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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., (Copyright 2001 John Wiley & Sons, Ltd.)

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., (Copyright 1999 John Wiley & Sons, Ltd.)

Up to nine out of 10 male STR/ORT mice develop osteoarthritis (OA) of the medial tibial cartilage at an early age. This has now been shown to be related to changes in the activity and distribution of monoamine oxidase which is related to the metabolism of catecholamines. Treatment with diclofenac sodium tended to normalize this activity but there was no significant histological improvement. It was therefore postulated that two influences were involved in the development of OA: a cellular and an extracellular factor. The first was improved by diclofenac sodium; the second, namely oedema of the matrix, was improved by tribenoside. In very preliminary studies, feeding the two drugs simultaneously resulted in 7/9 mice having no sign of OA.

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.

New drugs are generally developed against animal models of the human disease. Before they are subjected to clinical trials it might be helpful to be able to test whether they are as effective against the disease in human tissue as they were in animals. It is proposed that this can be achieved by the use of organ maintenance culture of the human diseased tissue, the relevant biochemical parameters being measured by quantitative cytochemistry. In the present studies differences between the effect of indomethacin and of the 'chondroprotective' drug diclofenac sodium, on human osteoarthritic cartilage, have been measured.

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.

Most of the male STR/ORT mice develop osteoarthritis (OA) involving the medial tibial plateau. A peculiarity of two chondroprotective drugs is the presence of a nitrogen atom so that cleavage of the molecule could generate a molecule that might act as an inhibitor of monoamine oxidase (MAO). Direct examination showed abnormal localization of MAO in the potentially osteoarthritic cartilage indicating possible abnormal response to catecholamines. In normal cartilage, the direct effect of excessive concentration of adrenaline caused considerable oedema, as measured by microscopic interferometry. It is therefore suggested that the excess of water found in the matrix of osteoarthritic cartilage may be related to disturbance of the MAO activity.

It was shown previously that the experimentally induced arthritis in the rabbit can be largely nullified by subsequent treatment with menadione (by gavage). It is now shown that menadione epoxide, as is produced in the vitamin K cycle, also exerts a beneficial effect histologically and biochemically. Such treatment decreased both the glucose 6-phosphate dehydrogenase and the 6-phosphogluconolactonase activities in the synovial lining cells of the challenged joints towards values found in the unchallenged joints; it had only equivocal effects on the 6-phosphogluconate dehydrogenase activity. The results indicated that the epoxide might be interfering primarily with the lactonase activity.

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.

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 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 mechanism by which cytotoxic T-lymphocytes (Tc) induce the death of specific target cells is still controversial. We have used quantitative cytochemical methods to distinguish the metabolic activities of the target cells from those of the Tc, even when they are attached to each other. Early events following Tc-P8(15) target cell interaction were first, increased glucose 6-phosphate dehydrogenase activity and second, labilization of the lysosomes within the target cell: these changes could be mimicked, in part, by polyamines and could be inhibited by inhibiting ornithine decarboxylase (ODC) activity. The crucial role of ODC in the chain of events that led to cytolysis in this particular experimental system was shown first, by measuring ODC activity directly and secondly, by the inhibition of cytolysis by the presence of a selective inhibitor of ODC activity.

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.

At least 80% of male STR/ORT mice naturally develop osteoarthritis that predominantly affects the medial tibial cartilage. Overt osteoarthritic changes, as judged by radiological and histological abnormalities, become apparent after 30 weeks of age. Consequently, mice less than 30 weeks of age were used to investigate early changes in the cartilage matrix related to the natural development of osteoarthritis, without the need for experimental intervention to induce this condition. Quantitative Alcian blue staining showed little change in the total amount of proteoglycans in mice of this age. Polarized light microscopy of the birefringence induced by such staining demonstrated a progressive decline in the orientation of the proteoglycans in the medial cartilage of these mice. This decline was not found in CBA mice, which only very rarely develop osteoarthritis of this joint. Such progressive disorganization of the proteoglycans would be likely to permit the increase free water-content characteristic of osteoarthritic cartilage.

