A quantitative method for measuring labeled compounds with whole-body autoradiography in tissue sections

A Quantitative Method for Measuring labeled Compounds With Whole-Body Autoradiography in Tissue Sections

EEVA-LIISASAINIO AND PERT-~SAINIO

Whole-body autoradiography is an effective method for localizing labeled compounds in various organs. However, the technique is limited in its ability to quantify such material. Using tissue sections, this study investigated certain parameters involved in the quantitative estimation of labeled compounds by whole-body autoradiography. These included correlation between thickness of the section and radioactivity counted, the precision of such measurements, and the reproducibility of the autoradiographic films as tested by image analysis transmission. The precision of radioactivity measurements using tapes with a tissue section or a “punch biopsy” (punching off a piece of tissue from the section) was compared. The results revealed excellent linearity between the thickness of the section and the radioactivity counted (r = 0.97) when section thickness was IO-30 km. The measurement precision using tapes was better than with the “punch specimen” method. The reproducibility of photographic films was good when transmission was measured by image analysis. It was concluded that a thickness of 30 km is ideal for use in whole-body autoradiographic studies. It appeared that radioactivity measurement of tissue sections on tapes was superior to direct measurement from organs. Image analysis was employed and statistically evaluated for the first time in this study, and the promising findings suggest that it is likely to become the method of choice for future studies of this type. Key Words: Densitometric

Whole-body; Autoradiography; Quantitation; image analysis; Labeled compounds

Tissue thickness;

INTRODUCTION

Whole-body autoradiography (WBA), first described by Sven Ullberg in the early 195Os, has proved a very useful technique for localizing labeled chemical compounds in animal studies. WBA has been employed in pharmacology, toxicology, nutrition research, and occupational health, among other disciplines. The technique makes it easy to recognize the tissues with particular affinity for the compound and/ or its metabolites under study, so that further procedures like histopathological characterization or biochemical tests can be focused on relevant organs or tissues.

From the Departments of Pharmacology and Toxicology (E.L.S.) and Oral Pathology, Roentgenology and Forensic Odontology (P.S.), University of Kuopio, P.O. Box 6, SF-70211, Kuopio, Finland. Address reprint requests to: P. Sainio, Department of Oral Pathology, Roentgenology, and Forensic Odontology, University of Kuopio, P.O. Box 6, SF-70211 Kuopio, Finland. Received August 1990; revised and accepted December 1990. 53 journalof

Pharmacological

Methods

0 1991 Elsevier Science Publishing

26, 53-59 (1591) Co., Inc.. 655 Avenue of the Americas, New York, NY loo10

54

E.-L. Sainio and P. Sainio In some countries, WBA is now a mandatory technique in trials for drug registration. Despite its superiority in detecting labeled compounds in tissues, however, WBA is unable to quantify such compounds satisfactorily. Quantification of tissue loading has often been expressed in the form low (+), moderate (+ +) or high (+ + +), to indicate

the degree

of affinity

based on the use of standards

of a compound containing

al., 1974) or commercial methacrylate (Irons et al., 1981) have been reported.

for a tissue.

different

Quantitative

methods

levels of radioactivity

(Cross et

sources of increasing 14C-specific activity The radioactivity of tissue samples can also

be measured with a liquid scintillation counter following combustion of the tissue. These values are correlated to concentrations on the x-ray film (Busch, 1977). In the present study, a quantitative WBA method with particular attention focused on the following:

was developed

1. The degree

of varying

radioactivity, 2. Comparison

of linearity

between

of the coefficients

the tape or “punch 3. The reproducibility

tissue

of variation

biopsy” methods, of exposed photographic

tissue when transmission density metric image analysis of exposed first time. 4. Summarizing MATERIALS

and drawing

AND

sections

in measuring

and evaluated,

thickness

tissue

and their

radioactivity

films in serial sections

by

of the same

was determined by image analysis. Densitofilms was employed for this purpose for the

conclusions

on the use of this WBA method.

