Division; Dr. Gerard Smith of Cornell Medical College, White Plains,. NY for the rat brain samples; Klaus Lohse and Mona Zakaria for their valuable comments ...
CLIN. CHEM. 25/2, 235-24 1 (1979)
Reversed-Phase LiquidChromatographic Separation of 3’,5’-Cyclic Ribonucleotides Ante M. Krstulovic,1 Richard A. Hartwick,2 and Phyllis R. Brown2
A rapid, reversed-phase “high-performance” liquidchromatographic separation of the five naturally occurring
cyclic ribonucleotides is described. The separation, optimized for the measurement of these compounds in biological samples, is short (25 mm), sensitive (50-100 pmol), and requires no sample pre-concentration steps. We also report an alternative isocratic elution mode, optimized for a rapid and selective analysis for adenosine 3’,5’-cyclic phosphate. The identity of chromatographic peaks in biological extracts is confirmed by several methods: retention times, co-chromatography with the reference compounds, absorbance ratios, enzymatic peak-shift with cyclic nucleotide phosphodiesterase, and stopped-flow ultraviolet-scanning techniques. The great importance
of the five naturally
occurring
3’,5’-
cyclic ribonucleotides in biological chemistry has long been recognized (1-6). Recent reports show that cyclic nucleotides may mediate the action of several neurotransmitters and act as regulators of the cell activities (2, 3, 7-9). Adenosine 3’,5’-
cyclic phosphate
(cAMP),
which is formed from adenosine
5’-triphosphate (ATP) by action of the enzyme adenylate cyclase (EC 4.6.1.1), is already known to play an important role in many physiological functions of various peptide hormones, implying that it acts as a second messenger in some neuronal systems (2, 10). These findings are based on the observation that the cAMP concentrations in blood increase on administration of 1-adrenergic agents or hormones to humans or experimental animals (11, 12). This increase is a direct result of the increased intracellular concentrations caused by the activation of adenylate cyclase. In addition to cAMP, guanosine 3’,5’-cyclic phosphate (cGMP) has also been shown to be involved in the actions of some neurotransmitters in the nervous tissue (2, 3).
Because
of the great physiological
importance
of these
compounds, cyclic nucleotides have received considerable attention. The many papers concerning cyclic nucleotide methodology reflect the difficulty in measuring the exceedingly low endogenous concentrations of these compounds. Because they comprise less than one-millionth of the cell content (13), research has been directed at more sensitive detection and developing rapid and efficient separation pro-
cedures that would eliminate the need for purification and preconcentration steps before the assay. Analytical work on cyclic
1
nucleotides
Chemistry
has focused
Department,
on analyses
Manhattanville
for cAMP
College, Purchase,
and NY
10577. 2 Chemistry Department, University of Rhode Island, Kingston, RI 02881. Received Sept. 25, 1978; accepted Nov. 27, 1978.
cGMP, because much more is known about the physiological role of these two compounds than that of the other three. Several techniques have been used to measure cAMP. The
most common ones are those that rely on cAMP-dependent enzymes (14, 15), the luciferin-luciferase (EC 2.8.2.10) system (16), radioimmunoassay (18, 19), binding to specific proteins (20-22), thin-layer chromatography on polyethyleneiminecellulose (23), and gas-liquid chromatography (24). Cyclic GMP has been measured in body fluids mainly by protein-binding assays (20), radioimmunoassay (17, 18), and thin-layer chromatography (23). Most of these methods, although sensitive, are generally time-consuming and require
purification The
procedures
separations
mance”
or other special treatments.
of cyclic
liquid
chromatography The ease of quantitation
nucleotides
(24-29). and combined with the high sensitivity of currently used in liquid chromatography tages over other methods of analysis. such separations of cyclic nucleotides on ion-exchange columns with either tional packings. Because
of the
simplicity
by “high-perfor-
are of rather the
speed
recent
date
of analysis
the detection devices offer great advanHowever, thus far, all have been performed pellicular or conven-
of use of reversed-phase
packings,
as well as longer column lifetimes (400-500 analyses, with proper column care), the use of this mode was investigated in liquid chromatographic analysis for cyclic nucleotides. The
developed
method
was applied
in the analysis of these com-
pounds in biological samples. Although the method is illustrated with the analysis of rat-brain extracts, it can also be used for other types of cell extracts and physiological fluids.