The aim of this investigation was to determine whether some metabolic defect might be related to the propensity of osteoporotic femoral necks to fracture acutely. To this end, the activities of two dehydrogenases of the glycolytic Embden-Meyerhof pathway, two of the pentose phosphate pathway, two mitochondrial enzymes, and alkaline phosphatase were measured in the cortical and in the trabecular osteoblasts. Comparison was made with such activities in iliac crest biopsies from patients with these fractures and from the equivalent femoral and iliac crest samples from patients with osteoarthritis of the hip, in biopsies from the iliac crests from seven patients with no bony abnormality, and in specimens from the fracture site of six traumatic fractures. The results showed a highly significant decrease in the activities of the two enzymes of the pentose phosphate pathway (p less than 0.001) in the cortical, but not in the trabecular, osteoblasts in the osteoporotic fractures. This could not be attributed to the trauma of acute fracture since it was not found in traumatic fractures. Other experimental evidence has indicated that a relationship may occur between depressed activity of these enzymes and a retardation of bone formation.

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.

Specimens of human rheumatoid and nonrheumatoid synovial lining were maintained in nonproliferative organ culture for 20 hours. The culture fluids were then assayed for prostaglandin E(2) (PGE(2)), thromboxane B(2) (TXB(2)), and 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)) by specific radioimmunoassay. The presence of each of these substances was confirmed by gas chromatography and mass spectrometry. Rheumatoid tissue produced significantly more of each cyclo-oxygenase product than nonrheumatoid tissue.

Oedema following periods of ischaemic arrest and subsequent reperfusion has been shown experimentally and clinically to affect the functional state of the heart. Tissue water content has been measured in myocardial sections by microscopic interferometry and densitometry, and the results correlated with those obtained by wet and dry weight analysis (r = 0.87; p less than 0.001). Microscopic interferometry also revealed the distribution of the water in the tissue. Experimentally induced ischaemic arrest in isolated rat hearts resulted in predominantly intra-fibrillar oedema, whilst subsequent reperfusion resulted in interfibrillar oedema. Microscopic interferometry facilitates accurate measurement of water content in tissue samples as small as 2 mg wet weight and shows (as conventional wet/dry weight analysis cannot) the distribution of the water in the tissue.

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).

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 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.

Rabbits have been immunized with ovalbumin in Freund's incomplete adjuvant (FIA) followed by the intra-articular injection of ovalbumin, in order to follow the development of inflammation in the synovial lining. The kinetics of cell proliferation have been investigated using tritiated thymidine (3HTdR) autoradiography and Feulgen cytophotometry. Unexpectedly, marked histological changes were found in the synovium, with hyperplasia of synoviocytes, and of the connective-tissue cells of the subintima, being seen as early as 3 days after challenge. Large numbers of inflammatory cells, including many plasma cells, were found in the synovium at between 5 and 11 days. Labelling of synoviocytes and connective-tissue cells reached a maximum at 3 days and declined thereafter, reaching normal levels at 14 days. Three weeks after challenge the synovium was normal in appearance.

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.

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.

The synovial tissue in experimentally induced immune arthritis induced in the rabbit has been used as a model of rheumatoid arthritis to determine which cells may contribute to the growth of this tissue. Tissue from the challenged and from the unchallenged knee joints was taken, after the intra-articular injection of a small amount of tritiated thymidine, from rabbits up to 3 months after the arthritis was induced. DNA synthesis, as a measure of cell proliferative activity, was assessed firstly by measuring the labelling index in autoradiographs of sections of such tissue, and secondly by the DNA synthetic index obtained by Feulgen cytophotometry. These measurements were made separately on synoviocytes, on the structural cells of the stroma, on the cells lining the small blood vessels, and on the infiltrating inflammatory cells. The DNA synthetic activity of the synoviocytes, and of the stromal noninflammatory cells, was maximal between 3 and 7 days after challenge. The activity in the synoviocytes, in particular, remained raised for up to 84 days after the challenge. Thus these cells appear to be capable of contributing to the hyperplasia, but the contribution of other cells, deeper in the stroma, cannot be excluded.

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.