Liquid scintillation counting of 14C- and 3H-labeled samples on solid supports: A general solution of the problem

ANALYTICAL

Liquid

BIOCHEMISTRY

54,

Scintillation Samples

1731

(1973)

Counting

Received

and

“H-Labeled

on Solid Supports: A General Solution of the Problem1

R. M. MCKENZIE” Agricultural

of 14C-

AND

R. K. GHOLSON3

Experiment Station, Oklahoma State Stillwater, Oklahoma 74074 June

16, 1972;

accepted

January

University,

12, 1973

The data presented demonstrate the potential of surfactant-fortified scintillation cocktails in overcoming many of the problems encountered in the quantitation of radioactivity on solid supports. Using a broad range of representative ionic and polar biochemical compounds, the in vial elution and quantitative recovery of 3H and “C-labeled components from a wide assortment of common solid support media has been demonstrated. This methodology in combination with zero elution counting systems and in some circumstances gelled suspension counting should combine to overcome most of the problems associated with determination of isotopic activity on such solid support media.

Liquid scintillation counting of I%- and 3H-labeled compounds on solid supports is a common technique in biochemical research. Quantitation of such counting data is often complex, but if it is used in an uncorrected form, can be grossly inaccurate and misleading. Many approaches have been used in an effort to avoid these problems. These methods are often time consuming and difficult, or require the use of expensive ancillary equipment. Combustion techniques which convert the label to carbon dioxide or water allow excellent recoveries (1). Preelution of samples from the supporting media has also been widely used. If the samples are left on the supports, precise standards duplicating very constant counting conditions can be used; however, such uniformity is often difficult to obtain. Partial and/or variable elution of the labeled material(s) in the vial can cause differences in the results obt,ained be‘Journal Article 52520 of the Agricultural Experiment Station, University, Stillwater, Oklahoma. ‘Present address: Department of Dairy Science, University of Illinois 61801. ‘Reprint requests should be directed to this author. 17 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

Oklahoma Illinois,

State Urbana,

18

MCKENZIE

AND

GHOLSON

tween duplicates of a given sample or when comparing chemically different compounds which may behave differently in the same systems. Variation in the nature, quantity, and orientation of the support in the vial can cause marked changes in counting performance particularly when counting tritium (25). References 1 to 14 present some of the methods used and the pitfalls involved in this area, but by no means cover all the specific conditions and approaches that have been used in an effort to overcome the problems of quantitation of radioactive compounds on solid supports. If the labeled components can be completely solubilizecl, the problems associated with solid support counting are eliminated. In this case the solid support and its orientation have little or no influence on the counting rate observed. Therefore, channels ratio correction of the data against a single precise set of liquid standards is possible and only those precautions used in routine liquid scintillation counting are needed. A method which allows solubilization of a wide variety of compounds from the broad range of commonly used solid support materials would, therefore, be helpful to investigators faced with these counting difficulties. This paper describes such a method. Recently, Chakravarti and Thanassi (15) briefly reported an apparently promising system using a simple toluene-based cocktail containing an acidic surfactant (Bio-Solv BBS-3) and water. A technical bulletin is available describing BBS-3 and its use in a wide range of difficult liquid scintillation counting situations (16). MATERIALS

AND

METHODS

Bio-Solv BBS-3 was purchased from Beckmann Instruments, Inc. PPO (2,5-diphenyl-oxazole) and POPOP (p-bis (2- (5phenyloxazoly) -benzene) were purchased from Packard Instrument Co. Uridine diphosphate galactose ( [U1*C] galactose) (338 mCi/mmole and used at 133 @LX/ mmole) (UDP gal-W), AT-methyl-I%-nicotinamide chloride (3.86 mCi/ mmole), NMeNAm-I%) , cholesterol-l-*4C (59.4 mCi/mmole) , cholesterol-7-3H (16.7 Ci/mmole) , choline-methyl-3H-chloride (1 Ci/mmole) and Aquasol were purchased from New England Nuclear. Deoxyadenosine-5’-triphosphate tetralithium (8-3H) (5.7 Ci/mmole) (dATP3H) was purchased from Schwartz/Mann. Nicotinic acid-7-‘“C (59.1 mCi/mmole) (NA-‘“C) and D-ghlCOSe-l-'4C (41.5 mCi/mmole) (Glc‘“C) were purchased from Amersham/Searle Corp. DL Aspartic acid-3H(G) was obtained from Nuclear Chicago Corp. This latter compound had been stored for several years in aqueous solution and was heavily contaminated with labeled impurities. It was lyophylized to remove volatile labeled components (approx 32%)) reconstituted in water, and used with-

