Quantitative isolation of radiolabeled metabolites without chromatography: Measurements of the biosynthesis of purines, pyrimidines, and urea in isolated hepatocytes

ANALYTICAL

BIOCHEMISTRY

108,406-418

(1980)

Quantitative Isolation of Radiolabeled Metabolites without Chromatography: Measurements of the Bigsynthesis of Purines, Pyrimidines, and Urea in Isolated Hepatocytes’ PHILIP Department

of Biochemistry

A. WENDLER~ and Biophysics,

AND GEORGE University

of Rhode

C. TREMBLAY Island,

Kingston,

Rhode

Island

02881

Received April 14, 1980 Measurements of the incorporation of radiolabeled precursors into urea, erotic acid, uridine nucleotides, and adenine nucleotides were used to demonstrate the reliability of procedures for the isolation of metabolites by cocrystallization with carrier. Incorporation of precursor into product was carried out with suspensions of isolated hepatocytes. Direct isolation of each radiolabeled product was accomplished essentially by saturating aliquots of the acid-soluble fraction of the incubation mixture with carrier at elevated temperatures, and allowing the radiolabeled metabolite and carrier to cocrystallize as the solutions cooled. When [“C]NaHCOs was employed as the precursor, the procedure permitted isolation of radiolabeled urea, erotic acid, and uridine nucleotides from the same incubation mixture. Evidence that the radiolabeled metabolite isolated with carrier was of the same chemical identity as the carrier was obtained by recrystallization to constant specific activity and also by the use of enzymes to remove the putative metabolite prior to cocrystallization. Conditions were varied to provide a range of rates of 8.4fold, 180-fold, and 13-fold for the incorporation of ]i4C]NaHC0, into urea, erotic acid, and uridine nucleotides, respectively, and the results of duplicate assays at six different rates over these ranges of activity showed individual determinations to differ from their means by 3.2 r 1.O, 1.1 f 0.3, and 3.3 + 1.5% (average f SE), respectively. The method of isolation by cocrystallization with carrier has proved inexpensive, reliable, and well suited to the simultaneous analysis of multiple samples. The advantages and limitations of these procedures over more conventional methods are discussed.

A few years ago, we were confronted with the necessity of quantitatively recovering large fractions of a specific radiolabeled metabolite, free from contaminants, and with limited resources in the way of instrumentation. We were seeking to determine the capacity of minces or slices 1 This woFk was supported by a Public Health Service Biomedical Research Support Grant (5S07RR07086) and by a PHS Research Grant (AM 26166) from the National Institute of Arthritis, Metabolism, and Digestive Diseases. * The work presented herein will also be submitted in partial fulfillment of the requirements for the Ph.D. in Biological Sciences (Biochemistry). 0003-2697/80/160406-13$02.00/O Copyright All rights

0 1980 by Academic Ress, Inc. of reproduction in any form reserved.

406

of various mammalian tissues to synthesize pyrimidines de MIVO, via the six enzymatic steps of the erotic acid pathway. Since measurements of the activity of a biosynthetic pathway in the intact cell require the use of simple precursors, the number and quantity of the metabolic products are multiplied by comparison to enzyme assays which employ specific substrates and dilutions of cell-free extracts. Thus, such measurements require the isolation of a known fraction of product in sufficient quantity for accurate determination, and free from contamination with a potentially large number of metabolites of the simple precursor

METABOLITE

ISOLATION

employed. The task may be a particularly onerous one, requiring sequential steps of chromatographic resolution, some of which may only be suited to the analysis of small aliquots of sample, thereby sacrificing sensitivity. In addition, each chromatographic step compromises the uniform recovery of product, requiring that internal standards for recovery be used routinely for accurate results. The chromatographic analysis of product may also require a substantial investment in instrumentation and hardware designed specifically for the resolution of the class of compounds under study. The alternative method we offer below does not require chromatography, labeled internal standards to measure the recovery of product, or any specialized instrumentation. It is simple in principle and practice, suited to large numbers of simultaneous assays, easily learned, very reliable, and it has been generally inexpensive. The procedure is based upon one’s ability to isolate the metabolite of interest, or a derivative, in crystalline form. In our earlier studies on pyrimidine biosynthesis, we took advantage of the differential solubility of monosodium orotate in hot and cold water, and of the effectiveness of a drug (6azauridine) in preventing the conversion of orotate to UMP. Thus, the capacity of the intact cell to synthesize pyrimidines de now was estimated from measurements of the rate of incorporation of [L4C]NaHC03 into erotic acid in tissue slices incubated in a physiological medium containing 6azauridine (1). The accumulated [14C]orotate synthesized during the incubation period was readily isolated from the neutralized acid-soluble fraction of the reaction mixture simply by saturating the solution near the boiling point with carrier monosodium orotate and allowing the radiolabeled orotate and carrier to cocrystallize as the solution cooled slowly to 4°C. Once the reliability of this procedure had been adequately demonstrated, routine assays re-

