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In the previous communication we described a histochemical method for measuring soluble guanylate cyclase (sGC) activity in sections of rat liver. In theory, this method could be used to assess nitric oxide synthase (NOS) activity by the increased sGC activity induced by the additional presence of the substrates for NOS activity. We found that this was correct provided that the concentration of the colloid stabilizer in the reaction medium was decreased to just below the concentration required to fully stabilize the guanylate cyclase activity in the sections. This was related to the fact that the site of NOS activity was different from that of the sGC activity in the hepatocytes, so that the NO generated had to diffuse from the Kupffer cells to the hepatocytes as could occur only in partially unstabilized sections. Optimal concentrations of arginine and of NADPH have been determined for demonstrating NOS activity; the increased reaction was shown to be largely inhibited by methyl-arginine.

Guanylate cyclase liberates pyrophosphate from guanosine triphosphate (GTP). In studies published previously, this phosphate is trapped by lead ions even though it is known that free lead ions inactivate a considerable proportion of this enzymatic activity. To overcome the damaging effects of fixation, this study used fresh cryostat sections stabilized with a sufficient concentration of a collagen-derived polypeptide to ensure no measurable loss of guanylate cyclase activity. To avoid the damaging influence of free lead ions, we used a hidden metal capture reagent, i.e., a complex of lead ammonium citrate/acetate that does not react with GTP but which rapidly forms a precipitate with the pyrophosphate liberated by the enzyme. The lead precipitate is then converted into the colored sulfide which is measured in individual cells by microdensitometry. This system was used to measure guanylate cyclase activity in individual cells in unfixed sections of rat liver.

The immunological induction of arthritis in the knee of the rabbit is well established as a model for human rheumatoid arthritis. It has the special advantage of allowing the development of the condition, and the effect of disease-modifying agents, to be followed. Attention has been focussed on the activity of glucose 6-phosphate dehydrogenase in the synovial lining cells since the fourfold elevation of this activity was shown to be fundamental in the human condition. An equal elevation of this activity has now been demonstrated in the rabbit model. Furthermore, it has been shown that the oral administration of menadione decreases this activity towards normality with a concomitant decrease in the degree of inflammation.

The ultraviolet absorbing components of human cartilage have been measured by microspectrophotometry. The characteristics of the chondrocytes appeared to be identical, irrespective of the pathology. However the matrix of osteoarthritis cartilage contained components that absorbed maximally in the region of 270 to 250 nm; such components were not found in the matrix of cartilage of non-arthritic joints. Substances that absorb maximally in this region of the ultraviolet could generate free radicals.

1. Through the vitamin K1 cycle, phylloquinone is now known to play an active role, not only in relation to prothrombin, but also in the synthesis of bone peptides. 2. The recent development of a sensitive method allowed the demonstration of a deficit of vitamin K1 in the circulation of osteoporotic subjects. 3. Vitamin K2, namely the menaquinones of various chain-lengths, has been shown by others to be more effective than vitamin K1 in the curative rat bioassay. 4. Earlier reports had shown that the concentration of menaquinones in human liver may exceed that of vitamin K1. But previous methods were too insensitive for testing the normal circulating levels of menaquinones in the human. 5. The new sensitive method has now been applied to measuring the circulating levels of vitamin K1 and of two of the menaquinones, namely menaquinone-7 and menaquinone-8. 6. In normal individuals, the circulating levels of vitamin K1 were the same, irrespective of age. 7. In young normal subjects, the combined levels of menaquinone-7 and menaquinone-8 were at least the same as the level of vitamin K1. In elderly normal subjects, there was a marked deficit of menaquinone-8.

There has been little evidence to indicate that arginine is the natural substrate for generating nitric oxide synthase (NOS) activity. It is now shown that carnosine, which is widely distributed in tissues, is likely to be the true substrate. In tissue sections it gives a stronger NOS reaction than does arginine.