COUNTING

3H

AND

14c

ON

SOLID

SUPPORTS

19

out further purification. All other labeled compounds were essentially radiochemically pure. Phosphocellulose (P,,) and diethyl-aminoethyl cellulose (DE,,) ion-exchange papers were obtained through Reeve Angel Co. Macherey, Nagel and Co. cellulose powder MN 300 and Silica Gel G powder (act. to Stahl) both for thin-layer chromatography were obtained t’hrough Brinkman Instrument Co. Whatman No. 1 filter paper and all other reagents were obtained from general chemical supply houses. Liquid scintillation quenched sets for tritium and carbon-14 were obtained from Nuclear Chicago Corp. In each case the labeled compound used is toluene and the cocktail contains 4 g of PPO and 0.05 g of POPOP per liter of toluene. Acetone is the quenching agent.. Cocktail

Composition

Three cocktails were used for comparative purposes. These were: a toluene-ethanol cocktail composed of 4 g of PPO, 0.2 g of POPOP, 400 cc absolute ethanol, and 600 cc of toluene per liter; a toluene-BBS-3 cocktail containing the same fluor concentration as above with 170 cc of Bio-Solv-BBS-3, 60 cc of water, and 770 cc of toluene per liter; and an Aquasol cocktail containing 940 cc of Aquasol and 60 cc of water per liter. Sample Preparation Labeled compounds were used at the specific activities specified above and were stored in aqueous solution in small plastic vials, except cholesterol which was dissolved in p-dioxane. Carbon-14-labeled solutions contained 0.5-l pCi/cc and tritium-labeled solutions l-2 ,.&i/cc. All samples were run in triplicate using 0.02-cc volumes taken with an Eppendorf micropipet (Brinkman Instruments Inc.). The pipet tip was rinsed twice with water or p-dioxane, as required, after each sample was drawn, and the rinsings were added to the original sample. For paper-based solid supports, sample and washings were applied to the center of a 3 X 3-cm square of the paper supported by its edges on a stainless-steel test tube rack and allowed to air dry. The dry papers were cut in half and the halves placed vertically on their long edge in the bottom of a counting vial to form an almost continuous ring around the vial walls. Thereafter, no effort was made to maintain this initial geometry. For silica gel or cellulose powder, an amount equivalent to a 1 X 5-cm area scraped from a 500 pm thin-layer plate was preweighed into the counting vial (40 mg silica, 60 mg cellulose). The sample and washings was then carefully applied to the powder in the bottom of the vial and allowed to air dry. Samples in free solution were added directly to vials containing 10 cc of a given cockt,ail.

20

MCKENZIE

AND

GHOLSON

When cellulose and silica powders were suspended in an Aquasol gel, 10 cc of Aquasol (containing no water) were added to the vials followed by 2.0 cc of deionized water. After tightly capping the vials, a brief, vigorous shaking through their Ion g axis was used to suspend the supports in a uniform gel. Counting

