CLIN. CHEM.23/8, 1427-1435 (1977)
High-PerformanceLiquidChromatographicSeparationand
Quantitationof Nucleosidesin Urineand Some Other Biological
Fluids
George E. Davis,1 Robert D. Suits,1 Kenneth C. Kuo,2 Charles W. Gehrke,2 T. PhillIp Waalkes,3 and
Ernest Borek4
Precise, quantitative
high-performance
liquid chromatography of samples equivalent to 25 l of urine can be
done in less than 1 h with excellent resolution and recovery
of pseudouridine,
1-methyladenosine,
1-methylinosine,
N2-methylguanosine,
adenosine, and N2,N2-dimethylguanosine. These six nucleosides are present in normal
urine in concentrations ranging from 0.4 to 60 mg/liter. The
use of an affinity chromatograph
column with a bound
boronic acid functionality has enabled us to retain nudeosides selectively as boronate complexes from biological
samples as complex as urine. The nucleosides are readily
eluted with dilute formic acid from the boronate affinity
column, concentrated, and separated by reversed-phase
liquid chromatography on “hz Bondapak/C18” columns. In
addition, about 10 other nucleosides are resolved and
present in normal urine, 0.2 to 4 mg/liter. Isocratic chromatographic quantitation gave a minimum detection limit
of 5 pmol or less for each of the nine nucleosides examined; the relation between peak area and concentration
was linear with average CV’s of less than 3% for six nucleosides over the range of 100 to 1000 pmol for each
nucleoside injected. Analytical recoveries of nucleosides
from standard mixtures exceeded 90% at concentrations
comparable to those found in normal urine; those for eight
nucleosides added to urine exceeded 85% at a concentration of 5 nmol (about 1 tg) added per milliliter. The
method is being used to determine the concentrations, and
their ratios (to creatinine), of nucleosides in urine from
patients with different types of cancer. Data on urine
samples from normal persons and persons with cancer of
the colon are compared by gas chromatography
and the
present method. Application of the method to serum and
amniotic fluid is demonstrated.
AddItional Keyphrases: cancer
immobilized phenylboronic
acid
serum, amniotic fluid
binding of cis-diols as boronate
complexes
.
.
Urine contains many different types of metabolic end
products,
including low concentrations
of modified
nucleosides. These methylated or otherwise structurally
transformed
ribosyl purines and pyrimidines
are not
recycled through the salvage pathways but are excreted
in the urine (1-7). Their measurement in urine provides
an accurate indicator of the metabolism of RNA, espe-
cially tRNA. Altered patterns of excretion of these
compounds may offer a sensitive biomarker for diagnosis and treatment of cancer (4,5). The patterns would
either indicate the presence of cancer or would parallel
changes in tumor mass and be useful in following the
effects of chemotherapy.
Methods have been developed for rapid analysis of
these compounds in human urine (5, 8, 9). In addition,
Gehrke et al. (10) have now completed an extensive
study of variables that must be controlled for optimum
separation of nucleosides by reversed-phase
high-performance liquid chromatography
(HPLC); and recently
Hartwick
and Brown (11) evaluated
microparticle,
chemically bonded, reversed-phase
packings in the
HPLC analysis for nucleosides and their bases.5
To improve the selectivity and sensitivity of the rapid
reversed-phase
HPLC analysis of urinary nucleosides,
we have investigated the use of an affinity gel containing
an immobilized
phenylboronic
acid for a preliminary
group separation. This affinity support, introduced by
Uziel et al. (12), can selectively bind cis-diols, such as
those found in the ribose portion of ribonucleosides,
under mild conditions. With the demonstrated
HPLC
Contribution
from the Missouri Agricultural Experiment Station
by a contract from the National Cancer Institute, Bethesda, Md. (Contract No. NIH NOl CM 12323). Journal
Series No. 7876. Approved by the Director.
1 Department
of Biochemistry, University of Missouri, Columbia,
Mo. 65201.
2 Department of Biochemistry,
Experiment
Station Chemical
and supported
in part
Laboratories,
University
The Johns Hopkins
Md. 21205.
Department
of Missouri,
University
of Microbiology,
Columbia,
Mo. 65201.
School of Medicine,
University
of Colorado
Baltimore,
Medical
Center, Box 2498, 4200 East Ninth Ave., Denver, Cob. 80220.
5Nonstandard
abbreviations used: HPLC, high-performance
liquid
chromatography
(-ic); Nuc, nucleoside; ‘, pseudouridine; C, cytidine;
m3C, 3-methylcytidine; m5C, 5-methylcytidine;
U, uridine; I, inosine;
m’I, 1-methylinosine;
m71, 7-methylinosine;
G, guanosine; &G, 1methylguanosine;
m2G, N2-methylguanosine;
mG, N2,N2-dimethylguanosine; 2-Me2Gua, N2,N2-dimethylguanine,
internal standard;
A, adenosine; m1A, 1-methyladenosine;
dA, deoxyadenosine;
dC,
deoxycytidine;
dU, deoxyuridine;
dG, deoxyguanosine;
dT, 5methyldeoxyuridine;
m5U, 5-methyluridine.