Methods and Materials Instrument A Waters Associates (Waters Associates Inc., Milford, MA 01757) Model 6000 A Solvent Delivery System, Model 660 Solvent Programmer and Model U6K Universal Injector were used in all determinations. A variable-wavelength detector, SF 770 Spectroflow
Monitor (Kratos Inc., Schoeffel Instrument Division, Westwood, NJ 07675), with a deuterium lamp and a 5.0-sL cell volume
was used
to monitor
column
effluents.
This
instru-
ment is also equipped with a SF A 339 Wavelength Drive and MM 770 Memory Module (Kratos Inc.), which were used for obtaining stopped-flow ultraviolet spectra. The chromatographic column was prepacked pBondapak C18 (30 cm X 4.6 mm i.d.) from Waters Associates. This packing, in which the silanol groups of the silica gel have been modified to nonpolar moieties by addition of chemically bonded -(CH2)17CH;1 groups, has an average particle diameter of 10 m. CLINICAL CHEMISTRY,
Vol. 25, No. 2. 1979
235
For the preparation Sonic Dismembrator,
probe assembly 15219).
a.
of tissue homogenates we used a Fisher Model 300, with a 12.5 mm, titanium-tip
(Fisher
Scientific
Co., Pittsburgh,
‘I
PA a.
Mobile Phase 1
For the analysis of all five cyclic mononucleotides, the low-concentration eluent, 20 mmol/L KH2PO4, was prepared by dissolving the anhydrous salt (Mallinckrodt, Inc., St. Louis, MO 63147) in distilled de-ionized water, and adjusting the pH to 3.7 with a dilute phosphoric acid solution. The high-concentration eluent consisted of a 60/40 (by vol) mixture of anhydrous methanol (distilled in glass; Burdick & Jackson, Inc., Muskegon, MI 49442) and water. A linear gradient from 0% to 25% of the high-concentration eluent by volume in 30 mm was used. For the rapid analysis of cAMP, an isocratic elution mode was used. The eluent was a solution of 20 mmol/L KH2PO4, pH 5.5, containing anhydrous methanol, 120 mLIL. All eluents were filtered through membrane filters, Type HA, pore size
0.45 m (Millipore Corp., Bedford, MA 01730), and degassed under reduced pressure before use. In all cases, the flow rate was 1.5 mL/min and the temperature was ambient.
a. 4
a.
U
“5
E C
It,
Lii
z 4
a. 0
U)
4
Chemicals All reference
compounds
and
the 3’,S’-cyclic
nucleotide
phosphodiesterase (EC 3.1.4.17) were purchased from Sigma Chemical Co., St. Louis, MO 63178. Solutions of the reference compounds were prepared in distilled de-ionized water and kept frozen when pared in KH2PO4
Phosphoric were purchased NJ 08865.
not in use. The enzyme solution was presolution (100 mmol/L, pH 7.5). acid, perchloric acid, and sodium metabisulfate
from J. T. Baker Chemical Co., Phillipsburg,
Sample Preparation
0
Proteinaceous material must before analysis, because it would and impair its performance. Any can be used for this purpose. The
nique is illustrated
be removed from samples otherwise clog the column acidic protein precipitant described separation tech-
with the analysis of the cyclic nucleotide
content of rat brain, so only details brain extracts will be given.
Male Sprague-Dawley 60606),
weighing
200-400
of the preparation
rats (Hormone
of rat-
Assay, Chicago, IL
the scalp and quickly and placed on its dorsal surface on a glass plate over ice. Sections were removed by standard dissection procedures, with a sharp
cranium were opened, and
g, were decapitated, the
brain
was removed
razor blade. Average sample weights were generally 20 and
40 mg.
ferred
Individual
samples
to a test tube containing
were
weighed
between and
500 sL of a 400 mmol/L
trans-
solu-
tion of HCIO4. Because the same samples were also used in the analysis
of catecholamines
dant (NaHSO3)
in rat-brain
extracts,
was added to the perchloric
an antioxi-
acid solution
(4
10
20
30
TIME (mm)
Figure 1. Separation of reference compounds detected at 254 nm AMP. adenosine 5’-monophosphate; cCMP, cytidine 3’S’-cyclic phosphate; cUMP, uridine 3’,5’-cyclic phosphate; cGMP, guanosine 3’,5’-cyclic phosphate; cIMP, inosine 3’,5’-cyclic phosphate. Concentration: approximately 10 nmol
each; chromatogmaphic conditions: column: uBondapakC18; low-concentration etuent: 20 nmol/L KH2PO4, pH 3.7; high-concentration eluent: methanol/water (3/2 by vol); gradient: linear from 0% to 25% of the high-concentration eluent in 30 mm. flow rate: 1.5 mL/min; temperature: ambient; attenuation: 0.1 A full-scale
was confirmed by three additional absorbance ratios, enzymatic peak cyclic nucleotide traviolet scanning.