METHODS

Test Animals and Chemicals The experimental under

regularly

animals

alternating

were

male outbred

periods

Han:

NMRI

mice (30 g), maintained

of light (14 hr) and darkness

(IO hr) and with

free access to food (standard diet from Hankkija Ltd, Turku, Finland) and water. Carboxyl-14C nicotinic acid (specific activity 53 mCi/mmol, code CFA.197) was purchased from Amersham, U.K., carboxymethylcellulose Department of Pharmacy, Kuopio, Finland (Ph.Nord and ethylether from E. Merck, Darmstadt, Germany.

Experimental Carboxyl-14C vein into mice.

(CMC) from the University grade), and Hexan (n-hexane)

Procedures nicotinic acid (5 FCi) in 0.9% NaCl (100 ~1) was injected via a tail After 30 min, the animals were anesthetized with ethylether. They

were then laid right side down in a shallow container constructed frame containing 1.5% CMC (w/v). The container was immediately

of a floor and a lowered into a

chamber containing hexane cooled with dry ice. The temperature was -74°C and freezing was complete in about 30 min. The solidly frozen specimens were allowed to stabilize overnight at -20°C before sections were cut. Sagittal sections from the whole animal were taken at -20°C by microtome (PMV Cryo Microtome 450MP, Stockholm) of several tissue thicknesses, using adhesive tape (Scotch Brand Magic Transparent Tape, No. 810, 3M). The sections were al-

A Quantitative lowed

to dry for 24 hr at -20°C

(Hyperfilm TM-B 5-week exposure. Sample

they were

placed

max, code RPN. 9, Amersham, made The films were developed by normal

Preparation

A cylinder

before

Method for Whole-Body

for Radioactivity

with a round,

Determinations

sharp edge (diam.

55

Autoradiography

on X-ray sensitive

film

by CEA Ltd, Sweden) for a photographic processing. from Sections

on Tape

3.829 mm) was used to cut out circular

pieces of tissue section from the tape (Figure I). The cutting procedure was performed on a cork base plate. A magnification glass (4 x ) was used for small biopsy areas. The specimens were placed in a liquid scintillation bottle and digested with 0.5 mL Lumasolve

(Lumac,

Netherlands)

overnight

at +37”C.

The following

day,

scintillation liquid (4 mL) (Lipoluma, Lumac, Netherlands) was added to the bottles and the radioactivity was immediately counted with a LKB Wallac 1216 Rackbeta liquid

scintillation

Direct Small surgical

counter.

Determination

of Tissue Radioactivity

pieces of frozen knife into liquid

Processing

and counting

Densitometric

Image

animal tissue (about 5-10 mg) were punched off with a scintillation bottles for digestion with 0.5 mL Lumasolve. were

as described

above.

Analysis

The autoradiograms were digitalized through a CCD video camera (TK870E, JVC, Japan) fitted with a mace bus model H7-C611AF and joined to an IBAS image analyzer UBAS 1, 2, Kontron,

Munich,

Germany),

and were

displayed

on a monitor

resolution

of 512 x 512 pixels and 256 shades of grey. The IBAS analyzer

FIGURE 1. the tape.

Sample taking by the tape method.

with

a

evaluates

1 = cylinder used, 2 = piece of tissue on

56

E.-L. Sainio and P. Sainio the mean

value

of gray shades

in the measured

area.

ograms as a standard the IBAS analyzer was calibrated 100 (background). Other values were linearily scaled

Using

one of the autoradi-

to a scale of 0 (darkest) to between these values. The

autoradiograms were scanned on a light box and the focus and exposure kept standardized. The imbalance of the light source was corrected. The percentage transmission

was measured

using a standardized

circle on the screen

placed

within

the

area occupied by the tissue. The radius of the circle was 8 pixels with a mean transmission value of 105 pixels within the measuring frame. The measured tissue area was the same in all sections. Large blood vessels and capsular avoided. Four measurements were carried out for each point.

structures

were

Statistical Analysis The results were analyzed with Student’s independent two-tailed t-test. P < 0.05 was taken as significance point. The percentage coefficient of variation (CV%) was calculated

accordingto

determinations

the formula

CV%

= 100 x

SD/x,

where

n =

number

of

and X is the mean.