Procedures

All counting was done on a Packard Liquid Scintillation Spectrometer (Model 3320) using standard 22-mm low potassium glass vials with aluminum-lined plastic caps. The instrument was optimized for counting “C samples in the toluene-ethanol cocktail, and for counting tritiated samples in unquenched toluene cocktails. Liquid and gelled samples were counted without further manipulation. Samples on solid supports were handled in groups of 36 vials each. With samples in place, IO cc of cocktail was added to each vial and the vials were counted for 1 min without deliberate agitation. After counting each vial was removed from the spectrometer, held on a vortex mixer for 1 min, replaced in the instrument, and counted again. This was repeated until each vial had been shaken five times. Prior to each counting, the samples were left in the instrument for approximately 30-35 min during which time nonagitation-assisted elution could occur. After the final counting, the vials were subsampled (1 cc into 10 cc of toluene-ethanol cockt,ail) and counted to determine the amount of activity solubilized from the solid support. In some cases another shaking procedure was used. The vials were packed vert.ically and tightly into a reciprocal shaker, which was then operated at 240 strokes per minute for a specified period of time. The vials must be packed tightly, since free play permits the caps to loosen, resulting in sample losses. Data

Handling

Counting data were converted to disintegrations per minute using the channels ratio method. Sealed acetone-quenched standards were counted daily to const,ruct the channels ratio correction curves. A set of standards was prepared in each of t,he three cocktails used, to establish the relationship between the curves generated by the sealed standards and the three differing cocktails. These lat’ter standards were prepared using either L- [methyl-l’C] methionine (93,500 dpm/vial) or cholesterol-7-3H (132,000 dpm/vial) and CO.4 cc of chloroform (in 0.05-cc increments) as a quenching agent. In all instances, the experimental data presented fell within the limits of these standard curves.

COUNTING

3H REStJLTS

AND

14c

AND

ON

SOLID

SUPPORTS

21

DISCUSSION

This work was inititated as the result of a report by Chakravarti and Thanassi ( 15’). Their study was confined to Whatman ~MM filter paper bearing [ :‘H 1glycinc, 1:‘H 1alaninc, 1“C 1glycinr, [“Cl tyrosine or [‘“C] histidine. The BBS-3 cocktail usctl in this study was essentially the same as the one used by those authors (15). They showed that the counting rate of samples dried on filter paper equaled that of the same sample in free solution after vigorous manual shaking of the vials (“usually about 6 minutes”). They also stated that the counts went int’o solution as the paper disintegrated and only when disintegration occurred were the two counting rates tending toward equality. However, we have found that destruction of the paper structure is not required to accomplish solubilization, since no obvious breakdown of any of the papers used in the present study occurred during tht vortex mixing process. Samples of P,, in horizontally positioned vials containing 15 cc of cocktail did not break down completely after 30 min of shaking on a reciprocal shaker operating at approximately 240 strokes per minute. When the pallerr were presaturated with water or T-ICI solutions their disruption was essentially complete within 5-10 min. However, disintegration in itself was not sufficient to release the activity into solution. Relationships Between the Supports and Compounds Studied The supports selected include most chromatographic media commonly used in biochemical separations of small metabolites. The compounds were selected because they were of particular interest to us and/or to show specific interactions with the supporting media used. The interactions of the ionic compounds with the ion-exchange media were of particular interest. [‘“Cl NMcNAm and [“H] choline were not eluted from P,, by extensive washing with deionized water and [‘“GINA, (a weak anion), UDP-gal-ldC and dATP-3H (strong anions) were not eluted from DE,, by extensive washing with deionized water. It should be kept in mind that the “aspartate-“H” is a complex mixture of labeled compounds which may not behave like aspartic acid under any given set of conditions and that it probably represents a more severe test of the solubilization system than pure aspartate. Channels

Ratio Correction

The channels ratio correction curves obtained from six sealed [‘“Cltoluene acetone-quenched standards and from a set of chloroform-

22

MCKENZIE

AND

GHOLSON

quenched standards prepared in each of the cocktails used showed good agreement in the channels ratio regions used in these experiments. A similar set of five sealed [3H] toluene-acetone-quenched standards was used in calculation of the tritium data. The channels ratio curve obtained from these standards and from a set of chloroform-quenched standards showed good agreement with the toluene-ethanol and BBS-3 cocktails in the range of channel ratios observed. However, the curve for the Aquasol cockt.ail fell below the standard curve. This deviation indicated that the data obtained with Aquasol would be underestimated by about 5% and all data obtained with tritiated compounds in this cocktail have been corrected for this deviation. Solubilization