identification methods: shift on treatment with
phosphodiesterase, To obtain absorbance
and
stopped-flow ulratios, peak heights
were simultaneously measured at 254 and 280 nm and their ratios computed for the standards. These were compared with the ratios
computed
for the peaks
under
investigation
in rat-
mmol/L).
brain extracts.
Samples were then homogenized for 20 to 30s, and centrifuged for 10 mm at a speed of 630 X g. The supernate was
Cyclic nucleotide phosphodiesterase hydrolyzes the 3’-bond of 3’,5’-cyclic nucleotides, converting them to the corresponding 5’-mononucleotides. In carrying out the enzymatic reaction, a 50-75-pL aliquot of the rat-brain extract was first chromatographed. To another aliquot of the same volume, 5-10 sL of the enzyme solution was added after the extract was buffered to pH 7.5 with a solution containing 20 mmol of
transferred to a prechilled test tube, stored frozen, and analyzed as soon as possible. Periodically, samples were re-run to ensure that no decomposition was taking place. Under the storage conditions used, samples were stable for at least four
weeks. Peak Identification We tentatively identified cyclic nucleotides in biological samples on the basis of their retention times and co-chromatography with reference compounds. The identity of the peaks 236
CLINICAL CHEMISTRY, Vol. 25, No. 2, 1979
K2HPO4 per liter. The sample was then incubated
for 10 to
15 mm at 25 #{176}C and the reaction stopped by placing the sample test tube in boiling water for 30 s. An appropriate volume of the incubated sample was then rechromatographed,
taking into account the dilution
of the original solution.
UNC0UCCT(O
030
B*CKGROUNO
250
210
SPCCTRUN
290
UNCORRECTEOSPCCTKUM OF
CORNECTED
00
230
SACKONOUNO
230
C0KCCTCO
SAMP
270
Results and Discussion
SP(C?*UM
290
For the separation of the five naturally occurring cyclic nucleotides, a gradient-elution mode of the reversed-phase liquid chromatography was used. It should be noted that the pH of the mobile phase and the steepness of the gradient are critical, because retention times of the compounds under study vary considerably with changes in either of these two variables. The separation of the five cyclic nucleotides and AMP, detected at 254 nm, is shown in Figure 1. With the chromatographic system used, the detection limits for all compounds were found to be between 40 and 60 pmol. To determine the linearity of the detector response, we obtained calibration plots for all compounds under study. Before analysis for cyclic nucleotides in biological samples, the efficiency of the extraction procedure was tested by adding exogenous thymidine 3’,5’-cyclic phosphate to the rat-brain samples. Analytical recovery was 100%. For the analysis of cyclic nucleotides in rat brain extracts, the separation conditions were optimized,
3(0
SPECTKUM OF
to minimize
interferences
from
other
naturally
occurring
constituents. 230
230
270
290
230
310
2’O
230
290
Under the conditions we used, all tn- and diphosphate nucleotides elute near the void volume. Of the monophosphate nucleotides, AMP has the longest retention time; however, it is eluted before the cyclic nucleotides and presents no problems. Nucleosides, bases, and other less-polar compounds did not interfere in the analyses of rat brain-or rat liver extracts, which were also ahalyzed. If this separation
00
WAVCLENKTH(3,,,)
Fig. 2. Corrected vs. uncorrected ultraviolet spectra (Upper part) no compound in the detector sample cell; (tower part) cAMP arrested in the sample cell. Scanning rate: 100 nm/mm; attenuation: 0.4 A full-scale
technique
The UV spectra of the peaks under study were obtained by the stopped-flow scanning technique (30). With a deuterium lamp, a wavelength range of 190-400 nm can be scanned at a rate of 100 nm/mm. The spectra obtained at high sensitivities from diluted owing
samples
to changes
monochromator. spectrum
is stored
are distorted
in optical
by a spectral
To account
background,
the flow cells and the for this, the entire background
properties
in the memory
of
module
and
automatically
subtracted from the repeated scan to give a correct spectrum. The corrected spectra were obtained by arresting the flow of the mobile
scanning
phase
at the top of each peak
over the desired wavelength
under
study
ultraviolet
250
21o
290
3(0
and
range.
