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The possible relationship between pyridoxal phosphate-dependent ornithine decarboxylase (ODC) activity and glucose-6-phosphate dehydrogenase (G6PD) activity has been studied in the osteoblasts of the growthplate of metatarsals of rats fed a pyridoxine-deficient diet, which caused depressed G6PD levels. The G6PD activity was fully restored when it was assayed in the presence of putrescine. It is suggested that this relationship may account for the correlation generally found between growth and ODC activity.

Circulating parathyroid hormone was studied in patients with pseudohypoparathyroidism and compared with that in normal subjects and patients with hypocalcaemia due to postsurgical or idiopathic hypoparathyroidism or vitamin D deficiency. The cytochemical bioassay was used to measure bioactivity and an amino-terminal specific immunoradiometric assay was used to measure immunoreactivity. In normal subjects (n = 12) the concentration of bioactive parathyroid hormone was 1.1-5.9 pg/ml. It was higher in patients with newly diagnosed, untreated pseudohypoparathyroidism (n = 4, range 20-53 pg/ml) and similarly raised in patients with untreated vitamin D deficiency (n = 9, range 23-74 pg/ml). The degree of hypocalcaemia was similar in these two groups of patients. By contrast, the concentration of bioactive parathyroid hormone was low (less than 0.6 pg/ml) in four patients with untreated postsurgical or idiopathic hypoparathyroidism. Restoration of normocalcaemia reduced the concentrations of bioactive PTH in both pseudohypoparathyroidism and vitamin D deficiency. Thus, in this respect the parathyroid glands in both conditions appeared to respond to the circulating calcium concentration. Immunoreactive PTH was also raised in patients with untreated pseudo-hypoparathyroidism and vitamin D deficiency, but restoration of normocalcaemia did not always reduce immunoreactive PTH to normal in these patients. Thus, there can be dissociation between bioactivity and immunoreactivity even when the PTH is measured in an amino-terminal specific assay.

The quantitative changes in alkaline phosphatase activity in the periosteal cells close to the fracture in rat metatarsal bones has been measured during the first 5 days postfracture. This study has been made possible by two technological advances, firstly the development of cryostat microtomy for cutting unfixed, undemineralised bone, and secondly the use of scanning and integrating microdensitometry for quantifying the activity in each periosteal cell. The results showed a loss of alkaline phosphatase activity close to the fracture site, with activity rising to normal values 0.8–1.0 mm from the site. No alkaline phosphatase activity was found in the cells which proliferated from the periosteum. It is suggested that reduced glutathione could cause such inhibition.

The mechanism of the lysis of target cells by cytotoxic T-cells (Tc) is still obscure; there is no evidence for transfer of material from the Tc and prior to lysis, despite intimate contact, the plasma membranes of both types of cell appear to remain intact. The effects on the target cell lysosomes of brief contact between anti-viral Tc and targets bearing both the appropriate histocompatibility and viral antigens, have been examined cytochemically. Both the distribution of acid phosphatase activity and the percentage bound lysosomal naphthylamidase activity indicated that, in virus-infected target cells exposed to Tc, the lysosomal membranes became totally labilized. Thus the contact between Tc and targets appears to cause sufficient perturbation of the target plasma membrane as to cause the intracellular release of some agent that activates ‘suicide capsule’ lysosomes.