Received May 9, 1977; accepted June 6, 1977.
CLINICALCHEMISTRY,Vol. 23, No. 8, 1977
1427
polyacrylamide-boronate
separation
of the nucleosides
(10) and the selective
isolation utilizing the phenyl boronate gel, we have developed a rapid, selective, highly sensitive, nondestructive method of analysis for low concentrations
of
ribonucleosides.
We are now using this method routinely for the chromatographic
analysis of urine and
other biological fluids.
Materials and Methods
Apparatus
We used a Model 6000A Solvent Delivery System,
Model U6K Universal Injector, and Model 440 Absorbance Detector (all from Waters Associates, Milford,
Mass. 01757) in our HPLC system. Peak areas, retention
times, relative molar response values, and concentrations based on an internal standard were calculated with
a Model 3352B Laboratory
Data System (HewlettPackard, Avondale, Pa. 19311).
Columns
The columns used for reversed-phase
HPLC were 4
X 300 mm “iBondapak/C1s”
(Waters Associates).
These columns were prepacked with 10-am (av diameter) porous silica particles to which octadecyl groups
were covalently bonded through a Si-O-Si bond.
The glass 5 X 150 mm columns (Fischer and Porter,
Warminster,
Pa. 18974) used for the boronate gel were
modified by attaching a 50-ml spherical reservoir to the
top of the column.
The samples were lyophilized in 25-ml, screw-cap,
round-bottom
centrifuge tubes (Corex; Corning Glass
Works, Corning, N. Y. 14830) on a custom-built
lyophilizer, which was capable of maintaining
a pressure
of 6.6-13.3 Pa (0.05-0.1 Torr) while eight samples were
being lyophilized.
A Model 3200/30 microcentrifuge,
Model 3300 rotary
shaker, and various sizes of Eppendorf pipets (all from
Brinkmann
Instruments,
Inc., Westbury, N. Y. 11590)
were used in the sample-cleanup
procedure.
Reagents
Ammonium acetate and formic acid (A.C.S. certified
grade; Fisher Scientific Co., Fairlawn, N. J. 07410),
ammonium hydroxide (analytical reagent grade; Mallinckrodt,
Inc., St. Louis, Mo. 63147), ammonium
dihydrogen
phosphate
(J. T. Baker Chemical
Co.,
Phillipsburg,
N. J. 08865), methanol that had been
distilled in glass (Burdick and Jackson, Muskegon,
Mich. 49442), and glass-distilled
water were used in
preparing buffers and other aqueous solutions. All solutions used in the HPLC system were filtered through
a membrane
filter (av. pore size 0.22 tm; Millipore,
Bedford, Mass. 01730) immediately
before use.
“Hydrazide
Bio-Gel P-2” (200-400 mesh, lot No.
15569; Bio-Rad Laboratories,
Richmond, Calif. 94804),
m -aminophenylboronic
acid hemisulfate,
succinic anhydride,
and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide
hydrochloride
(Aldrich Chem. Co., Milwaukee, Wis. 53233) were used in the synthesis of the
1428
CLINICALCHEMISTRY,Vol. 23, No. 8, 1977
for initial experiments
Nucleoside
gel. A sample of this gel used
was a gift of Dr. M. Uziel.
Standards
Pseuduridine
(st’), cytidine (C), 3-methyl cytidine
(m3C), inosine (I), uridine (U), 5-aminoimidazole-4carboxamide
riboside, and N2,N2-dimethylguanine
(2-Me2Gua) were purchased from Sigma Chemical Co.
(St. Louis, Mo. 63178). 7-Methylinosine
(m7I), 1methyladenosine
(m1A), 5-methylcytidine
(m5C), 7methylguanosine
(m7G), 1-methylinosine
(m’I), 1methylguanosine
(m’G), N2-methylguanosine
(m2G),
and N2,N2-dimethylguanosine
(mG) were purchased
from Vega-Fox Biochemicals
(Tucson, Ariz. 85719).
Guanosine
(G), adenosine
(A), deoxycytidine
(dC),
deoxyuridine
(dU), deoxyguanosine
(dG), 5-methyldeoxyuridine
(dT), and deoxyadenosine
(dA) were
purchased
from Schwarz/Mann,
Orangeburg,
N. Y.
10962. 5-Methyluridine
(m5U) was purchased from P-L
Biochemicals, Inc., Milwaukee, Wis. 53205.
