Download Ribonucleic Acid in Plasma from Normal

CLIN. CHEM. 25/10,
1774-1779 (1979)
Ribonucleic Acid in Plasma from Normal Adults and Multiple
Myeloma Patients
Thomas C. Hamilton,1-4 Albert G. Smith,1 Charles A. Griffin,2 and Ralph J. Henderson, Jr.2’3
Reports of the presence of RNA in human plasma have
been numerous, often suggesting that RNA in plasma is
correlated with human disease. We critically examined the
methods for determination of RNA in plasma. Lack of
method specificity has caused previous workers to overestimate plasma RNA concentrations by more than 50-fold.
To isolate RNA from plasma, we used both a phenolchloroform
extraction
and a modified
SchmidtThannhauser procedure. We show that RNA in plasma can
be identified and quantified by alkaline hydrolysis of the
plasma extract and subsequent separation of the resulting
2’- and 3’-mononucieotides
by “high-performance”
liquid
chromatography. We could not detect RNA in plasma from
either apparently healthy, normal adults or multiple myeloma patients, but found 1.1 mg/L in the plasma of a patient
with WaldenstrOm’s macroglobulinemia. Our method is
useful for the specific determination of RNA in plasma and
will detect as little as 600 tg/L.
AdditIonal Keyphrases:
WaldenstrOm ‘s macroglobulinemia
intermethod comparison
sources of analytical
error
“high-performance”
liquid chromatography
fluorometry
-
The presence of RNA and DNA
ciated with several human disorders
in plasma has been asso(1-14). DNA in cell-free
human plasma has received much attention (1-5). However,
the existence
and possible roles of RNA in cell-free plasma
have not been investigated
as fully. The presence of RNA in
serum or plasma, first suggested
by Mandel and Metais (2),
has been reported
sporadically
(6-13).
There is much indirect evidence to suggest that RNA may
be found in blood plasma. Virus-related genetic material, including RNA, may be present in the plasma of patients with
cancer of the colon or rectum (14). Involvement
processes
of phagocytosis,
immunoglobulin
of RNA in the
synthesis,
and
immune cytolysis
has been suggested,and RNA metabolism
in leukemia reportedly is abnormal (15-21). RNA, administered intravenously,
is said to reverse immuno-depression
(22), and anti-RNA antibodies have been isolated from the
sera of patients of certain disease groups (23). An RNA-contaming plasma factor that can stimulate idiopathic immunoglobulin
synthesis
in vivo and in vitro has been reported
(10). The potential
for RNA to be present in plasma is sug1 Department
of Pathology, Louisiana State University Medical
Center, Shreveport, LA 71130.
2 Department
of Biochemistry and Molecular Biology, Louisiana
State
gested by many of these reports, but direct chemical proof is
lacking.
Accurate
information
on RNA concentrations
in
plasma might well be useful as a marker of certain disorders.
The reliability
and accuracy of fluorometric
and chemical
methods for measuring plasma DNA have been questioned
(3). Experience
in our laboratory suggests that there are
similar reliability
problems
with fluorometric
and chemical
RNA assays as applied to plasma (6-8).
Here, we report the use of a classic two-stage extraction with
phenol and chloroform to remove RNA from plasma, and our
results for 10 apparently
healthy adults, seven multiple
myeloma patients, and one patient with Waldenstr#{246}m’s
macroglobulinemia.
Our attempts to extract RNA from the
plasma of 10 apparently
healthy adults by use of the
Schmidt-Thannhauser
procedure are also reported.
Complications
associated with substances that co-extract
with RNA and interfere
with the identification
and quantification of RNA from plasma are discussed.
We show that
RNA, if present in plasma in concentrations
of 600 sg!L or
greater, can be identified and quantified by alkaline hydrolysis
of the plasma extract and separation
resulting 2’- and 3’-mononucleotides
raphy.
and measurement of the
by liquid chromatog-
Materials and Methods
Blood Collection
We sampled blood from apparently healthy male volunteers, from seven patients with multiple myeloma, and from
a patient
with Waldenstrom’s
macroglobulinemia.
