Path. Res. Pract. 170, 39-49 (1980)

Division of Cellular Biology, The Mathilda and Terence Kennedy Institute of Rheumatology, Bute Gardens, London W6 7DW, U.K.

The Application of Cytochemistry to the Highly Sensitive Bioassay of Polypeptide Hormones

J. CHA YEN and LUCILLE BITENSKY

General Introduction to the Function of Cytochemistry

Definition of Cytochemistry

There is a degree of confusion between the terms 'Histochemistry' and 'Cytochemistry', as there is between the aims of those who profess to practise either subject. Yet it is becoming increasingly clear that these subjects repre­sent quite different approaches to the sort of chemical information that a histopathologist may obtain from sections of tissue. The histochemist's attitude has been clearly defined by Barka and Anderson (1963) in words as relevant today as when they were first published: Histochemistry " ... is a system of chemical morphology that adds another dimension to histology but which shares the basically static character of the morphological sciences. Its contribution cannot be assessed in the dynamic, physiological terms of biochemistry ... finite measurement is not the immediate goal of microscopic histochemistry. Deriving its theoretical foundations from chemistry, his­tochemistry remains essentially a morphological tool." The aims of the his­tochemist include the following. Firstly, the use of a histochemical 'stain' to define a particular cell-type, for example, the esterase reactions for distin­guishing monocytic cells or the a-naphthol butyrate esterase procedure for differentiating between mature T- and B-lymphocytes (as reviewed by Stuart, 1979). Secondly, the possibility that a disease process will cause a changed pattern of staining. Thirdly, the hope that there will be an all-or-none response in histochemical staining for a particular enzymatic activity. For example, Niles et al. (1968) found a loss of stain, for glutamate dehydrogenase activity, in certain regions of the myocardium that corresponded to the later appear­ance of focal necrosis.

40 . J. Chayen and Lucille Bitensky

In marked contrast, cytochemistry is an extension of biochemistry literally to the level of the cell or of intracellular organelles. It is essentially biochemis­try and it is essentially quantitative. Its best known example is cytophotometry, of the Feulgen reaction or of the gallocyanin-chrome alum method, for quantifying the amount of deoxyribonucleic acid (DNA) in each individual nucleus in a section or in isolated cells and relating changes in the DNA-content in nuclei of a population of cells to a particular disease-process or condition or treatment (e.g. Sandritter, 1979). The application of cytophotometry or more rigorously, of scanning and integrating microden­sitometry, to stoicheiometric chromogenic reactions for enzymatic activities (or for other reactive groups) in sections or in isolated cells, is one of the bases of modern, quantitative cytochemistry. But, as will be discussed, several other techniques had to be developed before it became possible to perform rigorous biochemical estimations on sections. The cytochemical bioassays will be taken as examples for the contention that cytochemistry is capable of making such estimations. If this is conceded, then it is possible that histopathology can now analyse chemical pathology with a precision that is not available to conven­tional biochemistry.

Cytochemistry as an Extension of Biochemistry

Cytochemistry has the following advantages over conventional biochemis­try: firstly, its great sensitivity permits measurement of the biochemical activ­ity of individual cells; secondly, it can relate this cellular biochemical activity to the histology of the tissue sample; thirdly, being a relatively non-disruptive technique it can demonstrate delicate changes in the functional state of cellular and sub-cellular membranes that may control cellular metabolic activities; fourthly, also because of its essentially non-disruptive nature, it can be used to study the functional state of molecular complexes and of inter-acting sub­cellular systems. Examples of these features will be considered briefly.

Tissue dilution artifact

In any tissue, the cells that form the target for the action of a hormone, or of a toxic substance, or of a disease-process, may constitute only a small part of the tissue. For example, among its other actions parathyroid hormone stimu­lates the resorption of calcium by the renal distal convoluted tubules; this involves the activation of glucose 6-phosphate dehydrogenase in the cells of these tubules. But even though the activity of this enzyme may be increased to three times its basal level, this elevated activity will not be observed if one measures the glucose 6-phosphate dehydrogenase activity of the whole renal

Cytochemistry of Polypeptide Hormones . 41

cortex: it will be masked by the relatively unchanged activity of all the cells that occur within the cortex. To put the matter into quantitative but hypothet­ical terms: suppose that a unit weight of kidney cortex contains 1000 cells, each of which has one unit of glucose 6-phosphate dehydrogenase activity; if the hormone trebles the activity of 1 % of the cells (namely those of the distal convoluted tubules), the measurable activity, per unit weight, will rise from 1000 to 1020 units, which will be within the experimental error of most measurements (e.g. ± 5 % ). In contrast, the activity of the target -cells will have changed from 1 to 3 units, which is a marked change, assuming that you have a method that can measure one unit of activity with the same degree of precision (± 5%).