Buffer Preparation
A 2.00 mol/liter stock solution of NH4H2PO4 was
prepared, sterilized by filtration through a 0.22-Lm
Millipore filter, and stored at 5 #{176}C.
Buffers for daily use
were then prepared by diluting an appropriate
aliquot
of the stock concentrate with distilled-in-glass
water to
200 ml or more, followed by addition of the methanol,
and then completing
the dilution to 1.00 liter with
water.
The pH of the buffer was adjusted with a few drops
of either ammonium hydroxide or phosphoric acid (3
mol/liter). Finally, the buffer was filtered through a
0.22-gm membrane filter immediately before use. At the
end of the day, the buffer was removed from the HPLC
system and stored at 5 #{176}C;
if stored for 24 h or longer,
it was refiltered before use.
Sample Collection
and Storage
For analysis, 24-h urine specimens were collected,
being stored at 0-5 #{176}C
until the collection was completed, without preservative.
Portions were frozen and
stored at -50 #{176}C
or lower. Samples were thawed in
flowing water at 20-30 #{176}C
immediately
before the isolation of nucleosides on the boronate gel column.
Synthesis of the Boronate
Affinity
Gel
The gel was synthesized as described by Uziel et al.
for the following modifications.
We used
Hydrazide Bio-Gel P-2, 200-400 mesh, with a hydrazide
substitution
of 1.2 mmol/gram
dry weight. The succinylated gel was coupled with a 10% theoretical excess
of both m -aminophenylboronic
acid, and 1-ethyl3(3-dimethylaminopropyl)carbodiimide,
based on the
hydrazide content of the Hydrazide Bio-Gel P-2. After
3 h an additional amount of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide,
equivalent to 10% of the theoretical amount needed for the coupling, was added to
the slurry. Another such addition, equivalent to 60% of
the theoretical amount, was made after 5 h total reaction
(12), except
time. The reaction mixture was then allowed to warm
to room temperature
and stirred overnight.
The gel product contained
1.2 mmol of boronate
(calculated from the amount taken up) per gram (dry
weight) of starting hydrazide gel. The amount of unreacted m -aminophenylboronic
acid was estimated
from the absorbance of the clear supernatant
fluid of
the reaction mixture at 293 nm (molar absorptivity
=
1610 liter mol’ cm
at pH 7) (12).
Isolation of Nucleosides
Column
with Boronate
Affinity
HPLC Determination
lii
z
A 5 X 40 mm column of the polyacrylamide-boronate
affinity gel was equilibrated
with ammonium acetate
(0.25 mol/liter, pH 8.8). The sample (1 ml of urine or
synthetic
nucleoside mixture) was made about 0.25
mol/liter in ammonium acetate by adding 0.1 ml of 2.5
mol/liter stock solution of pH 8.8 ammonium acetate.
After this pH adjustment, the sample was shaken 5 mm
on a rotary shaker, then centrifuged (5 mm, 12 000 X g)
to remove insoluble material. The supernatant fluid was
then transferred
to the top of the boronate gel column
and allowed to flow through the column until the liquid
meniscus had just reached the top of the gel bed. The
pellet and centrifuge tube were washed with 1 ml of the
ammonium
acetate buffer (0.25 mol/liter pH 8.8) by
shaking, centrifuging, and transferring as before. When
this washing was completed, the gel column was further
washed with 7 ml of the same buffer. The ribonucleosides were then eluted with 5 ml of 0.1 mol/liter formic
acid, the eluate was collected in a 25-ml tube, and 0.500
ml of a 40 nmol/ml solution of 2-Me2Gua was added.
The eluate was then shell frozen and lyophilized. The
columns were then washed with 20 ml of 0.1 mol/liter
formic acid and stored at room temperature
in this
solvent. Before re-use, the columns were washed with
10 ml of 0.1 mol/liter ammonium
acetate (pH 8.8).
When synthetic mixtures of nucleosides were analyzed,
the shaking and centrifugation
steps were omitted.
Reversed-Phase
lm’A
‘p
4
0
Co
m5C
4
m’G
o
10
20
30
40
The lyophilized
urinary nucleoside samples were
reconstituted
to their original volume (1 ml) with
glass-distilled
water. A 25-zl aliquot was injected and
chromatographed
at 1.0 ml/min with NH4H2PO4 (10
mmol/liter, pH 5.07, and containing 60 ml of methanol
per liter) on a 4 X 300 mm tBondapak/C18
(Waters
Associates) column. In some of our earlier experiments
NH4H2PO4 (50 mmol/liter, pH 5.10, and containing 50
ml of methanol per liter) was used as the eluent. However, subsequent
experiments
showed that the higher
buffer concentration
was unnecessary and changing the
methanol concentration
from 50 to 60 mI/liter gave
comparable resolution with shorter retention times. The
slight difference in pH makes no detectable difference
in retention
times of the nucleosides,
but the pH is
maintained exactly at 5.07 to ensure good precision. The
compounds were quantitated by their absorbance at 254
nm. The areas under the peaks were integrated with a
60
TIME (MIN)
Fig. 1. Reversed-phase HPLC isocratic separation of a mixture
of 16 nucleosldes
Sample, standards 500 pmol of each nucleoside, column Bondapak C18 (4 X
300 m); buffer 0.01 mol/llter NH4H2PO4, pH 5.07, wIth 60 ml of methanol added
per liter; flow.rate 1.0 mI/mm; detector, 254 nm, 0.02 absorbanceunit full scale;
temperature 24 #{176}C.