The
specimens were collected by standard venipuncture
into
“Vacutainer
Tubes,” 100 X 16 mm (Becton-Dickinson
Co.,
‘Rutherford, NJ 07070), which contained 14 mg of sodium
ethylenediaminetetraacetate
(EDTA)
and which were
promptly placed in crushed ice, unless otherwise indicated.
Two-Stage
Extraction
Phenol-Chloroform
From
blood obtained
of RNA with Phenol and
Mixture
as described
above we separated
cell-free plasma within 30 mm of collection, by centrifugation
(4 #{176}C,
10 mm, 2000 X g) with use of “Seraclears”
to improve
the separation. The extraction was then begun (first stage)
without delay.
“Extraction mixture no. 1” was a mixture of one volume of
liquified phenol and one volume of buffer consisting of, per
liter, 5.0 mmol of Tris [tris(hydroxymethyl)aminomethane
chloridej,
0.50mmol ofEDTA, 50 mmol ofsodium chloride,
1.0 g of 8-hydroxyquinoline,
and 5.0 g of sodium dodecyl sul-
University
Medical Center, Shreveport,
LA 71130.
Address reprint requests
to this author.
Present
address:
Tenovus
Institute
for Cancer Research, The
Welsh National
School of Medicine,
The Heath, Cardiff CF4 4XX
fate (pH 7.4).
One volume of plasma
(20-40 mL) was mixed with two
volumes of “Extraction
mixture no. 1” on a magnetic
stirrer
Wales, U.K.
Received Mar. 26, 1979; accepted
at 4 DC for 30 mm. After this mixing, the resulting emulsion
was broken by centrifugation
(4 #{176}C,
10 mm, 39 000 X g) and
1774
CLINICAL CHEMISTRY,
July 11, 1979.
Vol. 25,
No. 10. 1979
A
0.015
B
0.015
0.010
0.010
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ii
0.005
0
Fig. 1. Liquld-chromatograms of samples
hydrolyzed with KOH
0.005
$
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2
INJECT
4
4
3
2
101214
ECT
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4
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IC
TIME IN MINUTES
TIME IN MINUTES
0.015
4
A. chromatogram of a hydrolyzed yeast RNA standard solution(20 mg/liter). B,Chromatogram of a
hydrolyzed RNA-enriched extract(plasma was enriched with yeast RNA, extracted by our two-stage
phenol-chloroform method, and the extract hydrolyzed with KOH). C, Chromatogram typical of that
obtained from hydrolyzed extracts of both normal
plasma and plasma from multiple myeloma patients;
extracted by our two-stage phenol-chloroform
method. 0, Chromatogram of the only hydrolyzed
plasma extract that was positivefor RNA (plasma
from a patient with WaldenstrOr’s macroglobulinemia). CMP, cytidlnemonophosphate; AA*’,
adenosine monophosphate; UMP. urldlne monophosphate; GMP, guanosine monophosphate. Retentiontimes were determined with known 2’- and
3’-nucleoside
monophosphates. Volumes for all
sample injections: 100 ML.
D
0.0I5
0.010
0.010
Ii
U
0.005
0.005
0
2
INJECT
4
0
3
10
12
$244
INJECT
14
TIM!IN MINUTES
0
10
12
14
TIMEIN MINUTES
the aqueous
phase was re-extracted
(second stage) once as
above, but with an equal volume of “Extraction
mixture no.
2,” which consisted
of two volumes of the buffer used in
solution.
The recovery of RNA from plasma (n = 6) was 56.8
± 3.4% (mean ± SD).
Figure 1, A and B, shows typical chromatograms
of a hy-
“Extraction
mixture no. 1,” one volume of liquified phenol,
and one volume of chloroform (10).
After this mixing, the emulsion was broken by centrifugation as above. The aqueous phase was mixed with three volumes of 95% ethanol at -20 #{176}C,
and held at -20 #{176}C
for 4 h, to
drolyzed yeast-RNA standard solution and a hydrolyzed extract of yeast-RNA-enriched
plasma, respectively. The between-run CV for three hydrolyzed RNA standard solutions
was 8.4%. We could detect a minimum of 0.2 sg of RNA as
hydrolyzed
RNA (2’- and 3’-nucleoside monophosphates)
injectedintothe liquidchromatograph.As little
as 600 ig
precipitate
RNA. The precipitate,
obtained by centrifugation
(-15 #{176}C,
10 mm, 39000 X g), was dissolved
in 3.0 mL of so-
dium chloride solution
(154 mmol/L)
to yield the plasma ex-
tract.