Vitamin K1 functions in the conversion of glutamate residues, present in certain bone peptides, into the putatively active gamma-carboxyglutamate form. We have shown previously that the circulating levels of vitamin K1 are depressed in osteoporotic patients. However, it is known that menaquinones (vitamin K2:MK) may be more effective than vitamin K1 in this conversion of the inactive to active form of glutamate residues. A procedure for measuring such menaquinones has now demonstrated a marked deficiency of MK-7 and MK-8 in patients with osteoporotic fractures. It is suggested that estimates of circulating levels of K1, MK-7, and MK-8 might provide a biochemical risk marker of osteoporotic fractures.

The previous quantitative histochemical method for measuring nitric oxide synthase (NOS) activity in tissue sections involved the loss of about 15 per cent of the NOS, presumably from the section into the reaction medium. Two changes are now described. The first is concerned with the preparation in the laboratory of the active reagent, lead ammonium citrate/acetate (LACA). The second change involves an improvement of the procedure for measuring NOS activity. The new method appears to retain all the measurable NOS activity inside the section.

Summary Living cells are in an unstable state, responding often rapidly to gentle physiological stimuli. To study them either after the slow fixation and dehydration required for electron microscopy or by homogenizing the cells ensures that the resulting cells or their components are virtually unable to demonstrate the effects of normal trauma or of low concentrations of toxic substances or hormones. Multiphase chemistry has been designed to allow the investigation of cellular biochemistry and biophysics without such disturbances. This has required the development of a special form of quantitative cellular biochemistry. Where it has been possible to compare the results with conventional methods, there has been good agreement. But whereas normal biochemical procedures require 10 6 cells for each sample, multiphase chemistry selects 20 cells even in a complex tissue. The advantages of this form of investigation are shown in a number of studies; on problems that arose with venous transplants; in cardiac surgery; and for the very sensitive micro-bioassays (10 −12 g/ml) of polypeptide hormones.

The normal amount of DNA in human diploid nuclei was determined by the use of the Feulgen reaction measured by microdensitometry. The DNA-content of nuclei in normal human articular cartilage was determined in nuclei of zones 3 and 4 of cartilage of the femoral head removed from osteoporotic fractured necks of femur. Analysis of the results indicated that a degree of synthesis of DNA occurred even in these zones of very elderly persons. Results on these zones in the articular cartilage of osteoarthritic joints indicated that different populations occurred. In some there was DNA-synthesis related to tetraploidy; in others, the DNA was very stable to acid hydrolysis with no sign of biosynthetic activity; in the last group, which contained erosions of the superficial zones, the DNA was unstable to hydrolysis.

The biochemical and quantitative cytochemical assays of the activity of uridine diphosphoglucose dehydrogenase (UDPG-D) have produced perplexing results. It is now shown that the perplexity may be due to the possibility that the coenzyme (NAD) required for UDPG-D activity, may be acting as a substrate for a second dehydrogenase, namely xanthine dehydrogenase, which may utilize NAD as its substrate. The activity of UDPG-D can be distinguished selectively by the pH of its optimal activity and by decreasing the concentration of the coenzyme used in the assay.

A cytochemical section-bioassay of gastrin-like activity is described in which such activity in plasma is assayed by its stimulation of carbonic anhydrase activity in serial, 18 micrometers-thick, sections of suitably prepared gastric fundus of the guinea-pig. The index of precision was 0.1 +/- 0.05 (mean +/- SEM; n=8). Fiducial limits were 75-134%. Intra-assay variation was +/- 6.4% (n=4); inter-assay variation was +/- 16.3% (n=3). The mean gastrin-like activity in the plasma of 15 fasting normal subjects was 5.1 +/- 0.49 x 10(-12)M (range 1.4-18.2 x 10(-12)M).

5'-Nucleotidase (EC 3.1.3.5), a plasma membrane-bound enzyme, has been assayed in unfixed tissue sections of human synovium, activity being measured by scanning and integrating microdensitometry. Activity was markedly increased in the lining cells of the rheumatoid synovial membranes.