The internal standard (IS) Is N2,N2.dimethylguanine (2Me2Gua)
HP-3352B Laboratory Data System (Hewlett-Packard)
and the amount of each nucleoside was calculated by the
computer as follows:
of
Nucleosides
50
Nucleoside,
nmol/ml
of sample
x
L
r
lareaNUC
=
I
L areals
1
RMRN/Is
sample
]XInm0lISl
I
l
where
RMRN/Is
The RMR
termined
calibration
RMR was
RMRNefIs,
mol/liter).
=
area,
Lnmol/mlrC
x nmol/mlisl
areals
J standard
values for each of the nucleosides were
by at least three independent
analyses
standards of the nucleoside; thereafter,
determined daily. In the above expression
the concentration
units are nmol/ml
“IS” is internal standard.
Identification
deof
the
for
(or
of Peaks
For most of the nucleosides, peaks were identified on
the basis of retention time and co-chromatography
of
standards in one or more solvent systems. Because of
CLINICAL CHEMISTRY,
Vol. 23, No. 8, 1977
1429
‘1’
“I,
Ui
C.)
z
m’A
4
0
Ui
C.)
U)
z
4
‘U
z
0
U)
0
4
IS
(dU)
10
20
30
40
50
60
TIME (MIN)
Fig. 3. Reversed-phase HPLC isocratic separation of nucleosides. This chromatogram demonstrates the sensitivity of HPLC
analysis. The internal standard (IS) is N2,N2-dimethyiguanine
(2-Me2Gua)
Sample, 5 I standards, about 5 pmol (1 ng) each; detector sensItIvity 0.001
absorbance unit full scale. All other conditions are the same as InFIgure 1
G
ration of 12 of the early eluting ribonucleosides
(Figure
2). The improved resolution of these nucleosides was
obtained by changing the concentration of the methanol
0
10
20
30
in the eluent from 60 to 10 mi/liter. Inosine and riboMINUTES
thymidine (m5U) as well as 7-methylinosine
and cytiFig. 2. Reversed-phase HPLC isocratic separation of 12 earlydine, which co-eluted in the system used for the sepaeluting nucleosides
ration shown in Figure 1, are now well separated.
In
Sample, 100 tl standards, approx. 3 nmol each; buffer, 0.01 mol/Ifter NH4H2PO4,
to the nineteen nucleosides shown in Figures
pH 5.07 with 10 ml methanol added per liter; detector sensitivIty 0.05absorbance addition
unit full scale; temperature 25 #{176}C.
All other conditions are the same as In Figure
1 and 2, we have determined retention times and RMR’s
for dG, dA, and dC.
Minimum
detection
limit. The high efficiency of the
HPLC separation
allows an extremely low detection
limit-<5
pmol for all nine of the nucleosides in the
the selectivity of the analysis, the chromatograms
are
chromatogram of the standard mixture shown in Figure
relatively simple and identification
is unambiguous.
In
3. The high resolution so achieved with nucleosides gives
addition, identity was confirmed by correlation between
a sensitivity that approaches that of many fluorescence
gas-liquid chromatographic
and HPLC analyses for
measurements,
a sensitivity
much more remarkable
m’I, and mG. Further confirmation
of identities of
when one considers that the absorbance
detection
peaks in urine samples and identification
of unknown
method is continuous,
nondestructive,
and requires
peaks is now being done by mass spectrometry of HPLC
neither radiolabeling
nor preparation
of derivatives.
fractions.
Precision.
The reversed-phase
HPLC internalstandard method for nucleoside analysis gave excellent
Results
precision at concentrations
easily obtained from small
Reversed-Phase HPLC Analysis for Nucleosides
samples of biological fluids (Table 1). Repeated injections of 0.1 to 1 nmol of six purine ribonucleosides
at
Separation.
Figure 1 shows the isocratic separation
of 16 major and minor nucleosides achieved in less than
each of four different concentrations
gave average CV’s
1 h by reversed-phase
liquid chromatography
on a
of 0.8 to 3.0%.