For quantification
of RNA in this extract, a
was combined
with 0.20 mL of potassium
mol/L) and incubated
at 50 #{176}C
for 16 h, to
drolyze RNA to 2’- and 3’-nucleotides.
After
1.8-mL portion
hydroxide
(10
completely
hyhydrolysis,
the
solutionwas neutralizedwith perchloricacid (6.0mol/L),
cooled in crushed ice for 15 mm, centrifuged to remove potassium
perchlorate,
and filtered
through
an 8.0-ftm
(av pore
size), 13-mm polycarbonate
membrane (Nucleopore Corp.,
Pleasanton, CA 94566). The filtered sample was then examined for the products of KOH-hydrolyzed
RNA-2’and 3’nucleoside monophosphates-by
liquid chromatography
as
described below.
The analytical recovery of RNA from plasma by the above
procedure was assessed by adding 100 zg of yeast RNA to
20 mL of plasma and then extracting the RNA according to
the two-stage procedure
described
above. A recovery of 100%
was established
for an RNA standard
solution (20 tg of yeast
RNA per milliliter of isotonic saline) with the extraction
process omitted. The percentage recovery of extracted RNA
was determined by comparing the total nucleotide peak areas
of hydrolyzed plasma extract and hydrolyzed RNA standard
of RNA per liter of plasma could be detected
by our analysis
scheme (20 mL of plasma extracted;
50% extraction efficiency
for RNA; extracted RNA dissolved in 3.0 mL volume, 0.1 mL
of which was injected into the chromatograph).
Modified Schmidt-Thannhauser
Blood
was collected
and
plasma
Method
prepared
as described
above, and 10 aliquots of plasma were stored at -20 #{176}C
until
analysis. Plasma, 2.0 mL, was mixed for 10 mm with 1.5 mL
of cold perchloric acid (0.60 mol/L) at 4 #{176}C
to precipitate
protein and RNA (24, 25). The precipitate was sedimented
by centrifugation,
and washed twice with 3.0-mL aliquots of
cold perchioric acid (0.20 mol/L). The precipitate was dissolved in 2.5 mL of potassium
hydroxide (0.30 mol/L) and
incubated
with occasional mixing at 37#{176}C
for 1 h, to hydrolyze
RNA into acid-soluble short-chain polynucleotides
and mononucleotides (6, 7,24-26). The solution was cooled on ice for
5 mm and 0.15 mL of perchioric acid (12 mol/L) was added to
precipitate protein, which was removed by centrilugation. The
acidic supernate was neutralized with potassium hydroxide
(10 mol/L), cooled for 15 mm in crushed ice, centrifuged to
remove potassium perchiorate, and then concentrated
by lyophilization.
The lyophilized material was dissolved in 0.50 mL of poCLINICAL CHEMISTRY, Vol.
25, No. 10, 1979
1775
1.4
1.4
1.4
I
A
C
I.!
1.2
1.2
1.0
I.e
1.0
04
04
Di
03
04
03
0.4
0.4
0.4
0.2
0.2
01
220
240 2*0 230 300 320
NAJIOMETEIS
220
240
260 230
NANOMETEIS
240 260 230 300 320
NAISOINETEIS
220
320
Fig. 2. A, Ultraviolet absorbance spectrum of polyvinyl sulfate dissolved in water. B, Ultraviolet absorbance spectrum of an extract
of 20 mL of plasma, extracted by Scherrer and Darnell’s method as modified by Chen et aI. (10), treated with proteiriase K, and
re-extracted with phenol. C, Ultraviolet absorbance spectrum of an extract of plasma prepared by Scherrer and Darnell’s method
as modified by Chen et at. (10)
tassium
hydroxide
(1.0 mol/L) and incubated
for 16 h at 50
#{176}C
to completely
hydrolyze
RNA fragments
to 2’- and 3’nucleoside
monophosphates
(24, 25). The samples were then
neutralizedwith a 6 mol/L solution of perchloric acid, cooled
for
15
mm
in crushed
ice, centrifuged
to remove
perchlorate, filtered,and chromatographed
below.