Changes in cellular and sub-cellular membranes

Generally the state of the membranes of sub-cellular organelles is assessed after the tissue has been homogenized and the sub-cellular organelles have been isolated into a foreign medium that is likely to alter the state of the membrane bounding the organelle. Early studies (Chayen and Bitensky, 1968) confirmed biochemical studies on the damage done to mitochondrial mem­branes by the process of homogenization (Bendall and de Duve, 1960), and showed that the alteration in the functional state of liver mitochondria was sufficient to mask damage done, in vivo, by a hepatotoxin; the effect of this hepatotoxin was marked when studied cytochemically. Similarly the beneficial influence of aspirin on lysosomal membranes, which could not be decisively shown by conventional biochemical procedures, could be demonstrated cytochemically (Chayen et al., 1972). The cytochemical bioassay of thyroid stimulators depends on measuring the change in lysosomal membrane-func­tion in the target cells of the thyroid gland.

Functional state of molecular complexes

Particularly in the past, biochemical procedures have been designed to iso­late and purify the components of molecular complexes such as those between lipids and proteins. Yet the significant effect in early cell damage may not be a change in total amount of phospholipids and of protein, but a change in the way that such lipid-protein complexes are bound. If the lipid component becomes less tightly bound, it can alter the nature of the structure in which it occurs. For example, one of the main indicators of early myocardial damage may be the increased lipophilia of the myofibrils, shown by the fact that the phospholipids of the lipid-protein complexes become available to cytochemi­cal reactions for phospholipids (e.g. Braimbridge et al., 1973; Niles and Barn-

42 . J. Chayen and Lucille Bitensky

house, 1967). Similarly, changes in the binding of DNA to nuclear proteins, even without quantitative differences in the amount of either, may indicate vital changes in the nature of the cells, and even changes related to malignancy (Millett and Husain, 1979).

Comparison of cytochemistry and convential biochemistry

Thus cytochemistry can be regarded as a relatively non-disruptive form of cellular biochemistry that emphasizes the inter-relationship of biochemical systems within cells (e.g. Chayen et aI., 1973a) and relies on conventional biochemistry for the precise analysis of the isolated components. It uses tissue slices, as are used in some forms of conventional biochemistry, except that these must be sufficiently thin for detailed histological analysis; they must also be free of measurable ice-artifact that could disturb membrane-function. The chemical activity of enzymes, or of reactive groups, depends on chromogenic reactions that must be optimized at least as rigorously as those of conventional biochemistry. The difference is that the coloured reaction-products of cytochemistry must be precipitated close to the site of the activity, so that the individual activities of neighbouring cells can be distinguished. This means that the chromophore cannot be measured by simple spectrophotometry; recourse must be made to scanning and integrating microspectrophotometry or micro densitometry (e.g. Fukuda et aI., 1978; Chayen, 1978a) if the precipi­tated, optically inhomogeneously distributed chromophore of the reaction­product is to be measured precisely.

Problems in Establishing Cytochemistry

Preparation of sections

There is much evidence to show that tissue can be chilled, for example to

-70°C or below, without ice forming inside the tissue (as reviewed by Chayen, 1978b). To preserve the supercooled state of the protoplasm, sections (5 to 20 Ilm thick) are cut in a cryostat with the temperature of the cabinet at - 25 to - 30°C and the knife cooled to -70°C to dissipate the heat, generated by the cutting, into the knife. The supercooled protoplasm of the section is stabilized by 'flash-drying' the section from the knife on to a warm slide (as described by Chayen et aI., 1973b). When such sections have been subjected to

ultra-thin sectioning and examined by electron microscopy, no signs of ice artifact have been discerned (Altman and Barrnett, 1975; Zoller and Weisz, 1980); the section-assays, to be described later, are also powerful evidence for the integrity of these tissue-sections.