Linearity.
Response curves were linear for all six
nonpolar bonded-phase
microparticulate
column. An
nucleosides used in the precision study (Table 1) over
internal
standard,
N2,N2-dimethylguanine
(2Me2Gua), was also included for accurate quantitation
the range of 0.1-1 nmol injected, similar to the one
shown in Figure 4 for 1-methylinosine.
This range inof the nucleosides. The eluent was NH4H2PO4 buffer
(10 mmol/liter,
pH 5.07, and containing
60 ml of
cludes amounts present in urine samples of less than 1
methanol per liter). A 4 X 300 mm 1.tBondapak/C18
ml.
column was used, with a flow rate of 1.0 mi/mm. The
Urine Sample Cleanup for HPLC Ribonucleoside
conditions were chosen to give optimum separation
Analysis
within 1 h of the methylated
purine nucleosides found
Due to the complex nature of biological fluids, a rapid
in urine, but are adequate for quantitation
of many of
preliminary
class separation
of ribonucleosides
was
the earlier eluting pyrimidine nucleosides as well. The
made before HPLC analysis by use of an affinity gel
flexibility of the system is demonstrated
by the sepa‘,
1430 CLINICALCHEMISTRY,Vol. 23, No. 8, 1977
Table 1. PrecIsion of HPLC Analysis for
Nucleosides by the Internal-Standard Method a
Av. CV,
m1A
CV, %
G
CV, %
m11
CV, %
m1G
CV, %
A
CV, %
mG
CV, %
a
1
2
3
4
19.50
1.69
18.68
1.50
19.42
1.18
19.31
1.04
20.23
1.19
19.52
0.77
10.06
2.39
9.42
1.17
9.51
0.84
9.81
0.92
10.28
1.36
10.06
1.69
4.99
0.80
4.76
0.84
5.15
0.39
5.03
0.80
5.26
0.57
5.25
0.95
2.66
7.14
2.40
1.25
2.68
0.75
2.54
0.39
2.66
1.50
2.76
4.34
0.79
0
H
H
Fig. 5. Top: Structure of boronate derivatized polymer. Bottom:
Formation of cis-diol boronate complex (12)
0.79
1.16
1.94
described in the Methods
section. It differs from the
procedure originally described by Uziel et #{224}l.
(12) in
several ways. At Uziel’s suggestion we synthesized the
boronate gel from 200-400 mesh Hydrazide Bio-Gel P-2
instead of the coarser 100-200 mesh material. The
amount of hydrazide substitution
of this starting material was also decreased to 1.2 mmol/g (dry wt). These
changes significantly decrease the shrinkage of the gel
when pH and ionic strength are decreased to elute ribonucleosides.
This shrinkage
occasionally
caused
channelling and incomplete elution of the column. The
columns were now washed with a total of only 8 ml of pH
8.8 ammonium
acetate, which completely
removes
material not retained by the gel and gives complete
analytical recovery of pseudouridine
((.‘)
in the formic
acid eluate. The nucleosides were eluted with 5 ml of 0.1
mol/liter formic acid or 10 to 30 ml of 1 mol/liter acetic
acid. The use of both the lower pH and ionic strength
of 0.1 mol/liter formic acid gives a much more efficient
Load 1 ml urine pH 8.8 ofl
phenylboronate
affinity column, 5 x
40 mm equilibrated with 0.25M pH
8.8 NH4Ac
0.8
+
Wash with 8 ml 0.25 M pH 8.8 NH4 Ac
0
U-
4,
0.6
E
0
C
(S
lI
1.19
1.0
5)
0
[2
3.01
column. The column was packed with a modified
polyacrylamide
gel having an immobiljzed phenylboronic acid functionality
covalently linked to the hydrocarbon polymer backbone by a spacer arm (Figure
5). This type of boronate resin was introduced by Uziel
et al. (12), who demonstrated
that it can selectively bind
cis-diols as boronate complexes under mildly alkaline
conditions.
The stability of the complex varies with
conformation,
but is maximal for diols having the same
conformation
as ribose (12, 13). The boronate complexes are readily decomposed under acidic conditions
and the ribonucleosides
are quantitatively
eluted.
Figure 6 outlines the urine sample cleanup procedure,
Co
H
%
Each value is the mean of five or more analyses.
C
2
BO
Nucleoslde stds., nmol/mI
(zmol/IIter)
Nucleoslde
r\-
Elute ribonucleosides
with 5ml 0.1M
HCOOH.
0.4
z
Add 20 nanomoles of l.S. (2-Me2Gua)
to eluate, shell freeze and lyophilize
to dryness.