A high incidence of natural osteoarthritis of the knee joint is found in male mice of the STR/ORT strain. The condition affects mainly the medial tibial cartilage and by the age of 27 weeks most male mice of this strain show some osteoarthritic change. Analysis of the oxidative metabolism of the chondrocytes during the development of the lesion has been facilitated by the techniques of quantitative cytochemistry. The activity of glucose-6-phosphate dehydrogenase (G6PD) has been investigated as indicative of the NADPH-generating pentose-phosphate pathway; the activities of glyceraldehyde-3-phosphate (G3PD) and lactate dehydrogenase (LDH) have been studied as indicators of glycolytic activity. In young STR/ORT mice the G6PD activity of the lateral tibial cartilage was greater and more variable than in the control mice of the CBA/HT6 strain. The activity in the medial cartilage, relative to that in the lateral cartilage, decreased with age; this change was not reflected in the activities of the other enzymes. In the lateral cartilage, the expected relationship was found between the G6PD and the G3PD activities and between the LDH and the G3PD activities. In the medial cartilage, the G6PD activities were not related to the G3PD activities. The decreased proportionality of the G6PD activities in the medial cartilage as against that in the lateral cartilage was detected in mice as young as 9 weeks; by 27 weeks of age nine of the 13 mice showed marked depression of medial as against lateral G6PD activities. In contrast, only four of the 13 mice showed any overt histological charge until up to the age of 28 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this study was to try to elucidate the increased susceptibility of the neck of femur to fracture. Quantitative polarised light microscopy has been applied to fresh, undecalcified sections of samples of bone taken from the site of fracture, in specimens taken at operation from patients with fractures of the femoral neck or osteoarthritic femoral heads or from the equivalent site from otherwise normal subjects at necropsy. In all 21 specimens of fractured necks of femur, but in none of the other specimens, relatively large crystals (up to 2.5 X 0.5 micrometres) were found close to the site of fracture; the properties of these crystals were compatible with their being apatite. Measurement of the natural birefringence of the collagen showed no difference in the orientation of the collagen in all three types of specimen. However, the orientation of acidic glycosaminoglycans, measured by the birefringence of alcian blue bound to these moieties, was 45 per cent lower in the specimens from fractured necks of femur than in the other specimens, even though the total content of acidic glycosaminoglycans was unchanged. Although the decreased orientation was most marked close to the site of fracture, it was still apparent 15 millimetres from that site. These changes were unlikely to be simply the sequelae of fracture since they were not found in traumatic fractures of other bones. Thus it is conceivable that changes in the orientation of the ground substance allow formation of relatively large crystals of apatite and that such crystals, in the microcrystalline mass of apatite, are the cause of the increased fragility of such bones.

By varying the time of hydrolysis for the Feulgen reaction, done under conditions that protect the backbone of the DNA, it is possible to distinguish three species of DNA that are characterised by their lability to acid hydrolysis. The most labile DNA was found, in greatest proportions, in malignant cells; this may be helpful in diagnostic cytology. The fact that the cytologically normal cells, in grade V smears, also show this labile DNA may well facilitate cytological screening even in those smears that contain very few neoplastic cells.

Left ventricular endomyocardial biopsy specimens were obtained at cardiac catheterisation in 11 patients with mitral-leaflet prolapse, chest pain, and normal coronary arteries. Specimens were examined for birefringence before and after contraction induced by adenosine triphosphate and were also subjected to histochemical analysis. Biopsy specimens from a control group of patients with rigorously defined normal left ventricular function and another control group with poor left ventricular function were also studied. Biopsy specimens from patients with mitral-leaflet prolapse showed a markedly subnormal birefringence response to adenosine triphosphate, with the group mean value intermediate between values for controls with normal and poor left ventricular function. Histochemical studies in the prolapse group also revealed an abnormal pattern, particularly for monoamine-oxidase activity. This is further evidence of a cardiomyopathy in this symptomatic subgroup of patients with mitral-leaflet prolapse.

The anticoagulant, dicumarol, inhibits the vitamin K cycle by blocking the conversion of the vitamin K epoxide. The effects of dicumarol on ossification have been tested by feeding it to rats in which a closed fracture of the metatarsals had been induced; the effects were studied up to 12 days postfracture. At 12 days, treatment with dicumarol caused a highly significant decrease in the amount of bone produced, without affecting the total size of the callus. Quantitative cytochemistry of unfixed, undemineralized sections showed that dicumarol also markedly affected the periosteal activities of glucose 6-phosphate dehydrogenase and of alkaline phosphatase in the first 2 mm from the fracture measured at 3 and 5 days postfracture when normally, new bone is first formed. In contrast, dicumarol had little effect on these activities in the fully formed callus.

In osteoporosis it is postulated that while the amount of bone is diminished, the quality of the bone is unaltered. Recently relatively novel methods of analysis have shown that, at the fracture site of osteoporotic subcapital fractures, there is a marked change in the molecular orientation of components of the non-collagenous bone matrix. These procedures, now applied to iliac crest biopsies, confirm earlier findings of altered orientation of the proteoglycans at the subcapital fracture site but show that very similar changes occur even in the iliac crests from patients with both types of osteoporotic proximal femoral fracture. Thus, whereas the amount of these acidic moieties of the non-collagenous bone matrix was unchanged, the molecular orientation was markedly altered, albeit not to the same extent as that found at the fracture site. These results imply that the quality of the bone, as well as the quantity, may be generally affected in osteoporosis.

Myocardial preservation by continuous coronary perfusion is compared with cold cardioplegia. The 1 L. of St. Thomas' cardioplegic protective solution used for infusion into the coronary arteries was at 4 degrees C. and had a normal electrolyte content except for raised concentrations of magnesium and potassium and the addition of procaine. Myocardial preservation was assessed by cytochemical grading of myocardial biopsy specimens and also by the requirement for significant inotropic support postoperatively. In aortic valve replacements with an aortic occlusion time of 1 1/2 hours, both techniques afforded a similar degree of preservation, except that the inner half of the myocardium was markedly better preserved by cold cardioplegia. After 2 hours in double valve replacements, a single cardioplegic infusion was not as effective as continuous coronary perfusion, but this could be corrected by a second infusion of the cardioplegic solution after an hour.