Cytochemistry of Polypeptide Hormones . 43

Constancy of thickness

A major obstacle to measuring the amount of a stain, or a cytochemical reaction, in two sections of nominally equal thickness taken, for example, one from a treated and one from a control block of tissue, has been the realization that even serial sections from the same block can vary considerably in thick­ness. However, it has now been shown that the variable thickness is due to the variable speed at which the tissue-block traverses the knife. With an automatic cutting device that ensures a constant speed of sectioning, variation in thick­ness (Butcher, 1972) or in the amount of a cytochemical reaction (Loveridge et ai., 1974; Bitensky et ai., 1974; Daly et ai., 1974) is less than ± 5%.

Need for colloid stabilizers

It is well known that when fresh sections are placed in a histochemical reaction-medium at pH values of between 7 and 8, as is required for the study of most oxidative enzymes, much of the protein of the section becomes dis­persed rapidly into the reaction-medium. Consequently, histochemists had a difficult choice: either they could use such sections, knowing that the localiza­tion of the reaction-product was virtually meaningless; or they could chemi­cally fix the sections before reacting them, in which case the localization might be more meaningful even if the chemical activity had been largely attenuated. This problem has been overcome in cytochemistry by the addition of a suffi­cient quantity of one or other colloid stabilizer to the reaction-medium. This procedure retains all the measurable nitrogenous matter (Altman and Chayen, 1965) and all the enzymatic activities that have been studied (Chayen, 1978b) within the sections. The colloid stabilizers used include particular grades of polyvinyl alcohol (Wacker Chemicals Ltd., London), a partially degraded collagen (Polypeptide 5115; Sigma) and gum tragacanth.

Summary

The armamentarium of quantitative cytochemistry therefore includes the following: a method of preparing suitably thin sections, of fairly constant thickness, without causing measurable preparative artifact; a way of protect­ing the undenatured protoplasm of such fresh sections while the sections are exposed to the chromogenic reaction-medium even at relatively physiological pH values; and scanning and integrating microdensitometry for measuring the chromophore, and therefore the biochemical activity, with considerable preci­sion and sensitivity.

44 . ]. Chayen and Lucille Bitensky

The Cytochemical Bioassays of Polypeptide Hormones

Radioimmunoassay and Bioassay

The circulating concentration of a polypeptide hormone can be measured in one of two ways. The analytical approach (Chayen et aI., 1976), exemplified by radioimmunoassay, measures the number of molecules that have the anti­genic properties of the specific polypeptide. The functional approach is that of bioassay in which the biological activity of the polypeptide molecules is mea­sured. It was to be hoped that the two approaches would yield concordant results and, in general, this seems correct. Initially radioimmunoassay was so much more sensitive than conventional bioassay that it became the main tool of assayists. However, it soon became clear that there could be considerable discrepancy between the results of radioimmunoassay and clinical findings due, apparently, to radioimmunoassay measuring fragments of the hormone that still retained antigenicity while lacking biological activity. Moreover, the remarkable increase in sensitivity afforded by radioimmunoassay was still insufficient to measure the lower range of normal circulating levels of some hormones (Table 1). For these reasons, there was a need for a very sensitive bioassay system.

Principle of the Cytochemical Bioassays

The functional approach to the assay of hormones (Chayen et aI., 1976; Chayen, 1978b) is that when a polypeptide hormone acts on its target-cells it is first 'recognized' by specific receptors at the cell-surface. This gives some measure of specificity to the subsequent responses. The binding of the hor­mone to the receptor then sets in motion a series of events that change the biochemical activities of the target-cell in such a way as to provoke the phy­siological response by which the hormone is normally recognized. It is possi-

Table 1. Examples of the relative sensitivities of the cytochemical bioassays (CBA) and radioim­munoassays (RIA)

Hormone

ACTH TSH LH PTH

Expected circulating levels

8 -50 0.1- 2.0

10 10 -20

Sensitivity of:

RIA CBA Units

10 5 x 10-3 pglml 0.5 10-4 !lU/ml 0.2 10-3 mU/ml

75 5 x 10-3 pglml

Cytochemistry of Polypeptide Hormones . 45

ble, therefore, to measure the concentration of the hormone that acts on the target-cells by measuring one of the characteristic biochemical changes induced by the hormone; this also increases the specificity of the measured response. This question, of measuring the change in a particular biochemical activity in a specified target-cell within a complex tissue, is the special pro­vince of modern cytochemistry, as has been discussed above.

