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Understanding of calcium metabolism in health and disease has been retarded by the lack of an adequately sensitive bioassay of parathyroid hormone. The problem of dissociation of bioactivity and immunoactivity, well recognized for other polypeptide hormones, is exaggerated in the case of parathyroid hormone by the disproportionately long half-time in the circulation of the immunoreactive fragments. A new method of assaying the biological activity of parathyroid hormone in plasma has been developed, based on the cytochemical methods which have yielded highly sensitive bioassays of other polypeptide hormones. It depends on the stimulation of glucose 6-phosphate dehydrogenase activity in the distal convoluted tubules of segments of guinea-pig kidney maintained in vitro, and measured by microdensitometry. The limit of sensitivity of the assay is 5 fh/ml (bPTH); the index of precision is 0.09 +/- 0.04 (mean +/- SEM; n = 11).

Despite the high circulating levels of immunoreactive PTH in patients with pseudohypoparathyroidism type I (PSPI) the levels of bioactive PTH (bioPTH) have been found to be close to the normal range. To elucidate this dissociation, we have studied the recovery of the biological activity of bovine PTH added to the plasma of patients with either PSPI, or with hypoparathyroidism (PTX), primary hyperparathyroidism (HPT) or of normal subjects. In PSPI (n = 10) the recovery of biological activity was 5.6% +/- 3.6 (mean +/- SEM) whereas in PTX (n = 7), in HPT (n = 4) and in normal subjects (n = 8) it was 79% +/- 5, 75% +/- 9 and 68% +/- 4, respectively. In another PSPI patient, who had undergone total parathyroidectomy, bioPTH was undetectable but the recovery from the plasma of added PTH was 84%. Thus we have found inhibition of PTH bioactivity by plasma of PSPI patients which was absent after parathyroidectomy.

Although many studies have attempted to elucidate how cytotoxic T (Tc) lymphocytes cause the death of target cells, the mechanism is still controversial. In the present study the effect on the integrity of the lysosomes of the target cell has been investigated. We show here that the specific recognition and attachment of cloned type A influenza-specific Tc cells to A/X31 influenza virus-infected target cells caused rapid change in the amount of lysosomal naphthylamidase activity that was bound within the lysosomes, indicating that the lysosomal membranes in the target cells had been totally labilized. Target cells infected with type B influenza virus served as controls. We therefore suggest that the viral specificity of Tc lymphocytes allows for recognition and intimate membrane contact with suitably infected targets. This intimate contact induces sufficient perturbation of the target cell plasma membrane so as to cause total labilization of the target cell lysosomes which could account for intracellular lysis.

It has been shown previously that both thyrotrophin (TSH), and also immunoglobulins (Ig) derived from patients with goitrous Graves' disease, stimulate DNA-synthesis in guinea-pig thyroid tissue maintained in vitro. Here we describe the use of the same in vitro system and methods of quantitative cytochemistry to test the effect of these substances on the generation of NADPH, which is another indicator of the potential for growth. As could be predicted by its trophic action, TSH stimulated the generation of NADPH by glucose 6-phosphate dehydrogenase. The Ig-fraction from normal subjects depressed this activity. The Ig-fraction from Graves' disease patients with goitres stimulated the generation of NADPH, whereas the Ig from patients with Graves' disease but with minimal enlargement of the thyroid gland behaved like normal Ig. A similar lack of stimulation was found with Ig from patients with Pendred's syndrome, other dyshormonogenetic goitres, and autonomous single adenomas. In all specimens tested, there was good correlation between the amount of DNA-synthesis, measured by Feulgen cytophotometry, and the activity of glucose 6-phosphate dehydrogenase activity that generated NADPH. These results support the concept that there is a distinct type of autoantibody that influences thyroid growth.

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.

In assessing the possible efficacy of drugs for the treatment of osteoarthritis (OA), it may be helpful to have a model, in animals, of the early development of the disease prior to the expression of secondary phenomena. It is also necessary that such effects are quantifiable. To this end, the natural development of OA in the STR/ORT mouse has been investigated. It has been shown that very early events in the development of the disease are disturbances in the activity of chondrocytic glucose 6-phosphate dehydrogenase, the initial step in the pentose-phosphate pathway, and in the orientation of the proteoglycans of the matrix of the articular cartilage. The study has been done by reference to the effect of diclofenac sodium, which previously has been reported to retard the destruction of articular cartilage. The results appear to indicate that these markers may provide quantitative measures for assessing potential therapeutic agents.