BY COCRYSTALLIZATION

407

quired only that the crystals be recrystallized to constant specific activity before the content of radioisotope be used to calculate the rate of erotic acid synthesis. Since the quantity of product synthesized by the cell is insignificantly small compared to the quantity of carrier employed, and given that the radiolabeled metabolite is uniformly distributed with carrier in the crystal structure, the yield of carrier and the recovery of radiolabeled metabolite are identical. While isotopically labeled compounds are commonly employed in the identification, purification, and quantification of natural substances (e.g., through cochromatography, cocrystallization, determination of recovery, isotope dilution), we are unaware of procedures in current use which employ carrier as the sole vehicle for the direct isolation of radiolabeled metabolites from complex reaction mixtures. Procedures for the entrapment of radiolabeled metabolites by coprecipitation with carrier, followed by purification of the precipitate through crystallization, were among the early applications of the use of isotopes in biochemistry (e.g., (2-4)), but such procedures have apparently been abandoned over the years in favor of rapidly developing, sophisticated, and sensitive chromatographic methods. In the present communication we describe a contemporary application of the use of carrier as a vehicle for the isolation of radiolabeled metabolites, with measurements of changes in the activities of three metabolic pathways in the isolated suspended hepatocyte. Our purpose is to illustrate the versatility and reliability of this technique in the study of metabolism, and to draw attention to the advantages it may hold in both execution and economy over more conventional methods. Results of simultaneous measurements of the rate of incorporation of [14C]NaHC0, into erotic acid, total uridine nucleotides, and urea in isolated hepatocytes, with all products being isolated from aliquots of the same reaction

408

WENDLER

AND TREMBLAY

mixture, are presented. A procedure for measuring the incorporation of [14C]formate into total adenine nucleotides is also described. The procedures for measuring the rate of pyrimidine synthesis have been reported (5,6) and are further established herein; the procedures for determination of the rate of urea synthesis and the synthesis of adenine nucleotides have not been reported previously. MATERIALS

AND METHODS

Chemicals. All radiolabeled chemicals and Aquasol (liquid scintillation cocktail) were purchased from New England Nuclear Cot-portion, Boston, Massachusetts. Acids and bases were purchased from Fisher Scientific Company, Boston, Massachusetts. Enzymes and all other chemicals were purchased from Sigma Chemical Company, St. Louis, Missouri. Isolation of liver cells. All experiments were done with 200- to 300-g male SpragueDawley rats, CD strain, obtained from the Charles River Colony, Boston, Massachusetts and fed laboratory chow ad libitum. Hepatocytes were isolated as follows, according to the method of Seglen (7). The liver was perfused for 15-20 min at 37°C with Ca2+-free buffer (7) at a flow rate of 50-60 ml/min, without recirculation. Then 100 ml of fresh perfusion fluid containing CaCl,, 5 mM, and collagenase, 7500 units (type IV, 150 units/mg protein, Sigma Chemical Company), were recirculated for 5-10 min at 37°C without aeration. The liver was then placed in a petri dish and gently shaken to disperse the cells. Large pieces of debris were removed with forceps and the remaining material was transferred to an Erlenmeyer flask for an additional incubation of 5-10 min at 37”C, with swirling, in the same perfusion fluid used in the preceding step, containing the collagenase and CaCl,. Following this, the cell suspension was filtered through gauze and the cells were harvested by centrifuga-

tion at 30g for 2 min. The isolated hepatocytes were washed with 40 ml of Krebs Improved Ringer II Solution (KR-II(S))” and resuspended in this medium to give approximately 12 mg (dry wt) cells/ml; 700-800 mg hepatocytes (dry wt) were routinely obtained by this procedure. The dry weight of the cells was given by the dry weight of the acid-insoluble, lipidfree residue, as described by Katz er al. (9); we determined that 1 g cells, dry wt, represented 4.26 g liver, wet wt. Cell viability was assessed by the exclusion of Trypan blue and routinely found to be greater than 90%. Measurement of the incorporation of [‘“Cl NaHC03 into erotic acid, uridine nucleotides, and urea. Cells were incubated with swirling at 37°C for 1 h in a final volume of 20 ml of KR-II solution made 25 mrvr in [14C]NaHC03 (100-300 PCi) and containing 2-3% bovine serum albumin (Fraction V) which had been dialyzed overnight against four changes of bicarbonate-free KR-II solution. The incubation mixture contained approximately 3 mg dry wt of cells/ml. Each reaction flask was sealed with a rubber cap fitted with a plastic center well (Kontes Glassware, Vineland, N. J.) and a filter paper wick. At the end of the incubation period, 0.5 ml of 6 N KOH was injected through the rubber cap into the center well; the reaction was then terminated by the injection of 5 ml of 1.5 N HClO, into the reaction medium. Following an additional 10 min incubation at 37°C to allow the unreacted 14C0, from the acidified reaction mixture to distill into the KOH, the flasks were opened and the center wells, containing radioactive waste, were 3 Abbreviations used: KR-II, Krebs Improved Ringer-II Solution (8); HUMP, sum of the uridine nucleotides converted to UMP by hydrolysis with HClO, (1 N) for 1 h in a boiling-water bath; OPRTase, orotidine-S-phosphate:pyrophosphate phosphoribosyltransferase (EC 2.4.2.10); ODCase, orotidine-5’. phosphate decarboxylase (EC 4.1.1.23); PRPP, S-phosphoribosyl-1-pyrophosphate.