and Recovery

of Activity

In several instances (Table 1) the liquid samples counted in the toluene-ethanol or Aquasol cocktails gave lower disintegration rates than the BBS-3 controls. In each case it was found that addition of a BBS3:toluene:water mixture which adjusted the composition of the given cocktail to 17% BBS-3 and 6% water resulted in counting rates equal to those obtained in the original BBS-3 controls. The discrepancy between the BBS-3 and toluene-ethanol cocktails when counting [‘“ClNMeNAm is due to binding of NMeNAm to the vial walls in tolueneethanol (unpublished data). BBS-3 appears to prevent this binding entirely, and such surfactant-fortified cocktails should, therefore, be useful in preventing binding where it may occur (17). Placement of strips of Whatman No. 1 filter paper around the inner walls of the vials resulted in little or no decrease in the disintegration rate relative to t.hat observed in the absence of the paper with either l*C or 3H-labeled samples. Data showing recovery and solubilization of activity from solid supports is presented in Table 2. Only the data obtained at 0-, I-, and 5min shaking are presented. Samples which showed a marked increase in recovery with time did so gradually. When no large difference between 1 and 5 min occurred the values at intermediate times were essentially constant. The BBS-3 cocktail yielded over 90% recovery of activity for all compounds tested on all supports with few exceptions, and in some instances essentially no agitation was required. The toluene-ethanol cocktail was inferior to BBS-3 with the exception of [‘“Clcholesterol samples where all three cocktails gave equal recovery of activity on all supports tested. The Aquasol cocktail was, in general, inferior to the BBS-3 cocktail, particularly where strong ionic binding of the compounds to the support occurred. When *4C-labeled compounds on cellulose and silica powders were suspended in gelled Aquasol, the recovery of activity was generally

T B A

Cocktaild

T B A

Cocktaild

99.5 100.0 97.1

None

from

99.5 99.8 94.6

+BBS-3c

[W]Cholesterol (19,874)

75.3 100.0 78.5

None

NMeNAmW ;32,968)

of Activity

Various

75.4 100.0 100.4

None

[3H]ASP (144,023)

102.5 99.5 103.3

+BBS-3~

98.2 99.6 97.7

+ BBS-3”

TABLE aH-Labeled

UDP-gal-W (25,367)

and

92.9 100.0 98.9

None

‘“C-

1 Compounds

74.0 100.0 101.3

None

d-ATP-3H (56,555)

98.3 100.0 99.4

None

98.2 97.5 102.8

+BBS-3~

Solution

[WINA (28,252)

in Free

in Three

56.6 100.0 60.7

None

98.5 100.0 99.3

None

[W]Glc (26,745)

[3H]Choline (54,767)

Cocktails”

102.6 97.7 102.0

+BBS-b

a All activity data are expressed as a percentage of the BBS-3 liquid samples. The dpm values observed in these controls are recorded under each compound. The same values apply to the data in Table 2. All values represent the average of three samples. b Abbreviations used are defined in Materials. c After first counting the vials with no additions to obtain the data in the column headed “None ” 5 CC of a mixture of BBS-3: toluene: Hz0 of a composition which would yield a final concentration of 1770 BBS-3 and 6% water were added to each vial. The vials were mixed on a vortex mixer for 1 min and recounted. d T, toulene-ethanol; B, BBS-3 and A, Aquasol cocktails respectively.

Addition

Compoundb

Addition

Compoundb

Recovery

z

3 g

8

8

g

x

5

6

z 3 3

8

Whatman

1

Compoundb

No.

1

Diethyaminoethyl cellulose (DE,,)

gel

powder

Cellulose

Silica

No.