A microspectrophotometric method for assaying cytochrome P -450 in fresh 24 μm unfixed cryostat sections of rat liver has been developed. When used to assay this cytochrome in sections of microsomal preparations it has yielded results equivalent to those obtained by the conventional spectrophotometric assay of the same preparations. Random measurements made throughout sections of liver have given mean values for cytochrome P -450 concentrations which are twice those measured in microsomes prepared from the livers of the same animals (not corrected for the yield in the homogenate). Measurements of the cytochrome P -450 content of liver cells by the microspectrophotometric method show that in liver from male Wistar rats, cells nearer to the central veins contain up to twice as much cytochrome P -450 as those nearer to the portal tract (mean cell concentrations of 26.4 (±4.4) μmol/l and 17.5 (±3.0) μmol/l respectively). In the livers from similar rats, killed at the same time, but which had received 1 mg/ml sodium phenobarbitone in their drinking water for one week, the cells near the central vein contained up to five times as much cytochrome P -450 as those near the portal tract (mean cell concentrations of 77.3 (±25.0) μmol/l and 28.3 (±9.6) μmol/l respectively). The results show a selective increase in cytochrome P -450 content by the cells in the centrilobular region after treatment with sodium phenobarbitone and a smaller increase by some of the cells in the periportal region.

The load-carrying capacity of the skeleton is achieved and maintained as the result of a continued functional stimulus to the cell populations responsible for bone remodeling. Although some bone cells have been assumed to be influenced by the load-induced changes in strain throughout the matrix, no evidence is available to indicate which cells are susceptible to such strain change or how such transient events provide a sustained influence on cell behaviour. In the present study, we showed that a short period of dynamic loading in vivo affects the orientation of proteoglycan within bone tissue. This reorientation declines only slowly, thus providing a persistent record of the tissue's recent strain history. Such a record has the ability not only to "capture" strain transients but also to "update" and "average" them. In this way, the bone cells could be presented with a sustained and coherent stimulus directly related to dynamic strain transients. These transients are the tissue's principal function variable.

On the basis of studies with bone-seeking isotopes, it is generally believed that the metabolic activity of osteoblasts of the trabecular bone is greater than that of the osteoblasts of the cortex. This implies that the oxidative activity, which provides the energy for biosyntheses, will also be greater in the former than in the latter. In the present study, direct measurement of the activities of representative enzymes of the major oxidative pathways, as well as of alkaline phosphatase, showed that the reverse pertained. However, owing to the greater cellularity of trabecular bone, the activity per unit mass of bone may be higher in the trabecular bone.

A method for the measurement of cytochrome P-450 in unfixed cryostat sections is described. The sections are incubated for 10 minutes at room temperature in a buffered solution containing polyvinyl alcohol and sodium dithionite. Two incubations are performed on serial sections, one in nitrogen and the other in carbon monoxide. Readings are taken on a Vickers M85 microdensitometer fitted with a high sensitivity photomultiplier amplifier system, the measurements being made on corresponding fields in the serial sections. Subtraction of the nitrogen values from the carbon monoxide values, after allowing for an absorption shift, gives the absolute spectrum of cytochrome P450. The subtraction corrects for the tissue content of other haem-containing proteins. The cytochrome P450 spectrum shows a sharp maximum at 450 nm, and two other minor components absorbing at 444 nm and 458 nm. The content of cytochrome P450 in animals fed with phenobarbitone was 2.4 times greater than in control animals.

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.

SUMMARY Development of a highly sensitive bioassay for ACTH has enabled bioassayable ACTH to be measured in plasma during the course of the insulin hypoglycaemia test for the first time. Comparison with radioimmunoassay (RIA) on the same plasma samples shows that the latter consistently overstimates the true, or biologically active, ACTH level. This overstimate appears to be most marked when ACTH levels are highest and during the fall in plasma levels which occurs following the ACTH peak. This may limit the value of the RIA of ACTH, in some situations.