METABOLITE

ISOLATION

appropriately discarded. The contents of the reaction flasks were homogenized using a Kinematica Polytron tissue homogenizer (Brinkmann Instruments, Westbury, N. Y.), and the acid-insoluble material was removed by centrifugation. The acid-soluble fraction was diluted with water to 42 ml and duplicate aliquots of the dilution were removed for the isolation of each of the radiolabeled metabolites, as described in the following sections. Isolation of [14C]orotic acid. Duplicate 9-ml aliquots of the acid-soluble fraction were neutralized with KOH and the resultant precipitate of KClO, was removed by centrifugation. The [14C]orotic acid synthesized during the l-h incubation was isolated from the neutralized acid-soluble fraction by cocrystallization with carrier monosodium orotate as described previously (5). Isolation of [14C]uridine nucleotides as [‘“C]UMp. Duplicate 9-ml aliquots of the acid-soluble fraction were adjusted to 1 N in HCIO, and heated for 1 h at 100°C to hydrolyze the uridine nucleotides to UMP. The hydrolysate was then neutralized with KOH, and the resultant precipitate of KClO, removed by centrifugation. The [‘4C]uridine nucleotides synthesized during the l-h incubation were isolated from the neutralized acid-soluble fraction as UMP by cocrystallization with carrier UMP as described previously (6). Isolation of [‘4C]ureu. Duplicate 2-ml aliquots of the acid-soluble fraction were adjusted to pH 7.0-7.5 by the addition of KOH and the precipitate of KClO, was removed by centrifugation. The neutralized solutions were lyophilized and the urea in the residue was dissolved in 2 ml of ethanol by warming the mixture to 50°C for lo-15 min. The alcoholic mixture was cooled to room temperature and diluted with 10 ml of ethanol containing 900 mg carrier urea in solution. The solutions were mixed thoroughly and the alcohol-insoluble residue was removed by filtration through Whatman No. 1

BY COCRYSTALLJZATION

409

paper. The alcoholic solutions were diluted with 10 ml CHC& and the resulting precipitates of urea were dissolved by heating to 55°C. The [14C]urea synthesized from [ 14C]NaHC0, during the incubation period was isolated by cocrystallization with carrier as the solution cooled slowly to 0°C. The crystals were harvested by suction filtration, washed with ice-cold chloroform: ethanol (1: l), and dried in a vacuum. An aliquot of 50 mg of crystals was transferred to a scintillation vial, dissolved in 2 ml water, and the solution was diluted with 6.5 ml Aquasol to determine the quantity of radioisotope with a Searle Isocap 300 liquid scintillation spectrometer. The remaining [14C]urea was recrystallized from chloroform:ethanol(8:5) to constant specific activity. The final specific activity of the [14C]urea was used to calculate the incorporation of [14C]NaHC0, into urea during the incubation period. Measurement of the incorporation of [‘4C]HCOONu into udenine nucleotides and the isolation of [14C]udenine nucleotides us [14C]udenine. Cells were incubated as described above, with the exception that [14C]HCOONa was used in place of [14C]NaHC0, as the radiolabeled precursor; the incubation remained 25 mM in bicarbonate. Duplicate aliquots of the acidsoluble fraction of the incubation mixture were made in 1 N in HClO, and heated in a boiling water bath for 1 h to convert all adenine nucleotides and adenosine to the free base, adenine. After cooling, the acidsoluble fraction was adjusted to pH 7.07.5 by the addition of KOH, and the precipitate of KClO, was removed by centrifugation. The neutralized solutions were diluted with water to 25 ml and 500 mg of carrier adenine was dissolved with heat. The [14C]adenine nucleotides synthesized from [*4C]HCOONa during the incubation period were isolated as [14C]adenine by cocrystallization with carrier as the solution cooled slowly to 4°C. The crystals were harvested by suction filtration, washed

410

WENDLER

AND

with copious quantities of cold water and dissolved in 10 ml of 0.5 N HCI. The recovery of adenine was determined from the optical density at 260 nm of a suitable dilution and the quantity of radioisotope was measured in a 2-ml aliquot with a Searle Isocap 300 liquid scintillation spectrometer. The adenine was recrystallized to constant specific activity from the remaining solution after neutralization and dilution with water to 25 ml. Determination of the specific activity of [W]NaHC03. Measurements of any changes

in the specific activity of [14C]NaHC0, during the course of the incubation period were monitored after conversion to [‘“ClBaC0,.4 After the designated period of incubation, 0.50 ml of 6 N KOH was injected into the plastic center well suspended from the rubber stopper sealing the reaction vessel. The reaction mixture was then acidified by injecting 5 ml of 1.5 N HCLO, through the rubber stopper into the main

TREMBLAY

chamber, and the [14C]C0, generated was allowed to distill into the KOH for 1 h at 37°C in a reciprocating water bath. Following the distillation period, the contents of the plastic center well were quickly transferred to a centrifuge tube containing 8.5 ml of 1 M BaCl,. The tube was capped and the precipitate of BaCO, was collected by centrifugation. The precipitate was washed twice with 30-ml portions of ice-cold distilled water and dried to constant weight in vacuuo over P205. The dried [14C]BaC0, was ground to a fine powder and aliquots of 10 mg were suspended in the gel formed upon cooling the solution prepared by mixing 2.0 ml of water with 6.5 ml of Aquasol. The content of radioactivity was measured with a liquid scintillation spectrometer. Calculation of product formation. The quantity of precursor incorporated into product in a typical reaction mixture for the duration of the incubation period is given by:

(dpm observed \( units of carrier used \( 1 \ unit of carrier dpmlnmole precursor fraction of reaction mixture assayed

\

I\

I\

=

I

nanomoles precursor incorporated into product in the reaction mixture. The data may be expressed in terms of product formation per unit wet weight of liver by the following calculation: nanomoles

precursor incorporated

into product

mg cells, dry weight, in 4.26 mg liver, wet weight mg cells, dry weight [ i the reaction mixture I( )I nanomoles

precursor incorporated

i looogmg)

=

into product per gram tissue, wet weight.