Whatman

Diethylaminoethyl cellulose (DE&,)

(p81)

Phosphocellulose

support

time

Recovery

Compoundb

Shaking

Relative

T B A T

Ag

Ag T B A

T B A T B A T B A T B A

Cocktail

(min)

and Degree

64.5 85.9 70.7 70.5

73.9 97.3 75.8 70.3 98.1 79.5 70.8 100.1 76.3 82.1 98.1 88.9 94.7 80.8 88.7 80.7 84.1

0

1

recovery

of Activity

65.8 93.1 73.0 71.9

[XJNA

81.6 101.1 84.8

75.4 101.0 77.3 72.2 99.6 83.3 70.6 99.4 82.5 83.5 98.9 89.0

NMeNAm-‘“C

Relative

of Solubilization

68.4 95.1 79.7 75.3

79.5 98.2 81.1

73.9 100.6 78.3 70.7 100.6 88.3 69.6 100.2 89.6 82.6 97.7 89.3

5

TABLE 2 in laC- and

0 80.2 26.6 24.8

0 91.5 15.4

0 98.7 18.0 12.1 96.6 80.7 5.6 98.3 36.6 3.8 101.3 38.7

y. in solution

3H-Labeled

1

recovery

Various

73.7

72.1

[“C]Glc

79.6 98.2 88.0

71.6 86.9 82.1 64.7 88.0 73.2 70.3 87.6 79.4 80.1 94.8 89.1

uDP-gal-‘4c

-

69.9 81.5 78.2 65.5 84.5 71.8 69.3 81.5 76.4 79.5 90.0 86.7 97.6 78.5 93.6 85.1 95.8

0

from

Relative

Compounds

76.6

-

79.8 97.4 90.2

71.1 93.1 89.2 66.4 93.2 76.7 71.3 92.4 87.9 80.5 95.9 91.5

5

Solid

43.1

-

0 94.6 46.3

0 75.0 68.8 0 70.7 3.2 0 71.0 54.8 0 75.4 49.8

YOin solution

Supports”

-

Whatman

1

I

Compoundb

No.

Diethyaminoethyl (D&I)

(PSI)

Phosphocellulose

gel

powder

Cellulose

Silica

No.

gel

powder

Whatman

Silica

Cellulose

T B A T B A T B A

Ag T B A A!?

B A T B A Ag T B A Ag T B A T B A

4.8 33.4 19.5 5.1 29.7 13.9 10.4 33.1 25.8

91.5 90.5 80.7 93.6 95.0 97.9 81.4 90.8 89.8 97.1 97.3 99.5 98.1 97.8 100.0 9S.6 99.0 97.6 98.5 97.6 98.3

5.5 55.7 28.2 7.5 51.3 15.8 10.6 50.1 22.3

97.2 99.2 98.4

98.0 99.8 97.6 97.5 100.4 98.6

79.9 95.2 95.2

96.7 94.7 81.8 96.8 96.6


97.5 98.1 98.2

97.1 99.8 97.5 98.0 99.5 98.8

80.7 95.7 97.1

97.3 96.6 82.3 96.6 96.3

5.5 71.2 57.3 6.1 67.1 13.2 7.6 58.4 39.3

9.5.8 95.6 95.7

98.8 97.4 94.4 98.8 97.4 94.4

22.7 91.2 82.7

57.5 84.6 74.3 85.3 92.8

8.3 86.6 10.6 6.5 77.4 21.3 18.7 91.7 31.3

92.8 S2.7 S2.8 95.0 90.3 99.6 79.6 92.0 89.1 98.3 9.9 62.1 33.2 27.1 67.6 52.1 93.6 42.2 67.3 47.6 R2.1

8.6 94.6 10.8 11.7 78.6 32.3 15.4 94.9 44.0

33.8 83.2 57.6

11.6 78.8 45.0 26.9 90.7 63.7

79.4 95.1 93.4

97.4 89.9 83.1 97.6 93 4

13.9 110.5 17.1 20.5 95.4 65.2 37.1 113.2 SO.6

40.6 90.0 78.S

12.5 87.1 62.4 27.4 95.0 71.2

79.1 95.4 94.0

97.0 94.Fi so.7 99.3 95,s

0 93.3 6.0 11.8 78.8 29,s 15.5 89.4 24.9

6.6 81.6 37.9

6.6 64.6 45.4 8.S 74.0 54.3

14.3 93.2 90.4

91.0 87.5 33.2 92.6 90.9

gel

powder

a See a, Table b See b, Table c See d, Table

Silica

Cellulose

Support

time

Aquasol.