It was conceivable that the previously reported elevated pentose-shunt activity in human rheumatoid synoviocytes could be at the expense of glycolytic activity. To test this possibility the activities of glyceraldehyde 3-phosphate and lactate dehydrogenase, the two dehydrogenase enzymes of the latter pathway, have been investigated in the synovial lining cells in fresh sections of nonrheumatoid and rheumatoid synovial tissue. To measure the activity solely in the lining cells, apart from that in underlying infiltrating cells, quantitative cytochemical reactions have been used; the activities were measured by microdensitometry. The results showed highly and significantly increased activity of both enzymes in the rheumatoid cells. Increased activity was also found in synoviocytes in nonrheumatoid synovial tissue after trauma, so that the increased activity of these enzymes is not characteristic of the rheumatoid condition. However, the results indicate that the increased pentose shunt activity in rheumatoid synovial lining cells is not at the expense of glycolytic activity but may be part of an enhanced potential for utilising glucose 6-phosphate in these cells.

The experimental model of inflammatory arthritis in the rabbit has been used to study the possible origin of the apparent synovial lining cell hyperplasia. A small amount of 3H-thymidine was injected into the joints and the fate of the labelled cells investigated by autoradiography. At times up to 24 h after the injection, most of the labelled cells occurred in the sub-lining region; this proportionality was reversed when the joints were sampled at later times. These results indicate that at least much of the increase of synovial lining cells may be derived from cells from deeper in the synovial tissue which move to the synovial surface.

It has been shown that some of the controversy over the structure of articular cartilage may be due to slight differences in the orientation of the sample that has been studied. As our decisive criterion we have used the simple physical fact that elongate proteins, such as collagen micelles, that can exhibit form-birefringence, had to show virtually straight extinction when viewed under crossed polars. The use of a variably adjustable microtome chuck facilitated small adjustments in the orientation of the cartilage to meet this criterion. Under these conditions, the collagen of the matrix has been shown to be aligned mainly perpendicularly to the surface which was bounded by a thin lamina in which the collagen showed birefringence at 90 degrees to that of the matrix. The conventionally described zonation of articular cartilage has been shown to be inadequate for that of the dog tibial plateau. The conventional Zone 2 has been shown to consist of two zones, Zones 2a and 2b, with different cell sizes, cell concentrations, and concentration of matrix components.

Publisher Summary This chapter discusses hyperkinetic features of Graves' disease. It describes two categories of assays for thyroid-stimulating antibody (TSAb)—(1) the assay in which there is a more or less direct measure of thyroid stimulation; and (2) the assay in which TSAb interferes with binding of radioactive thyrotropin to its receptor in a preparation of human thyroid membranes. Although it has not been directly proven beyond doubt, it should be accepted that TSAb inhibits the binding of thyrotropin to its receptor and should be positive in a thyrotropin-binding inhibition (TBI) assay. It seems likely that the combination of an antibody with an antigen on the plasma membrane, at a site remote from the receptor for thyrotropin, may perturb the entire membrane so that the binding of thyrotropin is affected. The chapter also presents the results of studies regarding the clinical significance of TSAb.

Changes in the cellularity and in the nature of the matrix were studied in the cartilages of the tibial plateau in experimentally induced arthritis in the dog, 7 and 14 days after section of the anterior cruciate ligament. Samples from the different regions of these cartilages were chilled and sectioned in a cryostat, with a variable microtome chuck to allow precise orientation of the specimens. The samples were examined by normal light microscopy, by microscopic interferometry, and by quantitative polarized light microscopy. The orientation of the glycosaminoglycans was assessed by the new "induced birefringence" method. The results indicated that only the region of the medial tibial cartilage that was unprotected by the meniscus was affected, showing increased water content, loss of superficial cells, and a decrease in orientation of the glycosaminoglycans. Whereas the birefringence of the collagen was unaffected, the superficial area that lacked oriented glycosaminoglycans was markedly increased; this may be a useful indicator of early osteoarthritic changes.