Consequently the target-tissue is removed from a suitably responsive ani­mal, normally a guinea-pig, and cut into segments. Each segment is main­tained individually in vitro for 5 h in non-proliferative adult organ-culture, at 37°C, to allow the tissue to recover from the trauma of excision and from the hormonal influence endogenous in the animal. At the end of this period, each of four segments is exposed to one of a graded series of concentrations of a standard reference preparation of the hormone for the optimal time: this gives the calibration-graph of how the target-cells of this particular animal respond to the hormone. Two other segments are exposed for the same time to the

90

85

80

fl 75 c d Ll

] <

70

'" > :g 65 Qj

x

a:

60

55

10-4 10-3 TSH }JU/ml

Fig. 1. An example of a cytochemical bioassay: the section-assay of thyrotrophin (TSH). The amount of activity (freely available lysosomal naphthylamidase activity), measured as the relative absorbance (or relative absorption) in unit field of thyroid-follicle cells, increases with increasing concentrations of a standard preparation of thyrotrophin, from 10-4 to 10-1 IlU/ml. In serial sections, the activities induced by two concentrations of the plasma (1 : 103 and 1 : 102) are parallel to those produced in response to the standard preparation. The concentration of thyro­trophin present in each dilution of the plasma is read off the standard calibration-graph. Crosses denote mean activities measured in 10 cells of each of two serial sections; a single cross implies that the values for the two means were identical.

46 . ] . Chayen and Lucille Bitensky

plasma, appropriately diluted, e.g. either 11100 or 111000. The response in these target-cells will be compared with that produced by the standard refer­ence preparation to ensure 'parallelism of response', indicative of similarity between the hormone in the plasma and the reference preparation; it is also a test ofreprodutibility (Fig. 1).

The segments are then chilled to - 70°C, sectioned at - 25 °C and reacted for a biochemical change that is characteristic of the effect of the hormone. The activity is measured in the target-cells by scanning and integrating mi­crodensi tometry.

This principle has been applied to the assay of polypeptide hormones and of thyroid stimulating immunoglobulins (Table 2). At present it is being used to develop assays for other polypeptide hormones. The same general approach can be applied, in principle at least, to the assay of any pharmacologically or biologically active material.

Table 2. Mechanism of cytochemical bioassays

Hormone

Cortico­trophin

Luteinizing hormone

Target cells Biochemical activity

Adrenal zona Ascorbate­reticularis depletion for

steroidogenesis

Corpus luteum

Ascorbate­depletion for

steroidogenesis

Thyrotrophin Thyroid-folli- Endocytosis of

Thyroid­stimulating Immuno­globulins

Gastrin

Parathyroid hormone

cle cells colloid: lysosomal activity

Thyroid-folli- Endocytosis of cle cells colloid:

lysosomal activity

Parietal cells Gastric acid of gastric secretion fundus

Renal distal Transport of convoluted calcium: elevated tubules production of

NADPH

Cytochemical Range of assay Reference reaction

Prussian blue 5 fg-S pg/ml reaction for

Dalyetal., 1974, 1977

reducing groups

Prussian blue 10- 4-10- 1 mU/ml Buckingham et reaction for aI., 1979 reducing groups

Lysosomal naphthyl-amidase

Lysosomal naphthyl­amidase

Carbonic anhydrase

Glucose 6-phosphate dehy-drogenase

10- 4_10-1 !-lU/ml Bitensky et a!., 1974; Chayen et a!., 1980

5 fg-S pg/ml

5 fg-1 pg/ml

Bitensky et a!., 1974

Loveridge et a!., 1974

Chamberset aI. , 1978

Cytochemistry of Polypeptide Hormones· 47

Cytochemical Section-Bioassays

These assays (Alaghband-Zadeh et aI., 1974, for corticotrophin; Chayen et aI., 1980, for thyrotrophin) were developed to increase the rate at which the hormonal content of samples could be measured, without losing the advan­tage of 'within-animal' assays. In these section-assays, segments of the target­organ are maintained in vitro as for the segment-assays. At the end of the five­hour period the segments are chilled to - 70°C. Sections, just thicker than the critical dimension of the target-cells, are cut and these (in duplicate) are exposed to the standard reference preparation of the hormone, or to the dilutions of the plasmas. The interaction between the hormone and the target­cells requires a pH in the physiological range, so that the sections are pro­tected, during exposure to the hormone, by a concentration of a colloid­stabilizer that is carefully gauged to give adequate protection while leaving the target-cells still responsive to the hormone. The fact that these section-assays are as sensitive as the original segment-assays indicates that the processes of chilling, sectioning and flash-drying, and the use of the colloid-stabilizers, cause no appreciable damage.