0.2
0.4
Nanomoles
0.6
0.8
1.0
Injected
Fig. 4. Linearity of H’LC analysis of 1-methylinosine. This curve
is representative of those obtained for seven nucleosides tested
(pseudouridifle,
1-methyladenosirie,
1-methylinosine,
N2methylguanosne, adenosine, and N2,N2-dimethylguanosine)
Each point represents average of five or more analyses
4,
Dissolve in 1-2ml H20 or HPLC buffer
and inject 25-50 .tl on Bondapak
C18 HPLC column.
Fig. 6. Urine sample cleanup procedure before HPLC ribonucleoside analysis
CLINICAL CHEMISTRY, Vol. 23, No. 8, 1977
1431
Table 2. Isolation of Ribonucleosides from Phenylboronate Gel-Affinity Column
Nucleoslde
n
=
m1A
m7G
G
m11
100.7
4.7
88.1
6.6
97.6
2.8
99.4
2.7
10
CV, %
101.2
2.9
and recovery,
%
m1o
97.8
2.2
2G
A
mG
94.0
2.4
96.5
1.9
96.2
4.4
About 10 nmol of each nucleoside added.
elution and essentially quantitative
recoveries of all the
ribonucleosides
examined (Table 2). The boronate gel
columns are reusable; no carryover of material from
previous samples has been seen. Many of our columns
have been used three times a week for four months
without noticeable deterioration.
Analytical
Recovery
of Nucleoside
Standards
A synthetic mixture of nine nucleosides (tfi, m1A, m7G,
G, m1I, m’G, m2G, A, and mG) was repeatedly analyzed
using the boronate affinity column for isolation followed
by reversed-phase
HPLC, as described
above, for
quantitation.
Recoveries ranged from 88 to 101% for 10
nmol of each nucleoside placed on the affinity column
(Table 2). These mean recovery values were the average
of 10 analyses performed on 10 different affinity gel
columns by two different operators.
Pooled
Control
Urine
Urinary nucleosides were determined in 1-ml samples
of a pooled control urine. The nucleosides were isolated
using 5 X 40 mm boronate gel affinity columns as described above, then separated and quantitated
by the
HPLC system, with 2-Me2Gua as an internal standard.
Samples equivalent to 25 Ll of urine were injected for
each HPLC analysis. Figure 8 shows a chromatogram
for such an analysis. Table 3 demonstrates
the precision
obtained for four independent
analyses of a pooled
control urine on four different affinity columns.
Analytical Recovery of Nucleosides
Pooled Control Urine
Added to
These determinations
were made on equivalent 1-ml
samples of pooled control urine to which a known
amount of each of eight nucleosides had been added.
These analyses were compared
with those of 1-ml
samples of pooled control urine performed on the same
m’A
IS
w
C-)
z
IS
uJ
C-)
z
4
(mtcua)
4
0
0
U)
4
ng
U)
4
A
miG
I0
‘U
z
a
30
Sample, 50 l with 250 pmol each nucleoside; buffer 0.05 mol/liter NH4H2PO4,
pH 5.10, wIth 50 ml methanol added per liter; detector sensitivIty 0.01 absorbance unit full scale. All other conditions are the same as in Figure 1
1432 CLINICALCHEMISTRY,Vol. 23, No. 8. 1977
30
40
50
60
MINUTES
MINUTES
Fig. 7. Reversed-phase HPLC isocratic separation of standard
nucleosides after their isolation with boronate gel column
20
Fig. 8. Reversed-phase HPLC isocratic separation of nucleosides
in control urine after their isolation with a boronate gel column
Sample, 50 tl equivalent to 25 zl urine; temperature 26#{176}C.
All other conditions
are
the same as in Figure 7
Table 3. Precision of HPLC Analysis for Urinary
Nucleosides
Nucleoslde
a
Moan
SD
CV, %
m’A
nmol/ml
225.3
15.18
6.72
10.26
5.69
5.52
2.52
11.37
‘
mtA
m7Gb
m11
m1G
m2G
A
mG
a
4.07
0.54
0.31
0.35
0.16
0.23
0.15
0.82
1.8
3.5
4.7
3.4
2.8
4.1
6.0
7.2
m’I
m’G
I
(m’Gua)
w
C.)
z
4
90ng
Each value is the average of four independent runs with four different affinity
columns and a pooled urine control.
b Identity based on retention time only; needs further confirmation by other
methods.
affinity columns and the recovery of nucleosides added
to the urine was calculated
(Table 4). Each of the
standard nucleosides was added at a concentration
of
about 5 nmol/ml of urine, an amount resembling the
concentration
of the nucleoside in the original urine
sample. The range of the recoveries was 87 to 100%, with
standard deviations of 4% or less for the recoveries for
eight nucleosides; this demonstrates
the reliability of
the analytical method for urinary samples.