The best method of preserving myocardial integrity following aortic occlusion is still debatable 20 years after the beginning of open heart surgery. Occlusion of the aorta at normal temperatures1 and the original potassium arrest cardioplegic method of Melrose et al.2 have been abandoned because of unacceptable myocardial damage. Coronary artery perfusion with a beating heart has been the standard technique of myocardial preservation3 but pericardial cooling4, with or without the addition of a pump to circulate cold fluid faster5, has recently become more widely adopted. ‘Root cooling’ of the heart6, and the infusion of specially formulated cardioplegic solutions, such as those developed by Bretschneider7 and Kirsch8, have attracted much recent interest.

Articular cartilage is generally considered to be an homogeneous tissue. It has now been shown that, although different regions of the medial tibial cartilage of the dog have very similar oxidative enzymic activities, each region is heterogeneous with respect to these activities. The conventional histological delineation of this cartilage has been modified, to take into account a narrow band (designated zone 2a), just below the most superficial spindle-shaped cells, that has higher oxidative enzymic activity than any other. Changes in the activity in this zone might be diluted by the lack of change in other zones if measured by conventional biochemical procedures which could not measure the activities of the different zones separately.

Publisher Summary The evidence summarized in this chapter shows that it is possible to precisely measure the biochemical changes induced in target cells when the specific hormone acts on these cells with the modern techniques of cellular biology and cytochemistry. By measuring such effects, it is possible to assay the biological activity of the hormone with a degree of sensitivity that exceeds that of most currently available radioimmunoassays by two to three orders of magnitude. The increase in the use of cytochemical bioassays have resulted in the increase in their validity tests and applicability scope. The development of a cytochemical section assay for adrenocoticotropin (ACTH) has resulted in extension of these assays to routine purposes. It is a model for similar assays for other hormones. But the principle that the effect of bioreactive molecules on their target tissues can be measured very sensitively opens further vistas. These techniques can be applied to the detection, and assay, of other biologically active substances, such as pharmacologically active substances or toxic substances. These techniques can be readily used to study the analysis of end-organ sensitivity and the effects of hormones or their products on the other tissues in the body can be readily studied by these new techniques.

A cytochemical section-bioassay of thyrotropin has been developed which retains the sensitivity of the earlier segment-assay (10(-4) microunits/ml) and allows a considerable increase in the rate at which these within-animal bioassays can be done. The index of precision (n=23) was 0.11 +/- 0.06; intra-assay variation was 1.3%; inter-assay variation was 10.9 +/- 6%. Fiducial limits (p = 0.95) ranged from 65-150% to 78-127%. The system is also capable of detecting thyroid-stimulating immunoglobulins.

Human rheumatoid synovial lining cells have up to four times the capacity to oxidize glucose 6-phosphate, the first step of the hexose monophosphate pathway, as do the nonrheumatoid cells. The reducing equivalents produced by this system have many significant metabolic effects. Exposure of these cells by 10(-5) M prednisolone in vitro, or to 6 mg/day in vivo, causes some depression of this activity in the rheumatoid synovial lining cells; less than this dose of steroid, or the administration of nonsteroidal drugs in vivo, has little or no effect. The depression of activity produced by 6 mg/day does not bring this activity down to the value found in nonrheumatoid synoviocytes.

Cytochemistry now extends biochemistry down to the single-cell level. Special procedures have been developed for cutting sections of uniform thickness with no measurable or detectable artifact. The cytochemical bioassays use chromogenic reactions with the immediate precipitation of the resulting chromophore so that biochemical activity can be related to the individual cells constituting a tissue. This activity, in individual cells, is measured by scanning and integrating microdensitometry which also increases the sensitivity of cytochemical measurement over that of conventional biochemistry. A hormone, or indeed any biologically active substance, acting on its target cell, causes a change in the chemical activity of that cell that mediates the physiological effect of the hormone. By cytochemical methods one can assess such changes in the target cells even if these constitute only a small part of the target-organ; thus such methods are ideally suited to measuring chemical changes of this sort induced by the hormone. Such cytochemical bioassay of polypeptide hormones, done as "within-animal" assays, are about 1000-f0ld more sensitive than the equivalent radioimmunoassays and are as precise. Thus they have two advantages: (a) bioreactive hormone is measured rather than a composite of antigenic determinants characteristic of part of the hormone molecule and (b) their increased sensitivity allows discrimination between low normal and subnormal concentrations of the circulating hormone.

The activities of two mitochondrial enzymes, succinate dehydrogenase and cytochrome oxidase, have been measured by quantitative cytochemistry and microdensitometry in the synoviocytes of rheumatoid and non-rheumatoid synovial lining cells. Although both tended to be higher in the former, there was no statistically significant difference in the activities of either enzyme in these tissues. However, when cytochrome oxidase activity was measured without exogenous cytochrome c, the activity in the rheumatoid synoviocytes was highly significantly elevated. It is suggested that these findings may indicate only that the cytochrome c-cytochrome oxidase complex in the rheumatoid cells is more stable, possibly because of the increased availability of phospholipids in these cells.