230
250
to other
biological
samples,
the
spectra
of the five cyclic
nucleotides
and AMP
(Figure 3) were used for comparison with the spectra of the chromatographic peaks in the rat-brain extracts. As can be seen from Figure 3, the corrected spectra of AMP and cAMP
cGMP
1D
is to be applied
conditions can be optimized according to the complexity of the sample matrix, to avoid possible interferences. To confirm the peak identity based on retention times and co-chromatography with the reference compounds, ultraviolet spectra were obtained by the stopped-flow scanning technique. The background spectrum, which was scanned between 230 and 320 nm, is shown in the upper part of Figure 2, the corrected spectrum of cAMP in the lower part. The corrected
210
WAVELENGTH
cCMP
cUMP
cIMP
cAMP
290
310
230
‘
250
‘
200
290
tnm)
Fig. 3. UV spectra of some reference compounds obtained by the stopped-flow scanning technique Scanning
conditions
same as in Fig. 2
CLINICALCHEMISTRY,Vol. 25. No. 2, 1979
237
a.
a. (A)
(C)
3.
(B)
a.0, a. L)
-
4
0. D U
E C
‘8 8)
N
w 2 z 4
a.
0
0
UI 4
0
‘0
20
0
10
20
0
10
20
TIME (mm) Fig. 4. A, Chromatogram of a synthetic mixture of cyclic nucleotides, detected at 254 nm. B, Chromatogram of the mixture shown in Fig. 5A, incubated with diesterase for 5 mm. C, Chromatogram of the mixture shown in Fig. 5A, incubated with diesterase for 10 mm Chromatographicconditions in A, B. and C same as in Fig. 1 identical. This applies to all other cyclic nucleotides and their corresponding 5’-mononucleotides. However, because of the difference in retention times, these compounds can be differentiated on the basis of the combination of retention times and ultraviolet spectra. When the mixture of reference compounds (Figure 4A) is incubated with cyclic nucleotide phosphodiesterase for 5 mm, the chromatogram of the resulting mixture (Figure 4B) reveals that the rate of conversion of cCMP and cUMP is slower than for other cyclic nucleotides. However, prolonging the incubation to 10 mm results in complete removal of all cyclic fluare almost
cleotides, sponding
with the simultaneous formation 5’-mononucleotides (Figure 4C).
of the
corre-
Figure 5A illustrates the separation of the ultraviolet-absorbing constituents of rat brain with detection at 254 nm. Peaks 1 and 2 had the retention times of cGMP and cAMP, respectively. Co-injection with the corresponding reference compounds resulted in the increase of the peak areas, as shown in Figure 5B. To further confirm the identity of the two peaks, we incubated an aliquot of the rat-brain extract with the diesterase; the chromatogram of the resulting mixture (Figure 5C) shows the absence of the two peaks. The products of the 238
CLINICAL CHEMISTRY, Vol. 25. No. 2, 1979
enzymatic reaction are not resolved from the early eluting peaks in the rat-brain extract. The peak height ratios (254 nm/280 nm) for the cAMP and cGMP reference compounds were found to be 1.46 and 1.36, respectively; the ratios for peaks 1 and 2 in the extract were 1.45 and 1.34, respectively, adding further evidence to the elucidation of the chemical nature of the two peaks in the rat-brain extract. Next, the ultraviolet spectra of the two peaks in the ratbrain extract were compared with the spectra of the suspected compounds. Figure 6 illustrates the close similarity between the two sets of spectra. Therefore, from the evidence accumulated by several identification methods, we identified peaks 1 and 2 in the rat-brain extract as cGMP and cAMP, respectively. Because there is great demand especially for a simple, rapid, and reproducible assay for cAMP in biological samples, we developed such an assay for cAMP, using an isocratic elution mode with the same reversed-phase packings. By varying both the pH and the methanol concentration of the mobile phase, it is possible to alter the elution behavior of the cyclic nudeotides. Figure 7 shows the separation of a mixture of five cyclic
RAT
BRAIN EXTRACT
(A)
(B)
(C)
E C 8) C’)
Lii 0
z 4 0
3d, 4
Lk 0
5
0
5
20
25
0
5
0 TIME
5
20
25
0
5
10
5
25
20
(mm.)