Reversed-phase
HPLC chromatograms
of a standard
mixture of six nucleosides, a urine sample, and another
sample of the same urine to which this standard had
been added before analysis are shown in Figures 7, 8,
and 9.
Figure 10 shows a chromatogram
of the analysis of
urinary ribonucleosides
from a patient with advanced
colon cancer. Above-normal concentrations
of m’I, m’G,
m2G, A, and mG can be readily observed, whether
Table 4. Analytical Recovery of Nucleosides
Added to Pooled Control Urine
Av.
UrIne
+ suppl.
4
m’G
C.)
‘U
-,
z
0
10
20
30
MINUTES
40
50
60
70
Fig. 9. Reversed-phase HPLC isocratic separation of nucleosides
in pooled control urine with six nucleosides added
Mixture of nucleosides shown in Figure 7 addedto control urine shown in Figure
8 to obtain sample for analysis shown in this figure. Boronate gel cokimn cleanup
of sample before HPL.C.Sample, 50 l, equivalent to 25 zl urine, with 125 pmol
of each of six nucleosides added. All other conditions are the same as in Figure
7
in Urine from Colon-
Analysis for Ribonucleosides
Cancer Patients
Nucleoslde
0
U)
Suppi.
Urine
found
recovery,
%
measured either as total excretion of nucleoside per day
or as amount of nucleoside excreted per day per unit
weight of the patient. Further, we have found that a
more meaningful way to express the nucleoside marker
data is as a ratio of nanomoles of nucleoside per micromole of creatinine. Using this ratio, we have compared
a group of 10 normal adults to a group of 10 patients
with advanced colon cancer. Ratios exceeding 2 SD
above the average for a normal population were found
for all 10 of the cancer patients when the ratios for m’I,
mG to creatinine and the summation
of the ratios for
m1A, m7G, m11, m1G, m2G, A, and mG were compared
to the normal control values.
nmolImI8
m1A
m7G
23.30
10.05
17.38
5.69
5.92
4.36
92
88
0b
13.47
8.91c
4.56
92
m11
m1G
m2G
A
mG
15.46
10.66
10.69
7.88
15.72
10.48
5.64
5.46
2.55
11.28
4.98
5.02
5.23
4.83
4.44
99
101
100
98
87
a
b
C
Each value an average of three runs.
An unknown peak eluted with G and integrated together.
Identity of this peak based on retention time Only.
Analysis
for Nucleosides
in Other
Biological
Fluids
The analytical method developed for urine has been
used with other biological fluids, such as amniotic fluid
(Figure 11) and human blood serum (Figure 12). Neither of these samples was deproteinized before analysis.
Although the concentration
of the different nucleosides
are generally much lower in these fluids than in urine,
they can be readily quantitated
in samples of less than
1 mL Use of the boronate gel isolation of the nucleosides
provides a background-free
sample for HPLC analysis,
which contains the purified nucleosides. This eliminates
CLINICALCHEMISTRY,Vol. 23, No. 8, 1977 1433
m’A
m’I
m’G
mts
‘U
‘s
C)
z
I mtGua)
SOng
2-N.,
Gui (IS)
4
‘U
0
C)
z
U)
4
0
U)
m
4
m’G
mG
A
0
10
20
I..
0I
30
40
50
60
10
MINUTES
z
Fig. 11. Reversed-phase I-IPLC isocratic separation of nucleosides in amniotic fluid after sample cleanup
--
0
20
30
40
50
60
50 pI, equivalent to 0.2 ml amniotic fluid; detector sensitivIty 0.005
unit full scale; temperature. 23 #{176}C.
All other conditions the same
as in Figure 1
Sample,
-,
10
70
MINUTES
absorbance
Fig. 10. Reversed-’phase HPLC isocratic separation of nucleosides in urine of colon cancer patient after sample cleanup
Sample, 50 p1.equivalent to 25 p1 of urine. Conditions the same as in Figure
7
most of the difficulties encountered in quantitating
low
concentrations
of nucleosides
in biological samples.
Further, use of such cleaned samples for HPLC analysis
significantly prolongs the useful lifetime of the expensive HPLC columns.
‘U
C.)
z
4
0
Discussion
The reversed-phase
partition mode of liquid chromatography
for the separation of nucleosides with ultraviolet absorption detection is a rapid, efficient, selective, highly sensitive, nondestructive,
quantitative
method for the simultaneous
analysis of many nucleosides (10, 11). Isocratic elution eliminates the baseline
drift that occurs with gradient elution, thus improving
precision at high sensitivity.
Moreover, time is saved
because column equilibrium
does not have to be established with a lower strength eluent after each run.
The next sample can be injected immediately after the
last peak appears. This compensates
for the slightly
longer run time that is necessary in isocratic analysis.