Liquid
potassium
as described
Chromatography
We separated and quantified
the nucleotidesresultingfrom
the alkaline hydrolysis of RNA by chromatography on a
PXS-1025
SAX high-performance
anion-exchange
column
(Whatman, Inc.,Clifton, NJ 07014) at room temperature
(22
#{176}C),
with use of a Model LC-55 detector (Perkin-Ehner Corp.,
Norwalk, CT 06856) set at 260 nm. The eluant flow rate was
held constant at 1.12mL/min.
For nucleotidesto be eluted in a reproducible pattern as
shown in Figure 1,it was necessary to control elution condi-
tions as follows. Ammonium phosphate buffer (750 mmolfL,
pH 3.70) was used to regenerate the column for at least 15 mm
at the beginning
of each day and between
each sample
run.
Fourteen minutes (±1 mm) before sample injection, the eluent
was switched from the strong ammonium
phosphate buffer
(750 mmol/L, pH 3.70) to a more dilute ammonium
phosphate
buffer
(30 mmolfL,
pH 3.70). After the 0.100-mL
sample was
injected,
nucleotides
were eluted during the next 15 mm, as
illustrated
in Figure 1. Retention
times may vary with the
chromatographic
system used. After elution, the column was
cleared and regenerated
by again pumping
the strong ammonium phosphate
buffer through the column for 15 mm.
method that is based
on the enhanced fluorescence
of ethidium
bromide when it is
bound to RNA (8, 13, 27, 28). For these studies, a 20 mg/L
solution of ethidium bromide was prepared in phosphatebuffered
saline (per liter, 67 mmol of Na2HPO4,
13 mmol of
KH2PO4, and 154 mmol of NaCl), pH 7.4. Equal volumes of
diluted plasma and ethidium
bromide solution were mixed.
The fluorescence
intensity
of this solution was measured in
arbitraryunitswith an Aminco-Bowman
spectrofluorometer
(American Instrument Co., Inc.,SilverSpring, MD 30910),
the excitationand emission wavelengths being setat 546 and
590 nm, respectively.
1776
CLINICAL
used in this evaluation
CHEMISTRY,
above and processed
according
to the original
method (8) except forthe followingmodifications.Within 10
s of blood collection,
one volume of whole blood was diluted
with either two volumes of the pH 7.4 phosphate-buffered
salineor with two volumes of a 30 mmol/L solutionof iodoaceticacidin thisbuffer.Diluted cell-free
plasma was obtained
from thisdilutedwhole blood by centrifugation(4 #{176}C,
10 mm,
2000 X g). In some experiments, buffer-diluted
plasma was
treated with pancreatic ribonuclease (EC 3.1.27.5; 9.8 g/L;
Worthington
fluorometric
Biochemical
analysis
Co., Freehold,
NJ 07728) before
(see Results).
Results
We examined
several extraction
schemes in an attempt
to
finda procedure by which RNA could be isolated from plasma
in good yield. The possibility that plasma RNA could be destroyed by endogenous
ribonucleases
during a standard
phenol extraction similar to that described by Kirby (29)
prompted our examination of Scherrer and Darnell’s extraction method (30) as modified by Chen et al. (10). The extraction medium utilized in the method of Chen et al. contains
several ribonuclease
inhibitors,
including bentonite
and
polyvinyl sulfate. An ultraviolet absorbance spectrum of a
plasma extract prepared by this method (10) was suggestive
of multiple components, including protein (Figure 2C).
Our attempts to purify the plasma extract by treatment
with proteinase
K (EC 3.4.21.14), followed by re-extraction
with phenol, resulted
in an extract
having the absorbance
spectrum shown in Figure 2B, indicating the removal of substantialamounts of protein. Although the 260/280 nm ab-
sorbance ratio for this material is 1.5, suggestive of partly
purified RNA, the absorbance spectrum (Figure 2B) is not
Fluorometric
Assay
We evaluated an RNA quantification
Blood samples
described
were collected
Vol. 25, No. 10, 1979
as
characteristic
of
RNA.
260/280 nm absorbance
did not report spectral
Chen
et al. (10)
reported
similar
ratios in their plasma extracts, but
characteristics.
