Total Hydroxyproline Content Research Articles

Biochemical effects of formocresol on bovine pulp tissue

Calf pulp tissue was treated with full-strength formocresol, diluted formocresol, or saline for 4 hours. After washing and homogenization, the water extractable supernates were analyzed for total amino acid, carbohydrate, and hydroxyproline content. Additional samples were tested against trypsin, pepsin, collagenase, and hyaluronidase. Other tissue samples treated with 1 5 , 1 10 , and 1 25 dilutions of formocresol were subjected to trypsin and collagenase. The control tissue gave 50 per cent more extractable material, which contained over 300 per cent more total amino acids and hydroxyproline but only slightly more carbohydrate than the treated tissue. Formocresol treatment produced an 80 to 90 per cent reduction in reactivity to trypsin, pepsin, and collagenase but little change from hyaluronidase action. The increase in reactivity of the tissue to enzyme hydrolysis paralleled the increase in dilution of formocresol. These results indicate a profound effect on the protein fraction of pulp exposed to full-strength formocresol.

Glycosaminoglycans and Endochondral Calcification

Present controversy about endochondral calcification and ossification is concerned with changes in glycosaminoglycans. Some authors report a rise in amounts of glycosaminoglycans while others a fall. Cartilage was carefully sliced under microscopic control to provide samples of material from different functional zones of the developing tissues. The following zones were studied histochemically and analyzed for their content of total nitrogen, hydroxyproline, total hexosamines, uronic acid and phosphorus: the resting zone; the zone of proliferating and maturing cells; the calcifying zone, characterized by degenerating hypertrophic cells and early mineral deposition; the ossifying region, where early bone formation takes place. Serial analyses provided evidence that glycosaminoglycans increases before calcification starts. Afterwards, part of the glycosaminoglycan content is removed. This biphasic process appears to occur during the calcification of other tissues too, such as secondary bone and dentine.

The influence of fluocortolone treatment on the collagen content in guinea pig skin.

Specific pathogen free guinea pigs were treated with varying dosis of fluocortolone for 15 days. The treated animals showed the same per cent weight gain as the controls. The total hydroxyproline content of the skin and the hydroxyproline content in different collagen fractions was the same in treated and untreated animals. Thus fluocortolone seems to have no specific effect on the synthesis or the breakdown of collagen in the guinea pig skin. Also the physical development of the animals, kept under defined conditions, failed to show a catabolic effect of of fluorcortolone.

In vitro system for toxicological studies on the development of mammalian limb buds in a chemically defined medium.

In organ culture systems using the Trowell setup, morphogenetic differentiation (which largely mimics the development reached in vivo within 2--3 days) can be obtained in limb buds of mouse embryos during a culture period of 6 days. We succeeded in improving the technique and in achieving good differentiation of the limb buds in a chemically defined culture medium from which all additions of the heterologous serum used in previous studies were omitted. With this technique of using a chemically defined medium, the following results were obtained: (1) A high degree of reproducibility can be obtained in the grade of differentiation if the experimental conditions are standardized and limb buds of the same developmental stage (somite stage) are used. (2) The technique is applicable to species other than mice, such as rats and rabbits, however, the results obtained so far are not so satisfactory as those acquired with mouse limb buds. (3) In an attempt to offer some colloid osmotic pressure, macromolecules like polyvinylpyrrolidone (Periston), polydextrans (Macrodex) or polypeptides (Haemaccel) were added to the culture medium. None of these macromolecules had a beneficial effect on the differentiation of the limb buds in vitro; in the case of Macrodex the differentiation was even impaired. (4) In comparison with the development in a serum-containing medium, the results attained with a chemically defined medium are just as good when judged from microscopical and some biochemical studies (total content of DNA, RNA, protein, and protein-bound hydroxyproline in the explants). The applicability of the test system for the evaluation of embryotoxic effects is discussed.

Aging and Hydroxyproline Content in Human Heart Muscle

Hydroxproline content was chemically determined in 45 human hearts obtained at autopsy, in order to know possible correlations between aging and hydroxyproline content in the heart muscle. There were no significant differences in the mean total hydroxyproline content between the epicardium and the endocardium, between left and right ventricles, nor between young, middle and old age groups. The hydroxyproline content in NaCl-soluble fraction decreased, and that in TCA-soluble fraction and in residue fraction increased with age. There were significant differences in the mean hydroxyproline content in NaCl-soluble fraction between middle and old age groups, and in residue fraction between young and old age groups in the endocardium of the left ventricle. Accordingly, the ratio of the content in NaCl-soluble fraction to that in TCA-soluble fraction decreases with age. This tendency was more evident in the epicardium than in the endocardium on the left ventricle. These results indicate that total hydroxyproline content per 1 mg of lyophilized material does not show any quantitative changes, but does show qualitative changes with age.