The use of a class separation of the ribonucleosides
greatly improves the selectivity of the method and increases its reliability when nucleosides are being measured in the complex matrices of biological fluids. We
have confirmed the findings of Uziel et al. that nucleic
acid bases, deoxyribonucleosides,
deoxyribonucleotides,
urinary pigments, and most other interfering ultraviolet
light-absorbing
compounds
are not retained by the
boronate gel (12). Some of the ribonucleotides
are re1434 CLINICALCHEMISTRY,Vol. 23, No. 8, 1977
2-Me, Gui (Is)
35
ng
Ca
4
mG
0
10
20
30
40
50
60
70
MINUTES
Fig. 12. Reversed-phase HPLC isocratic separation of nucleosides in control serum after sample cleanup
Sample,
50 I, equivalent to 0.1 ml serum. Conditions the same as In Figure
11
tamed by the boronate column, and are eluted from it
along with the ribonucleosides.
However, they elute
rapidly from the HPLC column and do not interfere
with the quantitation
of the nucleosides.
We have
demonstrated
the advantages of this class separation
of nucleosides by the analysis of urine, serum, and amniotic fluid.
Analysis of urinary ribonucleosides by reversed-phase
HPLC chromatography
after sample purification on a
boronate gel is now routine in our laboratory. The values
obtained for
m’I, and mG are comparable to those
obtained by gas-liquid chromatography
(5,8). However,
with the HPLC method several other nucleosides and
related compounds can be quantitated
in one chromatographic step from the same sample and their total or
relative concentrations
compared.
This gives us a
powerful tool for identifying potential nucleoside biochemical markers of cancerous growth. In addition, this
rapid and selective method can be used to discover inborn errors in purine and pyrimidine metabolism as well
as determine
concentrations
of major and modified
nucleosides in enzyme hydrolyzates
of tRNA, cell extracts, and biological fluids.
,
2. Adams,
excretion
(1960).
W. S., Davis, F., and Nakatani,
M., Purine and pyrimidine
in normal and leukemic subjects. Am. J. Med. 28, 726
3. Park, R. W., Holland, J. F., and Jenkins, A., Urinary purines in
leukemia. Cancer Res. 22,469 (1962).
4. Waalkes, T. P., Gehrke, C. W., Bleyer, W. A., et al. Potential biologic markers in Burkitt’s lymphoma. Cancer Chemot her. Rep. 59,
721 (1975).
Waalkes, T. P., Gehrke,C. W., Zumwalt,
R. W., et al. The urinary
excretion of nucleosides of ribonucleic acid by patients with advanced
cancer. Cancer 36, 390 (1975).
6. Mandel, L. R., Srinivasan, P.R., and Borek, E., Origin of urinary
methylated purines.
Nature (London) 209, 586 (1966).
7. McFarlane, E. S., and Shaw, G. J., Observed increasein methylated
purines excreted by hamsters bearing adenovirus-12 induced tumors.
Can. J. Microbiol. 14, 185 (1968).
5.
8. Chang, S. Y., Lakings, D. B., Zumwalt,
R. W., et al., Quantitative
determination
of methylated nucleosides and pseudouridine
in urine.
J. Lab. Clin. Med. 83, 816 (1974).
9. Mrochek, J., Dinsmore, S. R., and Waalkes, T. P., Analytic techniques in the separation and identification
of specific purine and
pyrimidine degradation
products of tRNA: Application
to urine
samples from cancer patients. J. Nati. Cancer Inst. 53, 1553
(1974).
We thank Roy Wood of Bio-Rad Laboratories
for the gift of samples
of Bio-Gel-P2, 200-400-mesh, his help in securing the desired Hydrazide Bio-GeI P2, 200-400 mesh, and for valuable discussions
concerning the preparation and properties of these polymers. We also
wish to thank Debi Whisenand for her assistance in manuscript
preparation.
References
10. Gehrke,
of nucleosides.
C. W., Kuo, K. C., Suits,
R. D., et al., Chromatography
In preparation.
R. A., and Brown, P. R., Evaluation
11. Hartwick,
chemically
bonded
reversed-phase
of microparticle
packings
in the high-pressure
of nucleosides and their bases. J.
liquid chromatographic
analysis
Chromatogr. 126, 679 (1976).
12. Uziel, M., Smith, L. H., and Taylor, S. A., Modified nucleosides
in urine: Selective removal and analysis. Clin. Chem. 22, 1451
1. Weissman,
(1976).
bases of human
13. Boeseken, J., The use of boric acid for determination
of the configuration of carbohydrates.
Adv. Carbohydr. Chem. 4, 189 (1949).
224,407
D., Bromberg, P. A., and Guttman, A. B., The purine
urine. I. Separation and identification. J. Biol. Chem.
(1957).
CLINICAL CHEMISTRY, Vol. 23, No. 8, 1977
1435