Variations in site and levels of expression of chondrocyte nucleotide pyrophosphohydrolase with aging.

The aim of this study was to identify changes in cartilage intermediate layer protein/nucleotide pyrophosphohydrolase (CILP/NTPPH) expression in articular cartilage during aging. Adult (3-4 years old) and young (7-10 days old) porcine articular hyaline cartilage and fibrocartilage were studied by Northern blot analysis, in situ hybridization, and immunohistochemistry using a complementary DNA (cDNA) probe encoding porcine CILP/NTPPH and antibody to a synthetic peptide corresponding to a CILP/NTPPH sequence. Northern blot analysis of chondrocytes showed lower expression of CILP/NTPPH messenger RNA (mRNA) in young cartilage than in adult cartilage. In adult cartilage, extracellular matrix from the surface to the middeep zone was immunoreactive for CILP/NTPPH, especially in the pericellular matrix surrounding the middeep zone chondrocytes. In young cartilage, chondrocytes were moderately immunoreactive for CILP/NTPPH throughout all zones except the calcified zone. The matrix of young cartilage was negative except in the superficial zone. In young cartilage, CILP/NTPPH mRNA expression was undetectable. In adult cartilage, chondrocytes showed strong mRNA expression for CILP/NTPPH throughout middeep zones. Protein and mRNA signals were not detectable below the tidemark. CILP/NTPPH secretion into matrix around chondrocytes increases with aging. In this extracellular site it may generate inorganic pyrophosphate and contribute to age-related calcium pyrophosphate dihydrate crystal deposition disease.

Breakfasts high in monoglyceride or triglyceride: no differential effect on appetite or energy intake.

OBJECTIVE: To test whether isoenergetic isoenergetically-dense doses of dietary 1-monoglyceride or triglyceride differentially influence appetite and meal-to-meal energy intake in man. DESIGN: Six men and six women were each studied twice in a 2 d protocol. On day 1 (maintenance day) they were fed a medium fat (MF) maintenance diet (MF: 40% fat, 47% carbohydrate and 13% protein by energy) calculated at 1.6 x resting metabolic rate (RMR). On day 2 (manipulation day) at 08.30 h subjects consumed a high-fat breakfast designed to contain 80-85% of RMR, composition 10% protein, 56% fat and 34% carbohydrate by energy, with 65% of energy for fat derived as either 1-monoglyceride or triglyceride. Food and energy intake were monitored at lunch (given at 12.30pm) and throughout the remainder of the day. During this time subjects had ad libitum access to isoenergetic, isoenergetically dense MF (40:47:13) foods (550kJ/100g), until 22.30pm). Subjective hunger and satiety were tracked hourly, during waking hours. RESULTS: There was no significant effect of fat type on food or energy intake at lunch or during the ad libitum period. There was no diet effect on subjective hunger (F(1,10)0.00; P= 0.975) in the inter-meal periods of morning or afternoon, nor during the whole day. Subjects found both diets to be similarly pleasant (F(1,61)0.84;P= 0.364). Men and women responded similarly, except that men ate more on all occasions than women. CONCLUSIONS: This study suggests that when a large dose of l-monoglyceride or triglyceride is incorporated into a breakfast meal, it behaves in a manner that is very similar to triglyceride in terms of the effects on hunger, appetite or feeding behaviour.

Molecular cloning and expression of a porcine chondrocyte nucleotide pyrophosphohydrolase.

The porcine 127-kDa nucleotide pyrophosphohydrolase (NTPPHase) had been previously purified from the conditioned culture media of porcine articular cartilage. Protein sequencing of an internal 61-kDa proteolytic fragment of NTPPHase (61-kDa NTPPHase) determined the 26 N-terminal amino acids. This sequence was used to amplify a DNA fragment, which was used as a probe to clone the gene encoding the 61-kDa NTPPHase from a porcine chondrocyte cDNA library. DNA sequence analysis showed the cDNA insert to be 2509 bp, corresponding to a predicted open reading frame (ORF) encoding 599 amino acids. The 26 N-terminal amino acids of the 61-kDa NTPPHase were located within the ORF immediately downstream of a putative protease recognition region, RRKRR. This is consistent with this cDNA insert representing an internal proteolytic fragment of the full length 127-kDa NTPPHase. BLAST and FASTA analysis confirmed that the deduced amino acid sequence of 61-kDa NTPPHase was unique and did not possess a high degree of homology to sequence in the non-redundant protein and nucleotide databases. Proteins that possess limited homology (< 17%) with the 61-kDa NTTPPHase include several prokaryotic and eukaryotic ATP pyrophosphate-lyases (adenylate cyclase). Northern blot analysis of porcine chondrocyte RNA showed that the DNA encoding the 61-kDa NTPPHase hybridized to a single 4.0-kb RNA transcript. This DNA probe also hybridized to a single species of human chondrocyte RNA. Expression of a 61-kDa protein was detected by coupled in-vitro transcription/translation. Western blot analysis of this in-vitro transcription/translation reaction detected a 61-kDa protein, using an antibody raised against the peptide sequence that was originally used to clone the 61-kDa NTPPHase. These data indicate the successful in-vitro cloning and expression of the porcine chondrocyte 61-kDa NTPPHase. Future studies that utilize the gene encoding the 61-kDa NTPPHase may allow the characterization of the role of NTPPHase in calcium pyrophosphate dihydrate (CPPD) crystal deposition disease.

