts of lipids
(cholesteroL, triglycerides).
The extinction of the dried control serum which has the
addition according to the invention and has been recon-
stituted with distilled water was measured with a linespectrophotometer at 546 nm in a cuvette with a 1 cm
path length and compared with the extinction of the same
control serum without addition.
Reconstitution is defined as the dissolution of a dry
material in the amount of solvent which contained it
before drying.
The example which follows illustrates the invention.
E X A M P L E
Pooled human serum from healthy donors and bovine serum
was used as the basis for control serum. Egg-yolk
extract and bovine cholesterol concentrate were added to
part of the sera, resulting in concentrations of tri-
glycerides and cholesterol which were higher than the
figures indicated under the columns "addition of lipids"
in the table which follows. All the samples of sera
which were obtained in this way were in turn div;ded into
two portions, with the first portion being left without
addition, and to the second portion 3 g/l polyvinyl-
pyrrolidone, molecular weight 25,000-30,000 from Merck AG,
Darmstadt, Cat. No. 7443, being added in solid form and
dissolved therein.
All samples were then sterilized by filtration~ dispensed
into bottles and freeze-dried. Cholesterol, triglycer-
ides and the turbidity were me3sured after reconstitution
with distilled water.
The table shows the results of the measurements af
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cholesterol, triglycerides and, as a measure of the
turbidity, the extinction and the range of variation of
the extinction from bottle to bottle ~homogeneity) at
546 nm. It is evident from this that addition of poly-
S vinylpyrrolidone brings about with all samples a dis-
tinct reduction in the turbidity and in the coefficient of
variation (CV), a measure of the said range of variation.
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