Our examination
of
each component in the extractionmixture led to the observation that polyvinyl sulfatedissolved in water had an absorbance spectrum as seen in Figure 2A and a 260/280 nm
absorbance ratio of 1.5. The similarityof Figure 2A (polyvinyl
sulfate)and Figure 2B (plasma extract)suggested that the
character of the absorbance spectrum for plasma extract
prepared
in this laboratory
was possibly attributable
to
polyvinyl sulfate. Extraction of water rather than plasma, as
described above, resulted in an extract with an absorbance
spectrum identical to that obtained for the extract of plasma
(Figure 2B). This strongly suggests that the absorbance
spectrum of the plasma extract prepared by this method is
ascribable to polyvinyl sulfate from the extraction medium
rather than to any plasma component. Thus, the criteria used
by Chen eta!. (10)to assess the purity of their plasma extracts
may not be valid.
Phenol-Chloroform
0.7
0.6
Extraction
The plasma samples from the above-described persons were
extracted by the two-stage method, the extracts were hydrolyzed, and the hydrolysates were analyzed by chromatography
for 2’- and 3’-mononucleotides.
Small peaks characteristic of
mononucleotides
were seen in the chromatograms
of hydrolyzed extract of plasma from the patient with Waldenstr#{246}m’s
macroglobulinemia
(Figure 1D). On the basis of the amount
of plasma extracted (22 mL) and assuming a 50% analytical
recovery, we calculated the concentration of RNA
in the
plasma of thispatientto be 1.1mg/L, wellbelow the detection
limit of chemical or fluorometric plasma RNA methods. In
0.5
u.a
0.4
Li
z
0
vs
contrast, no hydrolysate of any of the other plasma extracts
exhibited characteristic mononucleotide peaks, although some
did exhibit small atypical peaks in the elution region of the
0.2
2’- and 3’-mononucleotides.
We established that these atypical peaks were in fact not
mononucleotides,
and thus were not derived from RNA, in
three ways. Firstly, under the defined chromatographic
elution scheme we used, the atypical peaks were not eluted in a
pattern characteristic
of nucleotides as seen in our controls
(Figure 1, A and B). Secondly, samples were chromatographed
three times with the detector set at 250, 260, and 280 nm, respectively. The 250/260 and 280/260 absorbance ratios of these
atypical peaks were not characteristic
of adenine-, guanine-,
uracil-,or cystosine-nucleotides.
Thirdly, the atypicalpeaks
were unaffected by treatingthe sample with alkalinephosphatase
(EC 3.1.3.1), which catalyzes the hydrolysis of nu-
cleotides to nucleosides and inorganic phosphate, thus eliminating the nucleotide peaks in the chromatograms.
In contrast, nucleotide peaks were eliminated from control samples
containing hydrolyzed yeast RNA when they were treated
with alkaline phosphatase.
A chromatogram
of a hydrolyzed
plasma extract devoid of mononucleotides
is seen in Figure
1C and a chromatogram
of a positive hydrolyzed plasma extract is seen in Figure 1D.
The isolation of RNA from plasma by a method not involving phenol (a modification of the Schmidt-Thannhauser
method) has been reported (6, 7). We attempted
to isolate
RNA from plasma by such a procedure. Ten different plasma
extracts obtained by the modified Schmidt-Thannhauser
method yielded absorbance
spectra typical of protein;a typical
spectrum for such an extract is shown in Figure 3. All such
spectra have an absorbance
mum
of 257 nm.
maximum
As a control,
at 280 nm and a mini-
we prepared
a Schmidt-
Thannhauser
extract of rat liver, and it exhibited a marked
absorbance maximum of 260 nm, a minimum at 230 nm, and
a 260/280 nm absorbance ratio of 1.9, all features characteristic
of RNA. In addition, a few Schmidt-Thannhauser
extracts
of normal plasma were reacted with orcinol (31). The absorbance spectrum of each orcinol-treated
extract exhibited
a peak at 550 nm, which is atypical of RNA. An absorbance
peak at 670 nm, characteristic
of RNA, was demonstrated in
orcinol-treated
extracts
of rat liver and yeast RNA
standards.