The response to the hormone in the section-assays occurs more rapidly than in the segment-assays: in the assay of corticotrophin the maximal response occurs at 60 sec; in that for thyrotrophin it is at 90 sec, with the slower response to the thyroid-stimulating immunoglobulins at 3.5 min (instead of 20-30 min as in the segment-assay). Many assays can be done on sections of the same region of the adrenal or thyroid gland. In the new section-assay of luteinizing hormone (Buckingham et aI., 1980), many assays are done on a single corpus luteum, so obviating differences between different animals which cause lack of precision in most in vivo bioassays.

Pathological Problems

These methods can be applied to investigate pathological effects of circulat­ing material. For example, the atrophic gastritis of pernicious anaemia has been attributed to the effect of circulating antibodies directed against the parietal cells even though the cytotoxicity of such antibodies has not been demonstrated. To test the effect of such antibodies, strips of guinea-pig gastric fundus were maintained in vitro, as for the cytochemical segment-assay of gastrin, in the presence of normal serum, of serum from patients with perni­cious anaemia and with the immunoglobulin fraction of such sera. The mate­rial was then removed and replaced with fresh culture-medium containing one of two concentrations of the heptadecapeptide human gastrin I. The cells

48 . J. Chayen and Lucille Bitensky

cultured in the presence of normal serum responded to the hormone normally whereas those maintained in the presence of serum, or immunoglobulins, from patients with pernicious anaemia did not respond (Bitensky et a1., 1979). Thus it seems that these antibodies make the parietal cells unresponsive to their trophic hormone so that the presumed 'cytotoxicity' may be simply a blockade of the trophic influence of the hormone.

References

Alaghband-Zadeh, J., Daly, J. R., Bitensky, L., and Chayen,].: The cytochemical section assay for corticotrophin. Clin. Endocr. 3, 319-327 (1974)

Altman, F. P., and Barrnett, R. J.: The ultra-structural localisation of enzyme activity in unfixed tissue sections. Histochemistry 41,179-183 (1975)

Altman, F. P., and Chayen, ].: Retention of nitrogenous material in unfixed sections during incubation for histochemical demonstration of enzymes. Nature 207, 1205-1206 (1965)

Barka, T., and Anderson, P.: Histochemistry, Harper and Row, New York (1963) Bendall, D. S., and Duve, C. de.: Tissue fractionation studies. 14. The activation of latent dehy­

drogenases in mitochondria from rat liver. Biochem. J. 74, 444-450 (1960) Bitensky, L., Alaghband-Zadeh,]., and Chayen, J.: Studies on thyroid stimulating hormone and

the long-acting thyroid stimulating hormone. Clin. Endocr. 3, 363-374 (1974) Bitensky, L., Loveridge, N., Chayen, J., Gardner, J. D., Bottazzo, G. F., and Doniach, D.: Inhibi­

tion of gastrin-responsiveness by parietal cell antibodies. Clin. Sci. Mol. Med. 56, 17P (1979) Braimbridge, M. V., Darracott, S. A. R., Bitensky, L., and Chayen, J.: Cytochemical analysis of

left ventricular biopsies in open-heart surgery: a pilot study. Beitr. Path. 148,255-264 (1973) Buckingham, J. c., Chayen, J., Hodges, J. R., Robertson, W. R., and Weisz, J.: A cytochemical

section assay method for the determination of luteinizing hormone. J. Endocr. 81, 160P (1979) Butcher, R. G.: Precise cytochemical measurement of neotetrazolium formazan by scanning and

integrating microdensitometry. Histochemie 32, 171-190 (1972) Chambers, D. ]., Dunham, J., ZaneUi, J. M., Parsons, J. A., Bitensky, L., and Chayen, J.: A

sensitive bioassay of parathyroid hormone in plasma. Clin. Endocr. 9,375-379 (1978) Chayen, J.: Microdensitometry. In Biochemical Mechanisms of Liver Injury. Ed. T. F. Slater,

pp. 257-291, Academic Press, London - New York (1978a) Chayen, J.: The cytochemical approach to hormone assay. Int. Rev. Cytol. 53, 333-396 (1978b) Chayen, J., and Bitensky, L.: Multiphase chemistry of cell injury. In The Biological Basis of