Differential pulse polarography, following solvent extraction, is used to monitor the clearance of vitamin K 1 (2-methyl-3-phytyl-1,4-naphthoquinone) from human plasma after a 20-mg intravenous injection. The average recovery of vitamin K 1 added to plasma (200–3000 ng ml -1 ) was 72.2%. The coefficient of variation was 3.0% at a concentration of 2.75 μg ml -1 of plasma. Measurements of vitamin K 1 in plasma from patients given an intravenous injection of the vitamin, support the idea that a metabolic cycle involving vitamin K 1 underlies calcification of bone.

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.

SUMMARY 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 fg/ml (bPTH); the index of precision is 0.09 ± 0.04 (mean ± SEM; n= 11).

To examine the reported beneficial effect of zinc in rheumatoid arthritis, rheumatoid synovial tissue has been maintained in vitro in non-proliferative culture with or without zinc sulphate in the culture medium. Alkaline phosphatase activity was measured by microdensitometry of the cytochemical reaction in cryostat sections; the activity in blood vessels was measured separately from that in the supporting tissue below the synovial surface. Zinc enhanced this activity optimally at concentrations of between 10(-5) and 10(-4) mol/l.

A method is described by which a numerical value can be assigned to the amount of DNA-synthesis shown graphically by population-histograms obtained by Feulgen cytophotometry. The index appeared to give a reasonable measure of DNA-synthetic activity in populations having very low, moderate and high mitotic indices and closely followed labelling indices obtained by autoradiography. Thus the advantage of a numerical value is that the DNA-synthetic activities in different populations can be compared for statistical analysis.

Normal myocardial function is dependent on the metabolic balance of a number of electrolytes and hormones. The calcium ion plays a major role in muscle contraction and is rigorously controlled within narrow limits. Open-heart surgery imposes metabolic disturbances on both electrolytes and hormones, especially ionized calcium. Normally, ionized calcium levels are controlled by parathyroid hormone with a negative feedback from the ionized calcium controlling the system, but the results from this study suggest that during open-heart procedures, ionized calcium does not impose its normal negative feedback on bioactive parathyroid hormone secretion. The low blood pH levels that occurred during the operative conditions of the patients studied and the level of hypothermia imposed on the circulating blood during cardiopulmonary bypass appeared to influence the control of parathyroid hormone secretion, causing high levels of hormone to be secreted during this period.

DNA synthesis has been measured both by Feulgen cytophotometry, quantified by the DNA synthesis index, and by tritiated thymidine autoradiography, quantified by the labelling index. In the early acute inflammation resulting from the intra-articular challenge of ovalbumin in sensitised rabbits both indices rose considerably, so that at least 1 in 10 synoviocytes was heavily labelled 3 days after challenge. The results are compatible with the concept that even such apparently differentiated synoviocytes are capable of cell division.

Circulating levels of parathyroid hormone (PTH) in six patients with pseudohypoparathyroidism type I (PSPI) have been measured by two immunoassays and by cytochemical bioassay and compared with measurements in normal subjects and patients with clinically defined hyper- and hypoparathyroidism. In all PSPI patients, the levels of immunoreactive PTH were in the hyperparathyroid range, whereas the bioactive levels were either in the normal or close to the normal range. In one patient from whom the dihydrotachysterol therapy was withdrawn, both immunoreactive and bioactive PTH concentrations increased. The finding that the PTH measured by RIA in these PSPI patients may have reduced biological activity may explain some of the clinical findings of hypoparathyroidism in this syndrome.

A method for making squash preparations of plant root tip cells is described. This entails the use of an inert preserving substance, polyvinyl alcohol. The advantages of the method are the preservation of the cytoplasmic bodies, including the mitochondria ; the retention of fat droplets inside the cells; the fact that the nucleus remains optically homogeneous; and the fixing of free aldehyde groups and of diffusible substances in the cytoplasm. It is a relatively rapid and simple technique, and the cells produced by it resemble closely those produced by the method for making living squash preparations of such cells.