RESULTS Kinetics of Incorporation into Product

of Precursor

The rate of product formation was determined from separate reaction mixtures 4 The authors are grateful to Dr. Y. Kobayashi of the New England Nuclear Corporation, Boston, for the analytical procedure to determine the specific activity of [%]bicarbonate as [“C]BaC03.

for each time period. The incorporation of [14C]HCOONa into adenine nucleotides was determined from one set of reaction mixtures and the incorporation of [14ClNaHCO, into urea, erotic acid, and uridine nucleotides was determined from aliquots of a second set. Reaction mixtures containing [14C]NaHC03 as the labeled precursor were made 5 mM in NH,Cl to ac-

METABOLITE

ISOLATION

BY COCRYSTALLIZATION

0

TIME OF INCUBATION

411

0.5

(h)

FIG. 1. Hepatocytes equivalent to 140 mg wet wt of liver were incubated at 37°C in 20 ml Kr-II solution (i) adjusted to 25 mM in [W]NaHCO, (200 &i) and 5 mM in NH&l for measurements of incorporation into urea, erotic acid (Oro), and uridine nucleotides (ZUMP), or (ii) adjusted to 25 mM in NaHCO, and made 3 mM in [W]HCOONa (100 &i) for measurements of incorporation into adenine nucleotides and adenosine (ZAde). Uridine nucleotides were converted to UMP and adenine nucleotides and adenosine to adenine by acid hydrolysis ofthe acid-soluble fraction. The radiolabeled products were isolated from the neutralized acid-soluble fraction by cocrystallization with carrier, as described under Materials and Methods.

celerate the synthesis of urea and pyrimidines (5). The rates of incorporation of [14C]NaHC03 into urea, erotic acid (Oro), and uridine nucleotides (ZUMP), and the rate of incorporation of [14C]HCOONa into adenine nucleotides (XAde) were all linear for at least 1.5 h and, except for incorporation into ZUMP, continued at the initial velocity for at least 2 h (Fig. 1). The amount of radioisotope isolated with UMP was essentially constant after 1.5 h, suggesting that incorporation and utilization had reached a steady state; the values at 2.5 and 3 h were 90 and 93% the value at 2 h, respectively (data not shown). Dilution of the specific activity of [14C]NaHC0, by CO,

generated by the isolated hepatocytes was minor. Assays of the bicarbonate, collected as [14C]BaC0,, after 0.5, 1.O, 1.5, and 2.0 h of incubation showed the specific activity to decline gradually, to 95, 92, 85, and 81% of the zero-time value, respectively. Reproducibility of the Isolation of Metabolites by Cocrystallization with Carrier

The reproducibility of each analytical procedure was examined by assaying duplicate aliquots of a series of reaction mixtures designed to monitor the biosynthetic pathways over a wide range of activities.

412

WENDLER

AND TREMBLAY TABLE

INCORPORATION

OF

[14C]NaHC03

INTO

UREA,

BY ISOLATED

Reaction No. 1.

Additions or deletions None (control)

1 OROTIC

Assay No. (1)

(2) 2.

-NH,Cl

(1)

(2) 3.

+ L-Omithine (10 mM)

(1)

(2) 4.

+6-Azauridine

(10

mM)

(1)

(2) 5.

+Norvaline

(5 mM)

(1)

(2) 6.

-NH&l

+ galactosamine (2 mM)

ACID,

AND

URIDINE

NUCLEOTIDE~

HEPATOCYTES”

(1)

(2)

Orotic acid

ZUMP

48.0, 51.7

2.58 2.64

1.07 1.00

13.8 14.4

0.02 0.02

0.11 0.09

119.0 117.0

0.49 0.51

0.43 0.40

41.1 43.8

3.35 3.28

0.08 0.08

17.0 19.8

3.63 3.56

0.92 0.97

14.4 14.9

0.45 0.46

0.22 0.22

Urea

* Hepatocytes equivalent to 326 mg wet wt of liver were incubated for 1 h at 3PC in 20 ml KR-II solution supplemented with NH&l, 10 mM and [“C]NaHC03, 25 mM (300 &i in reaction mixtures 2 and 6, 100 &i in each of the remaining reaction mixtures); other additions or deletions were made as indicated. Radiolabeled products were isolated from duplicate aliquots of the acid-soluble fraction of the same reaction mixture by cocrystallization with carrier as described under Materials and Methods. All values are given in micromoles [“C]NaHC03 incorporated into the designated product per gram wet weight of liver during l-h incubation.