Medicine. Eds. E. E. and N. Bittar, vol. 1, pp. 337-368, Academic Press, New York (1968) Chayen, J., Altman, F. P., and Butcher, R. G.: The effect of certain drugs on the production and

possible utilization of reducing equivalents outside the mitochondria. In Fundamentals of Cell Pharmacology. Ed. S. Dikstein, pp. 196-230, Chas. C. Thomas, Springfield, Illinois (1973a)

Chayen, )., Bitensky, L., and Butcher, R. G.: Practical Histochemistry, Wiley, New York -London (1973b)

Chayen,]., Bitensky, L., and Ubhi, G. S.: The experimental modification of lysosomal dysfunction by anti-inflammatory drugs acting in vitro. Beitr. Path. 147,6-20 (1972)

Chayen, J., Daly,). R., Loveridge, N., and Bitensky, L.: The cytochemical bioassay of hormones. Recent Progr. Hormone Res. 32, 33-79 (1976)

Chayen, J., Gilbert, D. M., Robertson, W. R., Bitensky, L., and Besser, G. M.: A cytochemical section-bioassay for thyrotropin.]. Immunoassay 1,1-13 (1980)

Cytochemistry of Polypeptide Hormones . 49

Daly, J. R., Loveridge, N., Bitensky, L., and Chayen, J.: The cytochemical bioassay of cortico­trophin. Clin. Endocr. 3, 311-318 (1974)

Daly, J. R., Alaghband-Zadeh, J., Loveridge, N., and Chayen, J.: The cytochemical bioassay of corticotropin (ACTH). Ann. NY Acad. Sci. 297, 242-259 (1977)

Fukuda, M., B6hm, N., and Fujita, S.: Cytophotometry and its biological application. Progr. Histochem. Cytochem. 11, 1-119 (1978)

Loveridge, N., Bloom, S. R., Wei bourn, R. B., and Chayen, J.: Quantitative cytochemical estima­tion of the effect of pentagastrin (0.005-5 pglml) and of plasma gastrin on the guinea-pig fundus in vitro Clin. Endocr. 3, 389-396 (1974)

Millett, J. A., and Husain, O. A. N.: Analysis of chromatin in carcinoma-in-situ. In Quantitative Cytochemistry and its Applications. Eds.]. R. Pattison, L. Bitensky and J. Chayen, pp. 37-42, Academic Press, London - New York (1979)

Niles, N. R., and Barnhouse, D. L.: The acid hematein stain and myocardial damage. Arch. Path. 83,407-410 (1967)

Niles, N. R., Zavin,]. D., and Morikado, R. N.: Histochemical study of effects of hypoxia and isoproterenol on rat myocardium. Amer. J. Cardio!' 22, 381-388 (1968)

Sandritter, W.: A review of nucleic acid cytophotometry in general pathology. In Quantitative Cytochemistry and its Applications. Eds. J. R. Pattison, L. Bitensky and J. Chayen, pp. 1-8, Academic Press, London - New York (1979)

Stuart, J.: Quantitative enzyme cytochemistry in acute leukaemia. In Quantitative Cytochemistry and its Applications. Eds. J. R. Pattison, L. Bitensky and]. Chayen, pp. 113-127, Academic Press, London - New York (1979)

Zoller, L. c., and Weisz, J.: A demonstration of regional differences in lysosome membrane permeability in the membrana granulosa of graaffian follicles in cryostat sections of rat ovary: a quantitative cytochemical study. Endocrinology 106, 871-877 (1980)

Received and Accepted· February 20, 1980

Key words: Cytochemistry - Cytophotometry - Polypeptide Hormones

J. Chayen, Division of Cellular Biology, The Mathilda and Terence Kennedy Institute of Rheumatology, Bute Gardens, London W6 7DW, U.K.

4 Path. Res. Pract. Vol. 170