The DNA content of individual cells in the root tip of Vicia faba has been measured by a cytochemical method. Roots were grown under standard conditions and divided into segments 0–2, 2–4, 4–8 and 12–20 mm behind the root cap, as in the biochemical experiments of Holmes et al. [7]. From the measurements on meristematic cells (0–2 mm) synthesis curves for DNA (from the 2C to the 4C value) are derived which are in broad agreement with that found earlier by the P 32 autoradiographic method [8]. Our results indicate that DNA synthesis occurs approximately during the middle third of interphase. Our results also indicate that there is negligible change in the total amount of DNA during the division of a cell. Measurements of the average DNA content per cell in each segment show an increase with distance from the root tip similar to that found by the biochemical method [7]. Cells in the elongation zone of the root appear to synthesise DNA from the 2C to the 4C value and from the 4C to the 8C value, without subsequent cell division.

A new technique for the quantitative estimation of glycolytic and respiratory enzyme activity in intact and unfixed bone marrow and peripheral blood cells is described. The method gives a two- to three-fold increase in demonstrable enzyme activity per cell compared with existing techniques using fixed marrow film preparations, and is particularly applicable to the study of relatively fragile cells such as the leukaemic lymphoblast.

Summary The dysfunction of lysosomal membranes has been investigated in a model system of inflammation in which human skin, maintained in vitro in non-proliferative maintenance culture, is subjected to the effect of histamine. The effect of histamine is time and dose related; 10 -7M histamine acting for 5–10 min. has been shown to have a significant effect on the permeability of lysosomal membranes in the epidermis. A comparison has been made between aspirin, salicylate and 4-acetamido-2-acetoxy-benzoate (benorylate) prepared by esterification of N-acetyl-p-aminophenol (paracetamol) with acetyl salicylic acid (aspirin). It has been shown that, in specimens from some subjects, aspirin alone has an activating effect whereas it has a stabilizing effect in other specimens. But in all cases aspirin retarded the effect of histamine on the lysosomal membranes. Benorylate had only a stabilizing effect and markedly inhibited the labilizing influence of histamine. This was not due to the properties of aspirin and paracetamol acting alone, but was a property of the benorylate molecule. It is suggested that the stabilizing effect may be related to the lipophilia of this molecule and its direct influence on the lysosomal membrane. Such studies, on the response of lysosomes in intact tissue, under the influence of a chemical mediator of inflammation, may clarify the controversial question of the effect of aspirin when studied on isolated lysosomes. The stabilizing influence of benorylate on lysosomal membranes found in the model experiments on human skin has been confirmed in studies on human rheumatoid synovial lining cells.

Summary This study has confirmed that the lysosomal membranes in the synovial lining cells of human rheumatoid synovial tissue are abnormally labile; that the cytoplasmic redox balance, as measured by the proportion of dithiol to disulphide groups, is exceptionally reductive; and that there is an excessive rate of generation of reduced NADP despite little change in its rate of oxidation. These results suggested that the increased reductive conditions in the cytoplasm might contribute to the state of the lysosomes. To test this, human synovial tissue was maintained in vitro , in adult maintenance culture, and hydrogendonating or hydrogen-accepting molecules were added to the culture medium. It was shown that ascorbate acts as a hydrogen-donor. In non-rheumatoid cells it increased the proportion of cytoplasmic sulphydryl groups (which indicates a more reductive redox balance) and also increased the lability of the lysosomal membranes. Hydrogen-accepting molecules, such as dehydroascorbate and particularly menadione, produced the reverse effect. Similar effects occurred in rheumatoid synovial lining cells, in which menadione also diminished the rate of generation of NADPH. It seems likely, therefore, that redox systems in the cytoplasm and in the extra-cellular space, can influence the cytoplasmic dithiol: disulphide balance and the functional state of lysosomal membranes in synovial lining cells. In rheumatoid synovial tissue, menadione diminishes the excessively reductive conditions in these cells and stabilizes lysosomal membranes.