Triple crystal disease: monosodium urate monohydrate, calcium pyrophosphate dihydrate, and basic calcium phosphate in a single joint.

A 49 year old man is described with a polyarticular arthritis. Synovial fluid aspirated from the knee joint showed monosodium urate monohydrate and calcium pyrophosphate dihydrate by polarised light microscopy. Additionally, diphosphonate binding and scanning electron microscopy with energy dispersive analysis showed that basic calcium phosphate crystals were also present. This appears to be the first report of three crystals occurring simultaneously in a single joint.

Arthritis associated with calcium oxalate crystals in an anephric patient treated with peritoneal dialysis.

We report a case of calcium oxalate arthropathy in a woman undergoing intermittent peritoneal dialysis who was not receiving pharmacologic doses of ascorbic acid. She developed acute arthritis, with calcium oxalate crystals in Heberden's and Bouchard's nodes, a phenomenon previously described in gout. Intermittent peritoneal dialysis may be less efficient than hemodialysis in clearing oxalate, and physicians should now consider calcium oxalate-associated arthritis in patients undergoing peritoneal dialysis who are not receiving large doses of ascorbic acid.

Inorganic pyrophosphate metabolism in arthritis.

Once thought of as a biosynthetic waste product, over the last 2 decades PPi has become understood as an entity with a variety of biologic roles (see Table 1). Documented roles include participation in intracellular Ca++ traffic, mediation of nucleotide and iron transport, storage of molecules in cellular granules, modification of enzyme function, and modulation of mineralization. Much has been established regarding plasma, urine, and synovial fluid levels (see Fig. 1) and urinary excretion in health and disease. Derangements in intracellular PPi content of skin fibroblasts have been noted in patients with CPPD deposition arthropathy (see Table 2). Mechanisms by which elevated PPi concentration develops in synovial fluid from joints with CPPD deposition and related arthropathies have come under scrutiny. The chondrocyte is now recognized as the probable cellular source of intra-articular extracellular PPi (see Figs. 3 and 4). Special attention has been focused on two basic pathways by which chondrocytes could generate extracellular PPi (see Fig. 2). In the first mechanism, chondrocytes demonstrate a set of ectoenzymes which could work in concert to directly produce extracellular PPi. The second pathway involves the major reactions by which PPi is formed within the cell and how intracellular PPi thus formed could be transported into the extracellular space. Much future research is needed regarding these two pathways and their relative importance in the pathogenesis of CPPD crystal deposition and related arthropathies.

Identification of collagen subtypes in synovial fluid sediments from arthritic patients.

Collagen fibers in synovial fluid sediment were described a decade ago. Since then, tissue-specific collagen molecules (types) have been characterized. Techniques were devised to identify the collagen types in joint fluid sediment. Collagens were found in 12 of 17 pellets prepared from fluid aspirates from 17 knee joints of patients with various forms of arthritis. Collagen types I and III and polypeptide chains A and B (basement membrane collagen) were specifically identified in four of seven fluids from patients with active systemic lupus erythematosus (SLE) and in a single fluid from a patient with severe septic arthritis. This "collagen profile" was identical to that of rheumatoid synovium. Type II collagen, characteristic of hyaline articular cartilage, was found in two of six fluids from osteoarthritic joints. The presence of sufficient collagen (about 5 micrograms) to permit typing was correlated with roentgenographic evidence of joint space narrowing; the presence of the "synovial" collagen profile was correlated with decreased joint fluid pH.