Analysisof Schmidt-Thannhauser plasma extractsby the
much more sensitive and specific “high-performance”
liquid
chromatographic
method yielded no chromatograms
with
characteristic
mononucleotide
peaks.
Plasma aliquots enriched with 10 zg of yeast RNA were
extracted according to the Schmidt-Thannhauser
procedure,
as were similar aliquots of normal plasma. No significant
difference was demonstrated
between the mean absorbance
0.1
220
240
260
300
280
NANOMETERS
Fig. 3. Ultraviolet absorbance spectrum of an extract of normal
plasma
according
to Schmidt-Thannhauser
at 260 nm (A = 0.146) of extracts of threenormal plasmas and
the mean absorbance (A = 0.162) of extracts
of three RNAenriched
plasmas (p> 0.01). However, use of the more sensitive “high-performance”
liquid chromatographic
technique.
gave chromatograms
with characteristic
mononucleotide
peaks when hydrolysates
of Schmidt-Thannhauser
extracts
of RNA-enriched
plasma were analyzed. No mononucleotide
peaks
were
tracts
of normal
seen
FluorometriC
in chromatograms
plasma
when
hydrolysates
of ex-
were so analyzed.
Assay
When plasma was subjected to the ethidium bromide-RNA
fluorometric
assay, there was fluorescence,
but it was not
decreased after ribonuclease treatment of plasma. Specifically,
blood from four normal adults, diluted
with buffer
scribed in Methods,
gave fluorescence
values (arbitrary
as de-
samples
for 2 h
units)
of 12, 17, 19, and 23, respectively, when mixed with an equal
volume of ethidium bromide reagent. However, no fluorescence decrease was observed when each of these plasma
was incubated
with pancreatic
at 37 #{176}C
before the fluorometric
ribonuclease
assay. Evidently
ribonucle-
ase-sensitive
RNA is absent from normal plasma. In the fluorometric
assay, yeast RNA (100 mg/L) in saline (154 mmol/
L) gave an arbitrary
fluorescence
value of greater than 80,
decreasing
to 1.0 after ribonuclease
treatment,
the fluorometric
assay was functioning
clease was active. We also observed
that
tive ribonuclease
inhibitor
(8, 13), had
served fluorescence,
and that plasma
added RNA) may be processed
at either
affecting their fluorescence
intensity.
showing that
and that the ribonuiodoacetate,
a puta-
no effect on the obspecimens
(without
4 or 25 #{176}C
without
CLINICALCHEMISTRY,Vol. 25, No. 10, 1979 1777
Using the fluorometric
assay, we studied the effects of endogenous plasma ribonuclease
on RNA by preparing
a solution of yeast RNA in buffer-diluted
plasma (30 mg/L). Portions of this solution were incubated
at 4 and 25 #{176}C,
respectively, for 4.5 h. Arbitrary
fluorescence
decreased
from 38 to
25, for the sample incubated
at 25 #{176}C
(with and without iodoacetate),
whereas the sample incubated
at 4 #{176}C
showed
virtually
no change in fluorescence.
These results for plasma
with added RNA indicated that any endogenous
RNA present
in blood would not have a chance to be degraded if the sample
is cooled in ice immediately
after it is obtained
from the test
subject and further
processed
within 30 mm as described
in
Methods.
Discussion
The procedure
described
here allows quantification
of as
little as 600 g of RNA per liter of human plasma. RNA is
removed from plasma by first extracting
it with phenol, then
with a phenol-chloroform
mixture. The extracted RNA is then
hydrolyzed
to 2’- and 3’-nucleotides,
which are separated and
quantified
by liquid chromatography.
The two-stage
extraction eliminates
most of the protein that may retard alkaline
hydrolysis
of RNA (24, 25). The chromatography
separates
the 2’- and 3’-nucleotides
from most interfering
substances
and provides
qualitative
identification
in the form of a reproducible
elution pattern characteristic
of KOH-hydrolyzed
RNA (Figure
1).