Duplicate assays of the incorporation of [14C]NaHC0, into urea, erotic acid, and uridine nucleotides were shown to vary from their means by an average of 3.2 k 1.0, 1.1 2 0.3, and 3.3 2 1.5% (average + SD, IZ = 6), respectively (Table 1). The fidelity of the measurements may be gauged by the predictable alterations brought about by additions or deletions to the reaction mixtures. Both the urea cycle and the orotate pathway are initiated by the synthesis of carbamoylphosphate from bicarbonate, ATP, and a nitrogen source. The carbamoylphosphate synthetase associated with the urea cycle requires ammonia as the source of nitrogen and is localized &th ornithine carbamoyltransferase in the mitochondrial matrix, while that associated with pyrimidine biosynthesis utilizes glutamine as its nitrogen source and is localized with aspartate carbamoyltransferase in the soluble cytoplasm. How-

ever, the intramitochondrial localization of the ammonia-requiring enzyme (which is much more active than its cytoplasmic counterpart) does not prevent it from contributing toward pyrimidine biosynthesis (5). Thus, deletion of NH,Cl from the reaction mixture sharply reduced the incorporation of [14C]NaHC0, into both urea and the pyrimidine metabolites (compare reaction mixtures 1 and 2, Table 1). The addition of omithine, which favors the incorporation of carbamoylphosphate into citmlline over its export into the soluble cytoplasm, more than doubled the incorporation of [14C]NaHC0, into urea while reducing incorporation into pyrimidines by 81% for erotic acid and 60% for ZUMP (compare reaction mixtures 1 and 3). Parenthetically, these data show the sum of the incorporation of [14C]NaHC03 into urea, erotic acid, and uridine nucleotides to be greatest by a wide margin in the

METABOLITE TABLE INCORPORATION NUCLEOTIDES

ISOLATION

2

OF [%~]FORMATE

INTO

ADENINE

BY ISOLATED

HEPATOCYTES”

Incorporation (nanomoleslg wet wt liver. h) Additions None Orotic acid (2

mM)

Assay 1

Assay 2

28.8 8.0

21.2 8.2

n Experimental conditions were the same as those described for Table 1 (including the addition of bicarbonate at 25 mM) except that NH,Cl was omitted and [W]HCOONa, 3 mM (100 &i) was added as the radiolabeled precursor.

presence of ornithine. Whether this same amount of carbamoylphosphate is synthesized in the absence of ornithine is not known, nor is it known what the metabolic fate of this fraction (>50%) of carbamoylphosphate is if it is indeed produced when ornithine is scarce. The addition of 6-azauridine, a known inhibitor of the conversion of erotic acid to UMP (lo), had no significant effect on ureagenesis but caused a marked reduction in the incorporation of [14C]NaHC0, into uridine nucleotides, and about three-fourths of this reduction could be accounted for in the increased pool of [‘“Cl erotic acid (compare reaction mixtures 1 and 4). Norvaline, an analog inhibitor of omithine carbamoyltransferase (11) which would decrease the intramitochondrial utilization of carbamoylphosphate and favor the export of carbamoylphosphate into the soluble cytoplasm, enhanced the incorporation of [‘“ClNaHCO, into erotic acid while limiting incorporation into urea to 37% of the control level (compare reaction mixtures 1 and 5). Note that norvaline did not enhance incorporation into uridine nucleotides beyond the rate observed with NH,Cl alone, suggesting that NH,Cl alone caused erotic acid to accumulate to saturating levels in the cell. Galactosamine has been shown to de-

BY COCRYSTALLIZATION

413

plete the liver of uridine nucleotides as it is metabolized to its UDP derivative, and galactosamine treatment has also been shown to lead to an acceleration in the de ~OVO biosynthesis of pyrimidines, presumably through the release of end-product inhibition of the cytoplasmic carbamoylphosphate synthetase (12,13). When NH4CI was omitted from the reaction mixture, to minimize the activity of the mitochondrial carbamoylphosphate synthetase, and galactosamine was added to deplete the cell of uridine nucleotides, the incorporation of [14C]NaHC0, into urea was reduced to the same level as was observed with the omission of NH,Cl only (i.e., galactosamine was without effect on ureagenesis), whereas incorporation into erotic acid and CUMP was accelerated 21-fold and 2.2-fold, respectively (compare reactions 2 and 6). Reproducibility of the procedure for isolating the adenine-containing metabolites of [ 14C]HCOONa by cocrystallization with carrier is illustrated in Table 2; the duplicate values deviate by less than 3% from their means. The influence of erotic acid on purine synthesis was readily detected by this procedure. Orotic acid inhibited the incorporation of [14C]HCOONa into adenine nucleotides by about 70%, presumably by depleting the PRPP-derived precursor for formate incorporation (12) since the metabolism of erotic acid to UMP consumes PRPP. These results are all consistent with reports in the literature and attest to the reliability of the procedures described above. Proof of the Identity of the Metabolites Cocrystallizing with Carrier The identity of the metabolite of [‘“ClNaHCO, cocrystallizing with carrier orotate was verified by specific enzymatic removal of the metabolite prior to cocrystallization. Six equal aliquots of the acid-soluble fraction of a typical incubation mixture were subjected to further treatment, in duplicate,

414

WENDLER

AND TREMBLAY TABLE

IDENTITYOFTHEMETABOLITEOF

[‘V]NaHCO,

3

WHICHCOCRYSTALLIZESWITH

CARRIER~ROTATE"

r4C content of carrier (dpm/50 mg monosodium orotate) Reaction

Additions

Assay 1

Assay 2

1. 2. 3.

