Elevated urinary excretion of free pyridinoline in Friesian horses suggests a breed-specific increase in collagen degradation.

BACKGROUND: Friesian horses are known for their high inbreeding rate resulting in several genetic diseases such as hydrocephaly and dwarfism. This last decade, several studies focused on two other presumed hereditary traits in Friesian horses: megaoesophagus and aortic rupture. The pathogenesis of these diseases remains obscure but an important role of collagen has been hypothesized. The purpose of this study was to examine possible breed-related differences in collagen catabolism. Urinary specimens from Friesian (n = 17, median age 10 years old) and Warmblood horses (n = 17, median age 10 years old) were assessed for mature collagen cross-links, i.e. pyridinoline (PYD) (=hydroxylysylpyridinoline/HP) and deoxypyridinoline (DPD) (lysylpyridinoline /LP). Solid-phase extraction was performed, followed by reversed-phase ion-paired liquid chromatography prior to tandem mass spectrometry (MS/MS) detection. RESULTS: Mean urinary concentrations of free PYD, expressed as fPYD/creatinine ratio, were significantly higher in Friesian horses compared to Warmblood horses (28.5 ± 5.2 versus 22.2 ± 9.6 nmol/mmol, p = 0.02) while mean fDPD/creatinine ratios were similar in both horse breeds (3.0 ± 0.7 versus 4.6 ± 3.7 nmol/mmol, p = 0.09). CONCLUSIONS: Since DPD is considered a specific bone degradation marker and PYD is more widely distributed in connective tissues, the significant elevation in the mean PYD/DPD ratio in Friesian versus Warmblood horses (9.6 ± 1.6 versus 5.7 ± 1.8, p < 0.0001) suggests a soft tissue origin for the increased fPYD levels. Considering that a previous study found no differences in total collagen content between Friesian and Warmblood horses for tendon and aortic tissue, this indicates a higher rate of collagen degradation. The latter might, at least in part, explain the predisposition of Friesians to connective tissue disorders.

[Urinary glycosaminoglycans in patients with non-insulin-dependent diabetes mellitus, collagen diseases and IgA nephropathy].

Urinary levels of glycosaminoglycans (U-GAG) were measured in 72 patients with non-insulin-dependent diabetes mellitus, 12 patients with collagen diseases, 14 patients with IgA nephropathy and 35 healthy subjects as controls to investigate the clinical significance of urinary GAG. The mean urinary GAG levels in diabetics and patients with collagen diseases were 72.4 +/- 36.2 and 147.8 +/- 59.2 mg/g.cr, respectively. These were significantly higher than the level in healthy subjects (46.7 +/- 11.3 mg/g.cr, p < 0.01). The mean urinary GAG level in patients with IgA nephropathy was 56.4 +/- 21.0 mg/g.cr and did not differ from that in healthy subjects. The mean urinary GAG level in 35 normoalbuminuric diabetic patients (U-A1b < 30 mg/g.cr) was 64.4 +/- 25.6 mg/g.cr and was significantly higher than that in healthy subjects (p < 0.01). The mean urinary GAG levels in 24 microalbuminuric patients (30 < U-A1b < or = 300 mg/g.cr) and 13 patients with overt albuminuria (U-Alb > or = 300 mg/g.cr) were 71.4 +/- 30.1 and 95.8 +/- 58.4 mg/g.cr, respectively and were also higher than the level in healthy subjects (both p < 0.01). Urinary GAG levels correlated positively with urinary albumin levels (r = 0.251, p < 0.05) and urinary N-acetyl-beta-D-glucosaminidase activities in diabetics (r = 0.491, p < 0.01). The prevalence of diabetic macroangiopathies in diabetic patients with elevated levels of urinary GAG was significantly higher than that in those with normal levels of urinary GAG (p < 0.05).

[A study of microproteinuria in patients with collagen disease].

To examine the subclinical renal damage in collagen disease, we analyzed the excretion pattern of microproteinuria. We studied 58 collagen disease patients including 25 RA (rheumatoid arthritis) patients, 15 SLE (systemic lupus erythematosus), 5 PSS (progressive systemic sclerosis), 4 MCTD (mixed connective tissue disease), and 9 others. Urinary protein was not detected by urine dipsticks in all patients. Urinary proteins, which were concentrated to 5 mg/ml, were subjected to linear gradient (4-30%) SDS-PAGE and then transferred to nitrocellulose membrane by electrophoretic blotting method. The membrane was stained with Auro Dye and the blotted proteins were identified by enzyme immunoassay using specific antibodies. The percentages of albumin of whole urinary proteins were 27.2 +/- 13.7% in RA and 25.8 +/- 12.6% in PSS, which were significantly lower than that of controls (42.1 +/- 15.3%). However no significant difference in the percentage of urinary albumin was noted between SLE and controls. The percentages of low molecular weight (MW) proteins (proteins having smaller MW than albumin) were higher in RA and PSS. Especially the bands with MW of 25,200 were prominent and these percentages were 11.3 +/- 6.1% in RA and 14.6 +/- 9.1% in PSS, which were significantly higher than controls (5.1 +/- 3.5%). These bands with MW of 25,200 were demonstrated to be free light chains of immunoglobulins by western blotting method. From the above observations, protein excretion patterns in RA or PSS patients were so-called tubular proteinuria, and especially free light chain excretion was increased. We proposed that tubular dysfunction and abnormal production of free light chain might exist frequently in collagen diseases, especially RA and PSS.

[The role of the urinary hydroxyproline level in the screening for collagen diseases]. / Importanza del dosaggio dell'idrossiprolina nelle urine come screening delle malattie del collagene.