T B A Ag T B A Ag

Cocktailc

(min)

1. 1. 1, Ag, gelled

Compound*

Shaking

24.4 38.3 37.7 94.0 16.0 33.2 31.0 41.5

0

1

recovery

25.2 73.5 55.3 36.4 69.2 37.2

56.9 45.0 26.0 44.0 31.8

5

2 (Continued)

23.3

NMeNAm-1%

Relat,ive

TABLE

69.5 16.9

13.1

68.2 39.2

8.8

y. in solution

50.4 26.6 47.6

100.4 39.0 92.7 37.1

24.2

0

1

recovery

79.1 30.0

32.5

105.2 52.8

25.1

UDP-gal-W

Relative

85.9 28.7

28.7

107.5 64.3

24.5

5

76.4 4.9

0

94.9 22.3

3.6

7c, in solution

8 F

5

E “w $ E L1

COUNTING

3H

AND

14c

ON

SOLID

27

SUPPORTS

good except with [14C]NMeNAm on silica gel. This procedure was inferior with tritiated compounds supported on silica gel but again gave reasonably good recovery of activity with compounds on suspended cellulose powder. The exact contribution of a limited solubilization to the success of gelled suspension counting was not determined. However, this is probably a contributing factor. In addition suspension of the support throughout. the counting medium tends toward an infinitely thin counting geometry as compared to a infinitely thick geometry when the support is resting on the bottom of the vial. Even the BBS-3 cocktail appeared to be unsatisfactory in the recovery of activity of tritiated compounds under some conditions. However, the results show the overall promise of the system. The specific problems presented by the results in Table 2 will be discussed further. The data in Table 2 indicate that recovery of activity is related to its release into solution, and that this is of particular importance for recovery of activity in tritiated compounds, where the effects of binding on counting efficiency are very large. It is probable that the effect of added BBS-3 on increasing the recovery of activity in the liquid controls (not originally containing BBS-3) shown in Table 1 was due to solubilization of material bound to vial walls. Further, it appeared probable that the same effect would be observed if BBS-3 were added to samples on solid supports in non-BBS-3 containing cocktails. However, to establish this, a new set of [*%]NMeNAm vials was prepared. The samples were shaken for 5 min on a reciprocal shaker and counted. Appropriate mixtures of BBS-3 : toluene: water were then added to each cocktail as described previously for liquid samples Effect

of Adding Recovery

TABLE 3 BBS-3 to Non-BBS-3 Cocktails of NMeNAm-14C Activity”

on

Cocktailb support None Phosphocellulose (PSI) Whatman No. Silica gel

T

1

T + BBS-3

B

B + BBS-3

A

A + BBS-3”

79.0 78.3

100.8 101.3

100 103.4

99.7 102.4

85.1 80

98.6 101.7

73 5 75.0

100.2 99.5d

102.4 100.5

102.6 99.0

85.3 83.2

101.0 98.gd

percentage

of recovery

a BBS-3 liq. control contained 33,444 dpm. b See d, Table 1. c See c, Table 1. d Samples were shaken 10 min; 5-min shaking of activity.

yielded

lower

28

MCKENZIE

AND

GHOLSON

(Table 1). The samples were again shaken for 5 min and recounted. The results are reported in Table 3. The data clearly indicate that the effective recovery of the activity is directly related to the presence of BBS-3 or the BBS-3: water mixture in the cocktail, which results, as demonstrated by t,he data in Table 2, in the release of the labeled component from the solid support int’o solution. El&ion

of Strong Anions

from Solid Supports

Obviously, solubilization of strong anions from the solid support media used is difficult (Table 2). This is most apparent with deoxy ATP-3H where total solubilization is not accomplished. Since the efficiency of counting bound tritium is much lower than for bound ‘“C a larger discrepancy is seen in the observed and predicted activities of [3H]dATP than of 14C-labeled molecules such as UDP-gal-14C. Since difficulty in solubilization appeared to be associated with anionic character protonation of this charge by acidification of the media prior to addition of the cocktail was attempted. The supports were just saturated with 3 M HCl Effects

Pretreat,ment Compounds

TABLE 4 on Recovery of Activity in I%from Selected Solid Supports

‘L,4sp-YH”

Compound” Support dpm

of Acid

paper (control)

What,man

No.