None (control) OPRTase + ODCase OPRTase + ODCase + PRPP

21,249 20,161 2,091

20,796 21,291 2,350

%Control 100 99 11

a Hepatocytes equivalent to 251 mg wet wt of liver were incubated for 3 h at 37°C in 20 ml KR-II solution adjusted to 25 mM in [i4C]NaHC0, (300 &i) and 10 mM in NH&J. Duplicate aliquots of 2 ml each were incubated for 4 h at room temperature under three sets of conditions: (a) without additions, (b) with the mixed enzymes OPRTase + ODCase (10 units), and (c) with the mixed enzymes, PRPP (5 mM), and MgCl, (5 mM). The reactions were terminated by transfer to a boiling water bath for 5 min and the precipitate was removed by centrifugation. The supematant fluid was diluted to 50 ml and the [“Cl orotate isolated with 375 mg carrier as described under Materials and Methods.

under three separate sets of conditions prior to cocrystallization with carrier. One pair of aliquots was incubated 4 h at room temperature with PRPP and the mixed enzymes orotate phosphoribosyltransferase (OPRTase) and orotidine monophosphate decarboxylase (ODCase). Incubation under these conditions should convert [i4C]orotic acid to UMP. A second pair of aliquots was treated in the same manner except that PRPP was omitted. The third pair of aliquots was incubated without any additions. At the end of the incubation period the reactions were terminated with heat, precipitated material was removed by centrifugation, and the soluble fraction was diluted to 50 ml with water for cocrystallization with carrier. About 90% of the metabolite cocrystallizing with carrier orotate was removed by treatment with PRPP and the mixed enzymes prior to the addition of carrier (Table 3), and the enzymatic removal was shown to be entirely dependent upon the addition of PRPP. Thus, the metabolite of [14C]NaHCO, cocrystallizing with carrier orotate was established to be [14C]orotate. Similarly, the metabolite of [14C]NaHC03 cocrystallizing with carrier urea was established to be [14C]urea. Treatment of the neutralized acid-soluble fraction with urease

prior to the addition of carrier removed 99% of the radioactivity cocrystallizing with carrier urea (Table 4), and more than 90% of this radioactivity was recovered by distillation into KOH following acidification (data not shown). The metabolite of [14C]NaHC0, which cocrystallizes with carrier UMP has previously been shown by chromatographic resolution in three different solvent systems to be [‘“C]UMP (6). In addition, by following a procedure similar to the others described above for enzymatic removal, we have also demonstrated that 98% of the radiolabeled metabolite of [14C]NaHC03 that cocrystallizes with carrier UMP can be removed by treatment with 5’-nucleotidase prior to the addition of carrier (data not shown). The metabolites of [14C]HCOONa yielding the derivative which cocrystallizes with carrier adenine have also been identified through selective enzymatic removal. Rather than convert the adenine nucleotides and adenosine directly to adenine by acid hydrolysis, as is routinely done, the purines in the acid-soluble fraction of a typical reaction mixture were adsorbed to and eluted from charcoal. The charcoal-extracted acid-soluble fraction was saved for

METABOLITE TABLE

ISOLATION

4

temperature, the charcoal-extracted acidsoluble fraction of the initial reaction mixture was added back to restore any other possible metabolites of [14C]HCOONa which might cocrystallize with carrier adenine. The [14C]adenine nucleotides and [14C]adenosine were then isolated as [14C]adenine after acid hydrolysis as described above. About 95% of the metabolites of [‘“ClHCOONa yielding the derivative that cocrystallizes with carrier adenine were removed by treatment with all three enzymes, and the removal was entirely dependent upon the inclusion of adenosine deaminase (Table 5). Thus, the radiolabeled derivative cocrystallizing with carrier adenine was shown to be [14C]adenine.

IDENTITYOFTHE METABOLITEOF [*4C]NaHC03 WHICH COCRYSTALLIZES WITH CARRIER UREA" 14Ccontent of carrier (dpm/SO mg urea) Reaction 1. 2.

Additions

Assay 1

Assay 2

None (control) Urease

29,905

28,071

100

349

205

1

415

BY COCRYSTALLIZATION

%Control

B Hepatocytes equivalent to 217 mg wet wt of liver were incubated for 3 h at 37°C in 20 ml KR-II solution adjusted to 25 mM in [“C]NaHC03 (100 &i) and 5 mM in NH,Cl. Duplicate 2-ml aliquots of the neutralized acid-soluble fraction were incubated for 1 h at 30°C with or without urease (60 units) prior to cocrystallization with 900 mg carrier urea. The reaction vessels were also fitted with a sealed stopper and plastic center well containing KOH to trap the [W]CO, released upon acidification after urease treatment.

Recovery of Standards by Cocrystallization

later use. The charcoal eluate was divided into equal aliquots for incubation under one of the following conditions: (i) without additions, (ii) with snake venom phosphodiesterase to hydrolyze all nucleotides to the monophosphates and 5’-nucleotidase to convert the monophosphates to the nucleosides, or (iii) with the phosphodiesterase, S-nucleotidase and adenosine deaminase to convert adenosine to inosine. After the incubation period of 1 h at room

Having established that radiolabeled metabolite and carrier were of the same chemical identity in each of the above procedures, we sought to estimate the reliability of each procedure in converting all of the designated radiolabeled metabolite contained in a typical incubation mixture to the structure of its carrier, and to determine the degree of recovery of each metabolite. This was accomplished by measuring the recovery of radiolabel from typical incubation mixtures which were fortified with

TABLE

5

i4C content of carrier (dpm/unit absorbance at 260 nm) Reaction 1. 2. 3.