Fig. 5. A, Chromatogram of a rat-brain extract. Sample weight corresponding to the volume injected: 3.0 mg; volume injected: 25 giL. B, Chromatogram of the sample shown in Fig. 5A, coinjected with cGMP and cAMP. C, CPwomatogram of the sample shown in Fig. 5A, incubated with diesterase. Incubation time: 10 mm. Attenuation for A, B, and C 0.04 full-scale Chromatogmaphic conditions In A. B, and C same as in Fig. 1
cAMP STANDARD
230
240
250
260
270
280
290
300
3)0
230
320
WAVELENGTH
240
250
260
270
280
290
300
3)0
320
(nm)
Fig. 6. Comparison of the con-ected ultraviolet spectra of peaks 1 and 2 in sample shown in Fig. 5A and the c(vlP and cAMP reference compounds Attenuation: 0.4 A full-scale. Scanning conditions same as in Fig. 2
CLINICALCHEMISTRY, Vol. 25, No. 2, 1979
239
(B)
(A)
(B)
(A)
(C)
a. 4
1’
a.
in r’J
4
Ui 0
E C
z
8) N
0
4
I’)
Ui 0
4
z 4 0
,.n 4
LLLL
LL 0
5
10
5
0
0
5
LL 100
5 TIME
5
100
5
10
(mm)
Fig. 8. A, Chromatogram of the rat-brain extract analyzed using an isocratic elution mode. B, Chromatogram of the rat-brain
0
TIME (mm)
extract of the rat-brain shown insample A, co-injected shown inwith A, incubated cAMP. C, with Chromatogram diesterase
Fig. 7. Isocratic elution of reference compounds detected at 254 nm
Sample weight corresponding to the volume injected: 2.0 mg: volume injected: 25 oL. Incubation time, 10 mm. Chromatographic conditions same as in Fig. 7B. Attenuation: 0.04 A full-scale
Separation conditions optimized for cAMP. A, Chromatographic conditions: column: zBondapak C18; mobile phase: anhydrous methanol in 20 mmol/L KH2PO4. pH 5.5(12/88): flow rate: 1.5 mL/min; temperature: ambient; attenuation: 0.1 A full-scale. B, Chromatographic conditions same as in A except for the mobile phase: anhydrous methanol in 20 mmol/L KH2PO4, pH 5.5 (15/
85)
nucleotides optimized for cAMP, (A) with a water/methanol (88/12 by vol) solution and (B) with a water/methanol (85/15 by vol) solution of the same pH. The retention time of cAMP in (B) is considerably reduced without any loss in its resolution from the remaining compounds in the mixture. Mono-, di-, and triphosphate nucleotides elute immediately after the void volume. All commonly occurring nucleosides and bases elute before cAMP under these chromatographic conditions. Adenosine
elutes
than
of cAMP
that
conditions chosen
with
for the rapid
time
rapid
analysis
minutes
longer
Therefore,
the
shown in Figure 7B were
of cAMP.
of the rat-brain
conditions,
several
not interfere.