30,652 recovery

102.0

87.1 98.3

[‘%]NA D81 28,213 Relative

Paper +3MHCl Silicab S&O Silicab +3MHCl

Choline-3H Phosphocellulose (PSL)

1,887,580 Relative

Paper f3MHCl Silicab +HzO Silicab +3MHCl Compound” Support paper dpm control

1

dATP-3H Diethylaminoethyl (Dsl) 52,291;

(yO of control) 104.0

100.5

87.3

55.3

97.3

79.0

NMeNAm-14C P81 26,751

IJDP-gaF4C D** 19,470 recovery

and 3H-Labeled

(%

of control)

97.4

94.7

98.9

95.1

94.0

97.0

96.8

89.5

96.8

a Abbreviation defined under Materials. b Quantity of silica gel was increased to 80 mg/vial.

cellulose

COUNTING

3H

AND

14c

ON

SOLID

SUPPORTS

29

or water and allowed to sit at room temperature for 20 min. The cocktail was then added, the vials shaken on a reciprocal shaker for IO min and counted. The results are shown in Table 4. Acid pretreatment resulted in total recovery of the dcoxy ATP-8H from DEAE-cellulose. Acid pretreatment had little or no influence on the previously successful recovery of the activity of cationic or nonionic compounds from paper supports. The recovery of [“Hlnspartate ia complex mixture) was complete from both paper and silica supports after acid treatment. On silica the recovery of [‘H]dATP was improved but, not complete. There was an apparent slight decrease in the recovery of UDP-gal-‘“C on silica gel, although this was not. marked relative to the previous data. In this experiment the amount of silica gel in the vials was doubled to 80 mg/vial, and this required the addition of extra cocktail to prevent separation of the added water from the system. Incubation of acid-saturated samples of deoxy ATP-“H and UDP-gal-14C in tightly sealed counting vials at 115°C for 15 min in an effort to clcavc the anhydride phosphate esters did not influence the recovery of activity in these compounds from silica gel and was not necessary mm-it11 paper supports where incubation with 1 M HCl at room temperature was as effective as 3 M HCl in releasing [“H]dATP. The recovery of strong anions from solid-support media, in particular, tritiated anions on silica gel, may prove somewhat difficult, especially when large quantities of gel are present. Effects

of Changing

Cocktnil

Composition

Cbakravarti and Thanassi (15) used essentially the Lame BBS-3 cocktail as described here at a level of 15 cc/vial. At this level the system is quite costly. A gallon of BBS-3 is sufficient to yield about 22 liters of the cocktail reported. The cost per vial is reasonably competitive with all commercially available cocktails used in equal volume, many of which, however, cannot be expected to have the same performance characteristics. If the cocktail volume used is reduced to 10 cc/vial as was successfully accomplished here, a 33% savings is realized. If BBS-3 were functional at lower concentrations, costs would be further reduced. A series of diluted cocktails was prepared, water content was maintained at S%,, and the BBS-3 content dropped by 2% increments. At a concentration of 7% the cocktail became cloudy, phased upon standing and was unsuitable for counting. At all higher concentrations the cocktail did not phase when refrigerated and proved satisfactory in its ability to remove [‘Z]NMeNAm from P,, (Table 5). A 10% BBS-3 cocktail would probably suffice in many instances where dry supports were being extracted and where no strong anions were present. An additional cost reduction

30

MCKENZIE

Effect BBS-3

of Changing

of NMeNAm-14C

from

PSI

y0 Control

dpm -0

7 9 11 13 15 17 phased,

GHOLSON

TABLE 5 Composition on Recovery

Cocktail

content of cocktail (76)

0 Cocktail

AND

36,149 34,425 35,153 34,651 34,564 not suitable

for

105.6 100.5 102.6 101.2 100.0

counting.