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.

A new method of measuring vasopressin activity is described. It depends on the finding that the Na+-K+-ATPase activity, measured cytochemically, in the thick ascending limb of the loop of Henle in rat renal tissue maintained in vitro, responded to increasing concentrations of synthetic arginine vasopressin in a log-dose related fashion. The limit of sensitivity was 0.002 pg/ml (2 x 10(-15) mol/l). The dose-responses were reproducible; the inter-assay coefficient of variation was 6.4% at a vasopressin concentration of 0.02 pg/ml. Normal plasma stimulated this Na+-K+-ATPase activity, the stimulation being reduced by 98% when the plasma had been treated with an antiserum specific for vasopressin. Measured in this system, the circulating levels of plasma vasopressin, in healthy adults after 18h dehydration, was 4.0 +/- 0.3 pg/ml (mean +/- SEM; n = 4) and fell to 0.6 +/- 0.1 pg/ml following a water load. Absolute plasma vasopressin values obtained by the cytochemical bioassay were comparable to those measured by radioimmunoassay (r = +0.97, p less than 0.001).

The original lead-trapping method for demonstrating Na+--K+-ATPase activity was discredited because of the effect that lead ions can have on the substrate and on the enzyme. Current methods, that measure this activity by the related K+-dependent phosphatase activity, do not appear to measure activity that is known, from microchemistry, to occur in proximal convoluted tubules. The disadvantages of using lead appear to have been overcome by the use of a new reagent in which the lead is complexed with ammonium citrate ions; phosphate, liberated enzymatically, successfully competes with these ions. The activities of total ATPase and of the ouabain sensitive Na+--K+-ATPase have been measured in three regions of the nephron in the guinea-pig and in the rat. The relative activities found, by this method, in the different regions of the latter, appear to be comparable with results found by others, using microchemical methods applied to isolated regions of the nephron.

The activity of pentose-shunt dehydrogenases is very low in periosteal cells of normal rat metatarsals, but increases one day post-fracture and rises linearly over the next two days. By four days post-fracture, the distribution of this activity along the bone shows two centres of high activity: the first in the region of proliferation to form callus and the second at the site where new bone is first seen, one day later. The high rate of generation of NADPH would be expected to reduce glutathione; reduced glutathione has been shown to inhibit alkaline phosphatase activity in these cells.

If gamma-carboxylation, by the vitamin K1 - cycle, of glutamate residues of bone-matrix peptides is essential for the formation of bone, the circulating levels of this vitamin might indicate the potential efficiency of this process. Methods involving HPLC with electrochemical detection have very recently been developed for assaying the low levels of vitamin K1 that occur in normal plasma. Using such methods, we found that the circulating levels of vitamin K1 in osteoporotic patients (who had sustained either spinal crush-fractures or fractures of the neck of the femur) were significantly lower than those of age-matched control subjects.

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.

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.

Reducing equivalents derived from the tissue re-oxidation of NADPH (NADPH-diaphorase) have been implicated in the peroxidation that is involved in the organification of iodine in the production of thyroid hormones. Immunoglobulin (Ig) fractions from patients with thyroid diseases and from normal controls, in a standard dose of 125 micrograms/ml and 0.3 microunits/ml thyrotrophin (TSH) were incubated with segments of guinea-pig thyroid gland maintained in vitro. A quantitative cytochemical study was made on how these fractions influenced the enzyme activity. A good correlation was found between the ability of such Ig fractions to stimulate the NADPH-diaphorase activity and (1) the degree of hyperthyroidism in the patients and (2) the amount of T3 secreted by the thyroid segments in vitro.

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.