Collagen Content of Lungs of Mice after X-Ray Irradiation

To investigate the development of late radiation fibrosis in lungs large single doses of X rays were given to the hemithoraxes of mice. Hydroxyproline, an amino acid specific to collagen, was estimated in both irradiated and shielded lungs up to 48 weeks after exposure. There was a dose dependent atrophy of the irradiated lung and a compensatory hypertrophy of the shielded lung. The hydroxyproline per gram of dry irradiated lung increased between 24 and 36 weeks after 2000-4000 rad with no further change up to 48 weeks. Both total hydroxyproline content and dry weight of irradiated lungs decreased but after 24 weeks the histological appearance was that of collagen deposition after edema and hyperplasia. It is concluded that collagen deposition occurs at a late stage in the reorganization after radiation injury to mouse lung and is therefore probably not the cause of 80-180 day death after exposure of the whole thorax.

Determination of hydroxyproline content in human heart muscle.

Hydroxyproline content was chemically determined on 45 human hearts obtained at autopsy in order to study whether hydroxyproline contents vary in proportion to heart weight or not. There were no significant differences in the mean hydroxyproline content between the epicardium and the endocardium, between the left ventricle and the right ventricle, or among cardiovascular and renal disease group, malignant disease group and miscellaneous disease group. Total hydroxyproline content per mg dry weight of the left ventricle increased proportionally with heart weight. Hydroxyproline content in neutral salt soluble fraction increased more proportionally than that in acid soluble fraction. This tendency was more evident in the endocardium than in the epicardium of the left ventricle. These results seem to be well consistent with those of histometrical estimation of scar tissue in human heart muscle. Hydroxyproline content in acid soluble fraction of the right ventricle was increased proportionally with heart weight.

Effect of Experimental Chronic Renal Insufficiency on Bone Mineral and Collagen Maturation

The effect of chronic renal disease on bone matrix and mineral maturation was evaluated in rats with experimental renal insufficiency of 2-11 wk duration utilizing bromoform-toluene gradient fractionation of bone powder, pulse labeling experiments with (45)Ca and proline-(3)H differential extraction, and X-ray diffraction techniques.Maturation defects in both collagen and mineral ((45)Ca) metabolism were documented as early as 2 wk after the induction of uremia, when total bone calcium, inorganic phosphate, and hydroxyproline content were unchanged. The maturational defect progressed with advancing uremia despite insignificant changes in plasma pH and calcium, and normal bone carbonate levels. Although circulating levels of 25-hydroxycholecalciferol were significantly lower than normal in the uremic animals, pretreatment with either this vitamin D metabolite or vitamin D(3) itself failed to alter the observed changes in skeletal maturation.

Injury and repair in arterial tissue in the rabbit. Analysis of DNA, RNA, hydroxyproline, and lactate dehydrogenase in experimental arteriosclerosis.

The aortic wall in rabbits was injured by pulling an inflated balloon catheter through the aorta. This was followed by an increase of the RNA-DNA ratio in the aortic wall 3 days after the injury. At the same time, the cathodic lactate dehydrogenase (LDH) isoenzymes composed of M subunits increased. The changes in LDH may reflect tissue hypoxia at the early phase after the injury. At 6 days protocollagen proline hydroxylase activity was increased, suggesting increased capacity for collagen formation in the injured aorta. Two weeks after injury the total content of hydroxyproline and DNA were increased, indicating connective tissue proliferation and hyperplasia in the injured aorta. The biochemical changes were interpreted as a repair response of the arterial connective tissue to injury. Gross arteriosclerosis of the aorta developed simultaneously with the biochemical alterations.

Effect of age on the collagen content of the normal rat myocardium.

AbstractThe development of the collagenous tissue in the myocardium was studied in rats aged one day to 18 months. During the first three weeks a rapid exponential increase of both the heart weight and the total amount of myocardial hydroxyproline occurred, although the concentration of total and insoluble hydroxyproline as estimated per mg of dry tissue showed no significant change. Thereafter the growth rates of the heart weight and total and insoluble hydroxyproline fell with increasing age. The growth rate of total hydroxyproline fell more slowly, however, and a slight increase in the content of total and insoluble hydroxyproline per dry tissue was noted with increasing age. The proportion of neutral salt‐soluble collagen hydroxyproline in the myocardium dropped from an initial value of 30 per cent of the total hydroxyproline in newborn rats to 10 per cent at the age of three weeks. After this the proportion of soluble hydroxyproline remained almost unchanged. At the same time the proportion of free hydroxyproline dropped from 25 per cent of the total hydroxyproline in newborn rats to 1.3 per cent at the age of 6 months. The results seem to suggest that during the postnatal period insoluble mature collagen develops rapidly, and therefore with increasing age constitutes a slowly rising proportion of the myocardial collagenous framework. This seems to be a response to the functional demand on the growing heart for increased tensile strength.