Modifications in urinary hydroxyproline concentrations were studied in various collagen degradation diseases. A significant increase in hydroxyprolinuria was found in both men and women with Paget's disease in all age groups considered. Significant increases were found in men with the same pathologies. Rheumatoid arthritis also increased urinary hydroxyproline in both males and females. It therefore seems that bone tissue pathologies are reflected in the metabolism of hydroxyproline as are articular connective tissue pathology strictly related to it.

Albumin to creatinine ratios in early morning samples of urine from healthy persons and patients without clinical proteinuria.

Urinary excretion of albumin was determined in 133 clinically healthy subjects and 236 patients suffering from diabetes mellitus (150 cases), collagen diseases (45 cases), or arterial hypertension (41 cases) in whom there was no clinical or laboratory evidence of renal involvement. Urine albumin was measured by an improved single-radial-immunodiffusion technique and results were expressed as urine albumin to creatinine concentration ratio (mg/g). The geometric mean values in the patients were about 2 to 2.5 times higher than in controls. When considered on an individual basis, at least 40 per cent of the patients had values above the 99th-percentile value for controls. The results suggest that quantitative determination of urinary albumin may be useful for detecting early structural functional alterations of the glomerular filter.

Simplified procedure for the analysis of 3- and 4-hydroxyproline.

A column chromatographic analysis for 3-hydroxyproline (3-Hyp), 4-hydroxyproline (4-Hyp), and gamma-carboxyglutamic acid (Gla) is described. The analyses of urine and plasma were performed with a JLC-6AH amino acid analyzer. A 0.15 M sodium citrate buffer, pH 2.1, was used for elution. Urinary Gla, 3-Hyp, and 4-Hyp were among the seventeen peaks eluted before aspartic acid. Hyp, Gla, glutamine, and asparagine in plasma were separated by elution with 0.2 M sodium citrate buffer, pH 3.25, containing 10% methanol. This single-column procedure achieves the sequential separation and quantitation of Gla, 3-Hyp, and 4-Hyp in urine as well as plasma, and is applicable to the diagnosis of collagen metabolism disorders.

Excretion of glucose-containing oligosaccharides in urines of orthopedic patients.

Urinary neutral oligosaccharides of various connective tissue diseases were studied by gel-filtration through Sephadex G--10 after treatments with cetylpyridinium chloride (CPC), Dowex 50 (H+ form) and Dowex 1 (Cl- form), in succession. Increased excretion of urinary glucose-containing oligosaccharides, specifically glucosylgalactose was observed in most of the patients with chondrosarcoma, rheumatoid arthritis, Werner's syndrome, Rothmund Thomson syndrome and Morquio's disease. However, urinary excretion of neutral oligosaccharides in the patients with osteosarcoma and other tumorous conditions, and some systemic disorders in the connective tissues, examined in the present study, showed almost normal values. It is indicated, therefore, that the activity of glucosidase in insufficient for the glucose-containing oligosaccharides produced from the ground substance(s) in the former type connective tissue diseases.

Radioimmunoassay of main urinary metabolite of prostaglandin F2alpha in patients with diffuse interstitial fibrosing pneumonitis (DIFP) and other respiratory diseases.

Radioimmunoassay of 5alpha, 7alpha-dihydroxy-11-keto-tetra-norprosta-1,16-dioic acid, main urinary metabolite of prostaglandin F2alpha (PGF2alpha-MUM), was performed in patients with various respiratory diseases including diffuse interstitial fibrosing pneumonitis (DIFP, fibrosing alveolitis). Twenty-four hr excretion of PGF2alpha-MUM in patients with primary lung cancer, pulmonary fibrosis secondary to collagen diseases and stationary DIFP was normal. On the other hand, 24 hr excretion of PGF2alpha-MUM in patients with carcinomatous pleuritis was high and that in patients with aggravating DIFP was markedly high. There was no correlation between serum LDH levels and 24 hr excretion of PGF2alpha-MUM.

Increased excretion of urinary glycosaminoglycans in a case of Rothmund Thomson syndrome.

The exercretion of urinary total glycosaminoglycans (GAG) in a case of Rothmund Thomson syndrome associated with osteosarcoma was increased about 2--3 times that of normal control. Since the excretion of urinary total GAG in two cases of osteosarcoma was within normal range, the increased excretion of total GAG might be on account of the metabolic disorder of GAG in Rothmund Thomson syndrome. The prominent fractions obtained by Dowex 1 column chromatography from this syndrome were 0.75 M Fr and 1.0 M Fr, in which the major GAG were indicated to be partially degraded forms of heparan sulfate and chondroitin sulfate, respectively.

The clinical significance of Bence Jones proteinuria.

The detection of monoclonal light chains in the urine by the Bence Jones heat test is a useful adjuvant to the diagnosis of myeloma and other related diseases. The test is particularly helpful when no serum spike is noted. Overall, it is positive in approximately half of all patients with myeloma. In order to assess the accuracy of the test, we reviewed the records of all patients with a positive heat test for Bence Jones proteinuria during a single calendar year. Myeloma accounted for 68%, but patients with amyloidosis, the adult Fanconi syndrome, and others also had positive results. One-fifth of the results were false positive in that urine protein electrophoresis showed no spike and immunoelectrophoresis, no monoclonal protein. This group consisted largely of patients with connective tissue diseases, chronic renal failure, or nonplasmacytic malignancies. We also have seen patients who had monoclonal light chains in their urine but failed to show positive results to the heat test and were thus considered false negative. Although the heat test for Bence Jones proteins is a useful clinical test, one must be aware of both false-positive and false-negative results. Electrophoresis and immunoelectrophoresis of concentrated urine are the methods of choice for detection of a monoclonal light chain in the urine.