Microdensitometry, or microspectrophotometry, is the measurement of the concentration or mass of a chromophore in microscopically defined regions, and is governed by well-established laws of physics. Initially it proved of value in Feulgen cytophotometry of the relative amounts of DNA in individual nuclei of isolated cells. It has now achieved wide applicability to the measurement of cellular biochemical activity by means of stoichiometric chromogenic reactions. The validity of some of these measurements has been confirmed by comparative biochemical and microdensitometric assays. Thus microdensitometry, even of heterogeneously distributed chromophores, can be precise, provided that the technique is operated with due regard to its limitations within the laws of physics. The potential errors include: variation in thickness of tissue sections (path-length); scatter; glare; diffraction; occlusion of light by optically dense particles; and the inhomogeneity error. However, under correct conditions for the cytochemical reactions and for operating the microdensitometer, these potential errors become small or negligible. Thus the highly sensitive cytochemical bioassay of thyrotropin exemplifies the precision that can be achieved by controlled use of microdensitometry.

Myosin form birefringence has been studied in cryostat sections of left ventricular myocardium from the dog and human. The muscle in such sections has been shown to demonstrate the sliding filament phenomenon. The sarcomere length of canine myocardium agreed with that found in comparable electron micrographs. Unexpectedly, it was found that glycerol, normally used as an inert and optically ideal mountant, caused profound change in myosin birefringence. This apparently invalidates results obtained with this mountant. The absolute birefringence found in these sections, whether mounted in glycerol or in an ATP-calcium buffer, corresponded to values found by other workers with skeletal muscle and isolated myosin. However, the birefringent properties (optical path difference: o.p.d.) of well functioning muscle was found to be low, the o.p.d. increasing when exposed to ATP and calcium. Poorly functioning muscle could be distinguished from well functioning muscle on the basis of its higher 'in air' o.p.d. This difference correlated well with physiological assessments of myocardial function or with clinical assessments of cardiac failure. Evidence is presented indicating that changes in apparent birefringence, caused by ATP-calcium or by anoxia, are due to altered orientation of the myosin micelles and can be inhibited by agents that inhibit myosin ATPase activity.

The skeleton's architecture is matched to the changing loads to which it is subjected because mechanical loading directly or indirectly influences the activity of cell populations to deposit, maintain, or remove bone tissue as appropriate. This responsiveness to load bearing presupposes that certain cells are sensitive to load itself or to its consequences within the tissue. The nature of this effect and the cells concerned have not yet been determined. In this series of experiments, bones were exposed in vivo to a single short period of dynamic loading, which if repeated daily had been shown to result in increased new bone formation. There was an increase in the activity of glucose 6-phosphate dehydrogenase (G6PD) in the periosteal cells adjacent to the bone surface 6 min after this single period of loading. This increase was proportional to the strain magnitude in the bone tissue in the immediate vicinity of the cells. In osteocytes, although the G6PD activity in each individual cell was unchanged by loading, the number of cells displaying activity was increased. This increase was also directly proportional to the applied strain in that area of the cortex (52% compared with 26% active osteocytes at a strain of 0.002). Activation of G6PD was unaccompanied by any equivalent changes in the activities of either glyceraldehyde 3-phosphate dehydrogenase (GA3PD) or lactate dehydrogenase (LDH). This finding is consistent with loading increasing the activity of the oxidative part of the pentose monophosphate shunt pathway. It is also consistent with stimulation of a synthetic process, such as the production of RNA from ribose 5-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

The apparent paradox of aerobic glycolysis has been investigated in bone and in cartilage. A new cytochemical procedure for hydroxyacyl dehydrogenase (HOAD) activity showed that the maximal activity of this enzyme in both tissues was equivalent to the maximal activity of glyceraldehyde 3-phosphate dehydrogenase (GAPD). The sum of these activities gave a measure of the maximum amount of acetyl-coenzyme A that could be produced. In these tissues, but not in liver which does not exhibit aerobic glycolysis, this summed value exceeded the maximal activity of succinate dehydrogenase (SDH). Consequently, it suggested that where fatty acid oxidation is sufficient to supply all the acetyl-coenzyme A required for the Krebs' cycle, that derived from fatty acid oxidation may inhibit pyruvate dehydrogenase causing accumulation of pyruvate which must be converted to lactate if pentose-shunt activity is to be maintained.