Additions None (control) Phosphodiesterase (1 unit) + 5’-nucleotidase Phosphodiesterase (1 unit) + 5’nucleotidase + adenosine deaminase (5 units)

(5 units) (5 units)

Assay 1

Assay 2

533 552 23

540 573 33

%Control 100 105 5

a Hepatocytes equivalent to 289 mg wet wt of liver were incubated for 1 h at 37°C in 20 ml KR-II solution adjusted to 25 mM in NaHCO, and supplemented with [W]HCOONa, 3 mM (100 &i). Equal aliquots of the acid-soluble fraction obtained at the end of the incubation period were treated with the enzymes designated above prior to cocrystallization with carrier (see text for details).

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authentic [14C]UTP, or [14C]ATP, or [‘“Cladenosine just prior to acidification. Each incubation mixture so prepared was then subject to the usual treatment for isolation of the radiolabeled compound with carrier. In two separate analyses for the isolation of each of these radiolabeled compounds by cocrystallization with the appropriate carrier (UMP or adenine), the recovery of radioactivity with carrier was always greater than 90%. Similarly, the recovery of authentic [14C]orotate and [14C]urea with carrier was also greater than 90%. These results show that small amounts of authentic metabolites were readily converted to and recovered with carrier. DISCUSSION Conventional chromatographic procedures for the isolation of radiolabeled metabolites have several disadvantages which may be circumvented with the method of isolation by cocrystallization with carrier. For example, the isolation of purines and pyrimidines is often initiated by adsorption to and elution from charcoal, followed by chromatography on at least two ion-exchange resins (15). Such a procedure suffers from incomplete elution from charcoal, a limitation on the amount of sample which can be applied to the resin bed, dilution of the metabolite in the volume of eluate collected from the column, and cochromatography of the metabolite of interest with contaminants. By comparison, isolation of a radiolabeled metabolite by cocrystallization with carrier allows recovery of virtually all of the metabolite from the acid-soluble fraction of the reaction mixture in a single step, requiring no transfers beyond decanting large volumes of supernatant fluid from small amounts of precipitated protein and nucleic acid. The data presented above provide evidence of the high degree of specificity and reproducibility that can be expected of this procedure. In addition, the use of carrier allows an exact determination of the amount

TREMBLAY

of radiolabeled metabolite recovered from every reaction mixture, since the percentage of carrier recovered is identical to the percentage of metabolite recovered. Cautious processing of sample to assure uniform recovery is not required in the isolation of metabolites by cocrystallization; from the point at which carrier is added, accurate determination of recovery is assured. Isolation of radiolabeled metabolites by cocrystallization with carrier may also offer advantages in both time and economy over conventional chromatographic procedures. The variety of alternative procedures available for each of the determinations presented herein, and differences of opinion on the acceptable allowances one might make for precision among these procedures, do not permit a meaningful summary comparison. However, to allow a comparison between our procedure and any given alternative one might consider, we provide the following statistics. The experiment described in Table 1 provided for duplicate assays of three separate measurements under six different experimental conditions; that is, 36 separate determinations in all. One full day of work by a single individual was required to obtain the neutralized acidsoluble fractions of the six reaction mixtures (the time required at this stage is independent of the method chosen for subsequent analysis of the quantity of each radiolabeled metabolite). The additions of carrier, crystallizations, and recrystallizations, and the measurements of radioisotope, and recovery of carrier, and metabolite required about l/2 day’s work by a single individual for each of the next 4 days. The results of all 36 determinations were available by the fifth day; the analytical procedures required intermittent effort totaling 17 man h. In our hands, the isolation of carrier at constant specific activity occurs after a single crystallization with urea and after an initial crystallization plus one or two recrystallizations for erotic acid and UMP. The only item of expense unique to our procedure is the cost of carrier, which is

METABOLITE

ISOLATION

consumed in appreciable quantities. The cost of urea, adenine, and erotic acid is very modest, but the price of UMP is, by comparison, somewhat expensive. While the amount of radioisotope employed determines the sensitivity of the assays, it is important to note that virtually all of the radiolabeled metabolite is recovered by cocrystallization with carrier, and a large fraction of this is available for measurement of radioactivity. Thus, assay by cocrystallization with carrier would require less radioactivity without compromising sensitivity than would other procedures analyzing smaller fractions of the reaction mixture. The amount of radioisotope employed in the experiment described in Table 1 was unusually high to allow for analysis of six aliquots of each reaction mixture. The results ranged from 5376 to 37,673 cpm/50 mg carrier urea (reactions 5 and 3, respectively), 208 to 12,430 cpm/ 50 mg carrier orotate (reactions 2 and 5, respectively), and 344 to 4469 cpm/unit absorbance at 260 nm for carrier UMP (reactions 4 and 1, respectively); these are the amounts of carrier routinely assayed for content of radioisotope. Total costs for radioisotope and carriers were $25 and $29, respectively, for all 36 determinations reported in Table 1. A reduction in the number of measurements required from a single reaction mixture, the use of larger aliquots of carrier for each determination, and the possibility that the same quantity of hepatocytes might perform equally well in a smaller incubation volume, would each reduce the requirement and expense for radioisotope. As with any technique employed in measuring the activity of a metabolic pathway by determining the rate of incorporation of radiolabeled precursor into product, such measurements are compromised to the extent that the endogenous pool sizes of intermediates in the pathway dilute the specific activity of the radiolabeled precursor, and to the extent that the product being measured is further metabolized during the incubation period. In