might also be obtainable by further reduction in the volume of cocktail used, with or without a reduction in BBS-3 content. Commercially prepared cocktails are available which should have characteristics similar to that of the BBS-3 cocktail used in this work since they contain the same or similar surfactants. These are ScintosolComplete (Iso Labs, Inc.) and Ready Solv Solution VI (Beckman). The surfactants used in these cocktails are available separately, allowing for great flexibility in cocktail composition. CONCLUSION

It is necessary that each system be checked to determine that the compounds of interest are being extracted from the given supporting medium. However, the data presented in this study demonstrate the potential for wide application of the technique described to the general problem of counting samples on solid supports. In our laboratory, this counting technique has been successfully used for quantitation of labeled urinary metabolites of [‘“Cl NA and 1°C ] nicotinamide chromatographed on Whatman No. 1 paper and to a P,,-based radioisotopic assay for methionine adenyltransferase (18). Radioactive compounds on solid supports are easily quantitated if the isotope either remains entirely in the support or is completely solubilized. When partial or variable elution occurs difficulties arise. Unfortunately, such partial elution is common with many compounds of biochemical interest in the usual counting cocktails. Under these conditions the technique described here is most valuable. ACKNOWLEDGMENTS The authors express assistance and to Drs. of the manuscript.

their appreciation R. E. Koeppe and

to Mr. Steve E. C. Nelson

Wash for his for their critical

technical reviews

COUNTING

This work Foundation.

was supported

3H

AND

in part

“c

by

Grant

ON

SOLID

SUPPORTS

GB-23042

from

31 the

National

Science

REFERENCES 1. DAVIDSON, J. D., OLIVERIO, V. T., AND PETERSON, J. I. (1970) in The Current Status of Liquid Scintillation Counting (Bransome, E. D., Jr., ed.), p. 222, Grune and Stratton, New York. 2. FURLONG, N. B. (1970) in The Current Status of Liquid Scintillation Counting (Bransome, E. D., Jr., ed.), p. 201, Grune and Stratton, New York and London. 3. SNYDER, F. (1970) in The Current Status of Liquid Scintillation Counting (Bransome, E. D., Jr., ed.), p. 248, Grune and Stratton, New York. 4. PENG, C. T. (1970) in The Current Status of Liquid Scintillation Counting (Bransome, E. D.. Jr., ed.), p. 283, Grune and Stratton, New York. 5. BRANSOME, E. D., JR., AND GROWER, M. F. (1970) And. B&hem. 38, 401. 6. NUNEZ, J., AND JACQUEMIN, CL. (1961) J. Chromatogr. 5, 272. 7. PINTER, K. G., HAMILTON, J. G., AND MILLER, 0. N. (1963) Anal. B&hem. 4, 458. 8. SHERMAN, J. R. (1963) Anal. Biochem. 5, 548. 9. JOHNSON, D. R., AND SMITH, J. W. (1963) Anal. Chem. 35, 1991. 10. SNPDER, F. (1964) Anal. Biochem. 9, 183. 11. FURLONG, N. B., WILLIAMS, N. L., AND WILLIS, D. P. (1965) Biochim. Biophys. Actn 103, 341. 12. DAVIES, J. W., AND COCKING, E. C. (1966) Biochim. Biophys. Acta 115, 511. 13. NEWSHOLME, E. A., ROBINSON, J., AND TAYLOR, K. (1967) Biochim. Biophys. Acta 132, 338. 14. GILL. D. M. (1967) Appl. Radiat. Zsotop. 18, 393. 15. CHAKRAVARTI, A., AND THANASSI, J. W. (1971) And. Biochem. 40, 484. 16. NEWMAN, F. M., Applications Research Technical Report No. 551. Beckman Instruments Inc. Sample Preparation Procedures for Liquid Scintillation Counting. 17. LITT, G. L., AND CARTER, H. (1970) in The Current Status of Liquid Scintillation Counting (Bransome, E. D., Jr., ed.), p. 156, Grune and Stratton, New York. 18. MCKENZIE, R. M., AND GHOLSON, R. K. (1973) Anal. Biochem. (in press).