used for the separation
A chromatogram these
a retention
and does
is shown
extract,
analyzed
8A. Peak
in Figure
under
1 had the
retention time of cAMP and therefore an aliquot of the sample was chromatographed 8B). Next, an aliquot the
diesterase;
the
with the reference sample
of the same disappearance
compound (Figure was incubated with
of peak
1 gave
further
in-
dication that it is cAMP. It should be noted that the enzymatic peak-shift not only confirms the peak identity, but also unmasks
the chromatogram,
other
ultraviolet-absorbing
240
CLINICAL CHEMISTRY,
which
is important
compounds
in assuring
are not hidden
Vol. 25, No. 2, 1979
that
under
the peak of interest. The spectral purity be confirmed by determining absorbance
of the peak can also ratios (31) and ul-
traviolet spectra. Because absorbances are additive, the resulting ultraviolet spectrum would look different from the spectrum of the pure reference compound if another compound eluted with the same retention time as the peak under study. Figure 9 shows the comparison of the corrected ultraviolet spectra of the material represented by peak 1 in the rat-brain extract and the cAMP reference compound. The close similarity between the two spectra tends to confirm the
identity
and the purity of peak 1 in the rat-brain
We conclude mode
of liquid
that
the use of the reversed-phase
chromatography
in the
analysis
extract. partition for cyclic
nu-
cleotides in biological samples is advantageous compared to other methods of analysis, in terms of speed, reproducibility, and accuracy. With the proper sample-preparation technique the column can be used for 400 to 500 analyses with no loss of efficiency. We saw no interferences from other naturally occurring constituents in the gradient elution separation of the five cyclic nucleotides in rat-brain and rat-liver extracts. The analysis does not require any pre-concentration and its sensitivity is of considerable importance in analyzing limited amounts of samples for the exceedingly low concentrations of these compounds. An alternative isocratic elution mode,
10. Goldberg, N. D., and O’Toole, A. G., Cyclic guanosine 3’,5’monophosphate in mammalian tissues and urine. J. Biol. Chem. 244, 3053 (1969). -
11. Strange, R. C., and Mj#{216}s, 0. D., The sources of plasma cyclic AMP: Studies in the rat using isoprenaline, nicotinic acid and glucagon. Ear. J. Clin. Invest. 5, 147 (1975). 12. Saitoh, V., Morita, S., Irie, Y., and Kohiri, H., Evaluation
of a new
beta-adrenergic blocking agent careteol, based on metabolic responses in rats. II. Blockade by carteolol of the epinephrineand isoproterePE
230
240
250
260
270
280
No. I
290
00
3)0
320
nol-induced increases of tissue and blood cyclic AMP in vivo. Biochem. Pharmacol. 25, 1843 (1976). 13. Brooker, G., Newer developments in the determination of cyclic AMP and other cyclic nucleotides, adenylate cyclase, and phosphodiesterase. Methods Biochem. Anal. 22,95 (1974). 14. Kuo, J. F., and Greengard, P., Cyclic nucleotide-dependent protein kinases. VI. Isolation and partial purification of a protein kinase activated by guanosine 3’,5’-monophosphate. J. Biol. Chem. 245,4067 (1970).
WAVELENGTH
lam)
Fig. 9. Comparison of the corrected spectra of peak 1 in sample shown in Fig. 8A and the cAMP reference compound Scanning conditions same as in Fig. 2
optimized
for the selective analysis of cAMP, offers a rapid
and reliable method for determination of this important compound in biological samples. Both methods of analysis lend themselves easily to several simple identification methods, which are necessary if the
identity of the components of complex mixtures of biological compounds is to be determined reliably and unambiguousAlthough the separation method is illustrated here with the analysis of rat-brain extracts, the analysis was found to be equally successful for rat-liver extracts. This method, coupled with an appropriate sample preparation, can be used for biological samples of different origin.
We thank
Waters
Associates
and Kratos Inc., Schoeffel Instrument Medical College, White Plains, Klaus Lohse and Mona Zakaria for their
Division; Dr. Gerard Smith of Cornell NY for the rat brain samples;
valuable
comments;
and Marion
Barry for preparing
the manu-
script.
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19. Farmer, R. W., Harrington, C. A., and Brown, D. H., A simple radioimmunoassay for 3’,S’-cyclic adenosine monophosphate. Anal. Biochem. 64,455 (1975). 20. Honma, M., Satoh, T., Takezawa, J., and Ui, M., An ultrasensitive method for the simultaneous determination of cyclic AMP and cyclic GMP in small-volume samples from blood and tissue. Biochem. Med. 18, 257 (1977).
21. Latner, tein-binding Chim. Acta 22. Tovey, assay for
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G., Determination
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displacement assays for cyclic Nucleotide Res. 2, 111 (1972).
AMP
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cagon on net splanchnic cyclic AMP production in normal and diabetic man. J. Clin. Invest. 53, 198 (1974). 8. Bloom, F. E., The role of cyclic nucleotides in central synaptic
30. Krstulovic, A. M., Hartwick, R. A., Brown, P. R., and Lohse, K., The use of UV scanning techniques in the identification of serum
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function.
(1974).
constituents
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liquid
chromatography.
CLINICAL CHEMISTRY, Vol. 25, No. 2, 1979
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