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the case of measurements of the incorporation of [14C]NaHC0, into erotic acid, the former problem is negligible since the intermediates do not normally accumulate to detectable levels, whereas in the case of measurement of the incorporation of [14C]NaHCO, into urea the latter problem does not apply since urea does not undergo further metabolism in the mammalian hepatocyte. The further metabolism of erotic acid or uridine nucleotides does, however, present a problem in the quantitative estimation of the incorporation of [14C]NaHC0, into these products. In the case of orotate, this problem was addressed with the use of 6-azauridine, which inhibited the incorporation of [14C]NaHC0, into uridine nucleotides about 90% by blocking the conversion of orotidine 5’-monophosphate to UMP (10). No doubt the use of an inhibitor results in some accumulation of all metabolites in equilibrium with the substrate of the inhibited enzyme, and one should be alert to the influence such an accumulation might have on the interpretation of results. In the case at hand, about three-fourths of the radioisotope prevented by 6-azauridine from appearing in uridine nucleotides could be accounted for in the accumulated erotic acid (Table 1). Thus, the use of selective inhibitors may enhance the accuracy of determinations by preventing the further metabolism of the product being measured, and the availability of such an inhibitor may influence the decision on which metabolite might be most suitable for measurement. In studies on pyrimidine synthesis, isolation of erotic acid with the aid of 6-azauridine is well suited to an examination of feedback control of the pathway at an early step, but obviously neglects the possibility of regulation at the level of OPRTase and ODCase, the enzymes which convert erotic acid to UMP (6). It is also important to consider the possibility that the endogenous levels of end products of the pathway might be sufficiently great to subject the pathway to appreciable feedback inhibition, in which case the capacity of the

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cell to synthesize the product would be underestimated. Evidence that such a situation influences measurements of the rate of pyrimidine synthesis in the intact cell was obtained in the assay containing galactosamine, which depletes the cell of free uridine nucleotides (compare reactions 2 and 6, Table 1). Problems unique to the isolation of radiolabeled metabolites by cocrystallization with carrier include the ease with which the metabolite can be crystallized, the chemical stability of the metabolite, the commercial availability of carrier at acceptable cost, and the availability of suitable analytical methods to demonstrate unequivocally that the radiolabeled metabolite cocrystallizing with carrier is chemically identical to the carrier (as opposed to a heterogenous molecular complex with carrier, for example). Thus far, we have had no difficulties in meeting these requirements and we have been able to obtain enzymes from commercial sources to verify the chemical identity of each of the metabolites we have isolated with carrier. In our experience the procedure has proved inexpensive, reliable, and well suited to the simultaneous analysis of multiple samples. REFERENCES 1. Smith, P. C., Knott, C. E., and Tremblay, G. C. (1973) Biochem. Biophys. Res. Commun. 55, 1141-1146.

2. Evans, E. A., Jr., and Slotin, L. (1940) J. Biof. Chem. 136,301-302. 3. Keston, A. S., Udenfriend, S., and Cannan, R. K. (1946) J. Amer. Chem. Sot. 68, 1390. 4. Kamen, M.D. (1948) Organic and Biological Chemistry (L. F. Fieser and M. Fieser, eds.), Vol. I, Radioactive Tracers in Biology, An Introduction to Tracer Methodology, Academic Press, New York. 5. Tremblay, G. C., Crandall, D. E., Knott, C. E., and Alfant, M. (1977) Arch. Biochem. Biophys. 178, 264-277. 6. Crandall, D. E., Lovatt, C. J., and Tremblay, G. C. (1978) Arch. Biochem. Biophys. 188, 194-205. 7. Seglen, P. 0. (1976) in Methods in Cell Biology (Prescott, D. M., ed.), Vol. 13, pp. 29-83, Academic Press, New York. 8. Dawson, R. M. C. (1%9) in Data for Biochemical Research (Dawson, R. M. C., Elliott, D. C., Elliott, W. H., and Jones, K. M., eds.), 2nd ed., p. 507, Oxford University Press, New York/ Oxford. 9. Katz, J., Wals, P. A., Golden, S., and Rognstad, R. (1975) Eur. .J.Biochem. 60,91-101. 10. Handschumacher, R. E., and Pastemak, C. A. (1958) Biochim. Biophys. Acta 30,451-452. 11. Marshall, M., and Cohen, P. P. (1972) J. Biol. Chem. 247, 1654- 1668. 12. Keppler, D. 0. R., Rudigier, J. F. M., Bischoff, E., and Decker, K. F. A. (1970) Eur. J. Biochem. 17,246-253. 13. Pausch, J., Wiikening, J., Nowack, J., and Decker, K. (1975) Eur. J. Biochem. 53, 349-356. 14. Von Euler, L. H., Rubin, R. J., and Handschumacher, R. E. (1%3) J. Biol. Chem. 238, 2464-2469. 15. Katz, S., and Comb, D. G. (1%3) J. Biol. Chem. 238,3065-3067.