RESULTS There was a strong linear relationship

between

the thickness

of sections

and their

radioactivity count (r = 0.97). The precision of the measurements is indicated by the coefficients of variation (CV%) shown in Table 1. The CV varied from 5.7% (liver cranial, 30 pm thickness) to 11.2% (salivary gland, IO pm). In addition, results from two different liver locations (liver cranial and liver caudal) were compared statistically, revealing no significant (P < 0.05) variation in radioactivity between them according to measurements performed on 10 different WBA sections from each location (results not tabulated). The reproducibility of radioactive tested

by measuring

the transmission

tions of the same thickness

TABLE 1

exposures

on the autoradiographic

percentages

from the same organ.

from five successively The transmission

Radioactivity in Different Tissues and the Corresponding

THICKNESS or SECTION (I&

10 IJ. 10 CL 10 I* 20 CL 20 P 20 P 30 W 30 CL 30 CL

SAMPLE/LOCALIZATION IN MICE Liver cranial Liver caudal Salivary gland Liver cranial Liver caudal Salivary gland Liver cranial Liver caudal Salivary gland

films was taken

CV% Values

MEAN + S.D.’ (DPM) 241 2 245 i 45 2 425 ? 461 ? 68 f 595 ? 624 ir 9426

19 20 5 32 38 7 34 12

sec-

measurements

CV% 7,6 8,1 11,2 7,4 8,1 IO,2 5,7 890 6,s

’ The measurements were performed according to the method described for determination of the radioactivity on tape, under material and methods (n = IO).

A Quantitative

Method for Whole-Body

57

Autoradiography

100 -

._ ; I ,m c-

40 20 .-

03

2

1

3

4

5

Organs FIGURE 2. Reproducibility of exposed autoradiographic films. Transmission was measured by densitometric image analysis on films obtained from five successively cut sections that were placed on films and exposed for 6 weeks. The value in bars are mean f S.D. Organs: 1 = liver, CV% = 2.6; 2 = testis, CV% = 5.2; 3 = salivary glands, CV% = 7.8; 4 = wall of small intestine, CV% = 4.6; 5 = seminal vesicle, CV% = 11.0. were

performed

CV variation

by densitometric

from 2.6%

(liver)

image

analysis.

to 11 .O% (seminal

The

result

(Figure

2) showed

a

vesicle).

The coefficient of radioactivity variation in three different organs was determined by punching off minute amounts of the tissue into a liquid scintillation bottle and measuring the radioactivity. The findings (Table 2) were CVs of 9.6% 8.6% in the small intestine and 30% in the salivary gland.

in the liver,

DISCUSSION Statistical

comparison

of radioactivity

measurements

by the two different

methods

of tissue collection revealed that the use of tape was accompanied by smaller coefficients of variation (5.7% for liver-11.2% for salivary gland) than when direct punching off was used (9.6%-30%, respectively). The better result with tape is due to the fact that a constant amount of tissue was always being measured. Direct determination of radioactivity in tissue samples can often suffer from problems such as variable

chemiluminescence,

which

means

that different

sizes of tissue sample

TABLE 2. Radioactivity Measured From a Punched Off Piece of Tissue. Details Described Under Material and Methods (n = 10) ORGAN Liver Small

intestine

Salivary

glands

DPM/~Y+GTISSUE

CV%

1890 f 182 226 c_ 26

986 II,5

152 + 46

30,o

58

E.-L. Sainio and P. Sainio can give non-comparable results. The use of a cylindrical cutting form for tissue sample-taking provides the advantage of accurate comparison of radioactivity between samples from different organs. The tissue biopsy cylinder of 3.8 mm in diameter was specially designed for our study purpose. Commercially manufactured skin biopsy punches of 2, 3, 4, and 6 mm in diameter are also available (Stiefel Laboratories Ltd, UK). The linear

relationship

between

tissue thickness

and radioactivity

means that sec-

tions thicker or thinner than 20 pm can be used. A thickness of 30 km seems ideal on the basis of our experience and findings. The use of thicker sections than 20 Frn shortens the advantage

the exposure

time needed,

of decreased

gives a more distinct

background.