In addition
to elimination
of most plasma
proteins,
the
reasonable
analytical
recoveries and recovery precision,
and is similar to a plasmaextraction method described by Chen et al. (10). Thus, results
by our method
can be compared
with those previously
reported (10). Moreover, our method eliminates
the inseparable
emulsion
that often is obtained
with the phenol-chloroform
method reported
by Chen et al. (10).
two-stage
extraction
Attempts
method
gives
to isolate RNA from plasma by the Chen et al.
yield
a color
processing
by Chen et a!. (10) in plasma
ex-
tracts may well have been the result of polyvinyl sulfate contamination
rather than being produced by any component
extracted
report
from plasma.
of idiopathic
We do not wish to imply that their (10)
immunoglobulin
synthesis
by normal
lymphocytes
after exposure
multiple myeloma patients
to phenol extracts of plasma from
is an artifact. However, we suggest
that if any RNA is present in the plasma extracts, it is much
less than is implied by the 260/280 nm absorbance
ratios reported
by Chen et al. (10), and that these ratios have no
bearing on the purity of preparations
with respect to RNA.
Our liquid-chromatographic
analysis of hydrolyzed extracts
from 18 plasma samples gave only one chromatogram
characteristic
of hydrolyzed
RNA; this was for plasma from a patient with Waldenstr#{246}m’s macroglobulmnemia.
This type of
plasma is viscous, making it difficult to separate
plasma and
cellular components.
Although the results must thus be viewed
with caution, they suggest that the concentration
of plasma
RNA we found may be a characteristic
of the disease.
Schmidt-Thannhauser
extracts of plasma appear to be
contaminated
with protein (Figure 3). Protein contributes
greatly to the extract’s absorbance at 260 nm, and some material other than RNA in the extract reacts with orcinol to
1778
CLINICAL CHEMISTRY, Vol. 25,
No. 10, 1979
to RNA. The
substances
iodoacetic
in plasma.
acid (at the concentration
used)
blood
after
it is removed
from
the
test
subject
mg of RNA per liter. Our work shows that the lowest
of these
reported values is at least 58-fold too high, based on our
minimum detection level of 0.6 mg of RNA per liter (34.9/0.6
= 58), although
RNA may be present
in normal plasma in
concentrations
that are below our detection limit. That RNA
is present in some unusual form protected from ribonuclease
or unextractable
by classic methods, or both, seems unlikely,
but is not excluded by our present study.
2. Mandel,
RNA). These data suggest that the high
attributed
makes it unlikely that endogenous RNA was destroyed. Because plasma specimens without added RNA could be processed at either 4 or 25 #{176}C
with no effect on ethidium bromide
fluorescence, we conclude that no measurable RNA was being
destroyed by endogenous ribonuclease in our experimental
work.
The concentration of RNA, as of DNA (3), in normal plasma
appears to be very low. Various workers (2, 6-8, 13) have reported plasma RNA concentrations
ranging from 34.9 to 144
tain any extractable
reported
be mistakenly
did not inhibit endogenous plasma ribonuclease. The possibility that RNA, present at the time of blood collection, was
destroyed by endogenous ribonuclease had to be considered.
However, studies with yeast RNA-enriched
plasma showed
that RNA, although considerably degraded in plasma at 25
#{176}C,
is not significantly degraded in plasma kept at 4#{176}C
for no
longer than 4.5 h. Thus, our practice of promptly cooling and
script.
ratios
can
with interfering
In our hands,
(10) modification
of Scherrer and Darnell’s extraction method
(30) resulted in an extract with complex absorbance characteristics
(Figure
2C), suggestive
of multiple
components.
Proteinase
K treatment
removes much of the material
absorbing in the 260-280 nm region of the spectrum
(Figure 2B),
and results in a spectrum
virtually
identical
to that of polyvinyl sulfate in water (Figure 2A). We showed that polyvinyl
sulfate is distributed
into the aqueous phase, precipitated
by
ethanol, and is present in the extract (which would also conabsorbance
that
absorbance
spectra of orcinol-treated
plasma extracts are
unlike the spectra of the RNA-orcinol product. Majumder et
al. (32) reported similar problems in estimating the RNA
content of another complex protein matrix, milk.
Our results with the fluorometric assay for plasma RNA (8,
13) suggest that it yields inaccurate results. The failure to see
a difference in fluorescence after treatment of buffer-diluted
plasma with ribonuclease strongly suggests that the fluorescence observed is the result of ethidium bromide’s interaction
We thank
Ms. Deborah
W. Cox for assistance
with
the manu-
reaction
of human
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