The determination of urinary hydroxyproline

Three methods of determining the total hydroxyproline content in urine were compared to check the inhibitory influence of some urinary constituents on the analysis. It is recommended to perform Chromatographic purification through an ion-exchange resin column prior to the determination of hydroxyproline.

Effect of growth hormone, an anabolic steroid, and cortisone upon various fractions of skin and bone hydroxyproline in mice.

Various fractions of hydroxyproline were determined in skin, femora, and humeri of DBA/2 mice treated for 2 weeks with one of three hormones: bovine growth hormone, anabolic steroid (17-β-hydroxy-17-α-methylandrostano[3,2-c]pyrazole), and cortisone acetate. The following fractions were studied: 0.15 M NaCl, 0.50 M NaCl, 0.50 M citrate, and total hydroxyproline. Since collagen is the only protein in the body containing hydroxyproline in significant amounts, this fractionation method allows an appraisal of the rates of synthesis, maturation, and breakdown of collagen. Growth hormone produced a significant increase in all extractable fractions and in the total hydroxyproline content of tissues studied. The action of anabolic steroid on hydroxyproline was generally comparable to that of growth hormone, but was less marked. Both total hydroxyproline and saline-extractable fractions were significantly reduced in skin and bone of cortisone-treated mice.

Effect of vitamin K on connective tissue metabolism

Radiosulphate incorporation into calf cartilage slices has been significantly inhibited by vitamins K 1 and K 3 respectively. In rats the uptake of intraperitoneal radiosulphate into costal cartilage has been strongly reduced by vitamin K treatment. The total hydroxyproline, hexosamine, and nitrogen content of inflamed granuloma tissue has been considerably reduced by treatment with vitamins K, while the cutaneous concentration of these components remained unchanged.

Role of pyrrolidine residues in the structure and stabilization of collagen

The physical properties of two neutral salt-soluble invertebrate collagens of markedly different total imino acid and hydroxyproline content have been compared. Measurements of sedimentation velocity, viscosity and optical rotation indicate that both Ascaris cuticle and earthworm cuticle collagens behave as rigid, asymmetric, rod-like particles in solution, with component polypeptide chains in the helical configuration of poly-L-proline II type and with molecular weights appreciably greater than those reported for the vertebrate tropo-collagens. Ascaris collagen (29% Pro; 2%Hypro) undergoes a thermal transition (collagen → gelatin) at 52°C (TD) whereas earthworm collagen (0·8% Pro; 17% Hypro) exhibits a TD of 22°C. When these results are considered with the thermal denaturation temperatures of other soluble collagens of known composition, it is concluded that the total pyrrolidine (Pro + Hypro) rather than the Hypro content alone, is the significant feature in the stabilization of the collagen structure. The distribution of pyrrolidine residues in various collagens has been estimated statistically and leads to the conclusion that triplets containing neighboring pyrrolidine residues are thermally stable. The re-naturation rates of Ascaris and earthworm gelatins have been measured at low temperature and their relationship to the pyrrolidine content and distribution considered.

Influence of the Parathyroids on Plasma Hydroxyproline Levels

Plasma levels of hydroxypro line were studied in rats subjected to dietary limitation, parathyroidectomy, nephrectomy and combinations of these conditions. Total hydroxyproline levels were determined on hydrolyzed samples and compared with the amounts observed without hydrolysis in aliquots of the same samples. Parathyroidectomy consistently lowered plasma hydroxyproline levels, although the changes were minimal in hydrolyzed plasma samples taken from animals maintained on normal diets. Nephrectomy lowered the values of nonhydrolyzed plasma samples, but increased markedly the total hydroxyproline content as determined by hydrolysis. It is suggested that one source of plasma hydroxyproline was collagen which was broken down as a result of parathyroid-bone interactions in the process of maintaining a constant plasma calcium level. Nephrectomy, which increases endogenous parathyroid activity, appears to increase the breakdown of bone collagen, transferring the hydroxyproline residues to plasma in a bound form.

Fibrinoid and hydroxyproline of the rheumatoid subcutaneous nodule.

SummaryThree subcutaneous nodules and one specimen of synovial membrane from patients with rheumatoid arthritis have been subjected to analysis for water, lipid, collagen, elastin and hydroxyproline. The hydroxyproline content of the collagen in these tissues was similar to that found in collagen from other sources. The total hydroxyproline content of the nodules and the synovial tissue was accounted for completely by the collagen and elastin fractions. The suggestion is made on this basis that the fibrinoid material of the rheumatoid nodule is not of collagenous origin.