Previously,

20 km

picture

and also has

has been

considered

ideal for such measurements (Irons and Gross, 1981). WBA has often been criticized for not being able to distinguish the administered compound from its metabolites. It may well be possible to use thicker sections and to apply analytical methods to identify and measure metabolites further. The densitometric measurements in serial sections made from the photographic films showed that the standard deviations fell within better earlier study (Yonekura et al., 1983). Our results conformed ings that, provided adequately tested hardware is used and strument calibration and adjustment are made, the analytic sitometry different

limits compared

to an

with earlier study findsuitable checks on inaccuracy of videoden-

is high (McEachron et al., 1988). Our study showed that radioactivity at tissue levels on separate films is totally comparable in the same animal.

The present study also agrees with the finding that the lower the absorbance are, the higher the coefficients of variation (Schweitzer et al., 1987). Our mental compound was nicotinic would give similar results. The mathematical relationship

acid.

However,

between

it is likely

radioactivity

that other

in tissues

and

values experi-

chemicals autoradi-

ographic image density has been shown to be linear using polymethacrylate standards (Irons and Gross, 1981). Thus, in principle it should be possible to obtain exact quantitation of radioactivity in $i/wt of tissue by measuring radioactivity in organs with this tape section method, correlating these values with the corresponding transmission values and drawing a standard curve. However, this is troublesome and time consuming and earlier results with this method have not been satisfactory (Franklin, 1983). Cross et al. (1974) have introduced

the idea of constructing

a scale of different

concentrations of radioactivities in tissues. This has promoted development of commercially manufactured standards (Amersham International plc, England). According to our experience these are quite suitable and easy to use. A good reproducibility of autoradiograms is an absolute prerequisite for using the standards. Our study showed that these requirements are fulfilled and the standards can be used for obtaining the tissue concentrations of labeled compounds in FCi/wt. Validity of brain paste standards or standards based on radioactive blood homogenates have also been reported suitable for quantitative WBA (Ito and Brill, 1990; Schweitzer et al., 1987). Our

technique

has

other

advantages

when

compared

with

the

traditional

“punched

off”

method.

A Quantitative

Method for Whole-Body

Autoradiography

measurement

of tissue radioactivity

from tapes can

Direct

serve as a preliminary guide for assessing the approximate exposure time for films. It also makes it possible to provide an estimate of the radioactivity in different organs point. This method histological It seems

so results can be treated could come to augment

realistically from the prescreening

examination or micro-autoradiography. clear that WBA will develop into the preferred

the distribution WBA has been

needed needed

the statistical viewof study targets in

method

and kinetics of chemical compounds in tissues. inhibited by the difficulties with quantification.

for determining

So far, adoption of Further advances

also depend on progress in developing more sensitive films with lower background. However, the basic principles of the method are already established. This is the first study involving examination based weight The

to further authors

thank

for preparation

statistically-handled on image analysis.

progress Mr.

quantification The promising

of results from densitometric findings will hopefully lend

in this field.

E. Pesonen

and Mr. A. Dunberg

for technical

assistance

and Mrs.

S. Oksman

of the manuscript.

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U

(1977)

(WBAR): its

Whole-body

autoradiography

use for pilot studies

of pharmacokinet-

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Cross

SAM,

titative

Groves

method

sues sectioned Int / Appl Franklin

films

RD, Gross

ibration

icol Appl Ito T,

Brill

autoradiography.

Factors

affecting

the sensitivity autoradiog-

EA (1981) Standardization autoradiography analysis

compounds

McEachron

DL,

(1988) The

autoradiog-

radionuclides.

Appl

Ra-

Gallistel

CR, Eilbert

JL, Tretiak

Oj

analytic and functional

video densitometry

Schweitzer

system.

accuracy of a

/ Neurosci

A, Fahr A, Niederberger

ple method

and cal-

333.

for rou-

Yonekura

of the distribution

in animal tissues.

Tox-

Pharmacol59:250-256. AB (1990) Validity

whole-body

short-lived

for

Methods

(1983)

paste stan-

AB,

Quantitative

opharmaceuticals, system

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Y, Brill

Appl

j Nucl

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Radiat

Som P, Bennett auoradiography Part

1:

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W (1987) A sim-

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quantitative

25~63-74.

used for whole-body

tine semiquantitative of “C-labeled

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/sot 25:381-386.

raphy. Xenobiotica Irons

T (1974) A quanradioactivity

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Radiat

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com-

59