Adult primary myxoedema is usually due to an autoimmune thryoiditis characterized by progressive shrinking of the thyroid gland, loss of epithelium, dense infiltration by sensitized lymphocytes and plasma cells with final replacement of the gland by a fibrous scar. Antibodies directed against thyroglobulin (TgHA) and microsomal antibodies (McHA), detectable years before the onset of hormonal failure, and cell-mediated immune mechanisms contribute to the pathogenesis. When 90% of the gland is destroyed, the secretion of thyroid hormones (T3/T4) falls below normal needs and pituitary thyrotrophs react by producing over 100 times the normal amount of thyroid-stimulating hormone (TSH) which should normally lead to regrowth of the gland as happens in goitrous Hashimoto thyroiditis. The inability of the thyroid in primary myxoedema to respond to the trophic action of TSH suggested that blocking antibodies exist in this disease which compete with TSH for its receptors. The trophic effect of TSH can be assayed in vitro by measuring DNA-synthesis by Feulgen cytophotometry; elevated pentose-shunt oxidative activity correlates with DNA synthesis. Here we report that immunoglobulins from patients with adult, primary myxoedema block the trophic effect of TSH, as assessed by this in vitro system. This is in marked contrast to the growth stimulation induced by immunoglobulins from patients with thyrotoxic Graves' disease with prominent goitres.

A quantitative cytochemical method was developed for measuring the GSH (reduced glutathione) content of hepatocytes in different regions of the rat liver lobule. Use of this method enabled us to show that GSH is not evenly distributed within the rat liver lobule. The hepatocytes located within 100 micrometer of the central vein contain much less GSH than do those in other regions of the rat liver lobule. We suggest that this partially explains the peculiar susceptibility of these cells to electrophilic attack by toxic metabolites formed via the microsomal cytochrome P-450 system.

Synovial tissue from 3 patients with villonodular synovitis has been examined by quantitative cytochemistry. Considerable changes in the metabolism of the synoviocytes have been observed. These included a massive rise in the activity of glucose 6-phosphate dehydrogenase to levels even greater than those found in rheumatoid arthritis, and a significant rise in the activity of 2 glycolytic enzymes, glyceraldehyde 3-phosphate and lactate dehydrogenase. Lysosomal napthhylamidase showed raised activity with no latency. The sulphydryl content in the synoviocytes was raised, as was the amount of available phospholipid. This pattern of metabolic alterations is similar to that found in human rheumatoid synoviocytes.

The procedure for the cytochemical bioassay of parathyroid hormone has been examined to improve the reliability and robustness of the assay. It has been shown that the control of the pH, at all stages of the assay, is essential. The use of the assay confirmed that the circulating level of the biologically active form of this hormone is less than one tenth of the values found by the more recent immunoassays, and is in good agreement with previously calculated values. The assay clearly distinguished between normal, hypoparathyroid and hyperparathyroid levels and allowed relative potencies to be ascribed to preparations of the hormone obtained from human, bovine and porcine sources.

Five different types of myocardial protection were employed in this series of 168 patients undergoing aortic valve replacement. Two methods of assessing myocardial preservation were used: cellular biological estimations and quantitative polarization measurements. Both parameters showed that either of two methods, continuous perfusion at 32 degrees C. with a beating heart or cardioplegic hypothermic arrest, protected the myocardum best. Intermittent perfusion at 30 degrees C. with a fibrillating heart was the worst means of preservation. Our investigations (both clinical and experimental) have also shown that changes in birefringence, indicative of deteriorating myocardial function, are often detectable before parallel cytochemical changes are apparent.

There are reports of an apparent dissociation between the responses of kidney and bone to parathyroid hormone (PTH): certain fragments or analogues have been shown to have differential activity in these two tissues. Consequently, cytochemical bioassay techniques have been applied to the development of an assay for PTH using the epiphyseal plate of the rat metatarsal as the target organ. Glucose 6-phosphate dehydrogenase activity in the zone of hypertrophic cartilage is stimulated in a log-linear fashion with increasing concentrations of parathyroid hormone over the range 0.001 - 1.0 pg/ml. A parallel response was found in the osteoblasts of the metaphysis. Measurements of a normal and a hyperparathyroid plasma sample showed good parallelism to the standard graph and good discrimination. Addition of a PTH - specific antibody inhibited both the effect of the standard preparation of the hormone, and the response induced by dilutions of a normal plasma.