Formation of proline metabolites in chick embryo bone: Interference with the measurement of free hydroxyproline by ion-exchange chromatography

Chojkier, Mario; Bateman, John F.; Phang, James M.; Peterkofsky, Beverly

doi:10.1016/0003-2697(82)90354-2

Cited by 18 publications

(10 citation statements)

References 23 publications

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“…Nonetheless, the marked increase of salt-soluble radioactive hydroxyproline in cirrhosis is a provocative finding. It should be noted, however, that contamination of this fraction is a possible source of error in the analysis of hydroxyproline (19). Lastly, these investigators (7, 36) did not attempt to eliminate environmental factors and therefore their studies may not be comparable to our study.…”

Section: Nd 321mentioning

confidence: 68%

“…After a 15-min preincubation period in the presence of 0.2 m M ascorbic acid, cofactor for prolyl and lysyl hydroxylases, approximately 25 pCi of ~-[5-~H]proline were added, and the incubation was continued for 4 hr (18). Before use, ~4 5 -3H]proline was purified on a 1 x 15 cm AG50W-X8 ionexchange column by elution of contaminants with 1.0 N HCl followed by elution of proline with 6.0 N HCl (19).…”

Section: Radiolabeling Of Cellsmentioning

confidence: 99%

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Acetaldehyde Stimulates Collagen and Noncollagen Protein Production by Human Fibroblasts†

1984

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The mechanisms responsible for the increased hepatic collagen deposition in alcoholic cirrhosis remain unknown. The question of whether ethanol or acetaldehyde has a direct effect on collagen and noncollagen protein production was investigated in human fibroblasts with no detectable activity of alcohol dehydrogenase to distinguish the effects of these metabolites. To eliminate environmental factors, protein production by confluent human skin, fetal and hepatic fibroblasts was studied after three passages. Cells were labeled with [5-3H]proline for 4 hr in the presence of 0.2 mM ascorbate alone or with addition of either ethanol (50 mM) or acetaldehyde (0 to 300 microM). Rates of protein production were calculated from the radioactivities of collagenase-sensitive and collagenase-resistant proteins. Skin fibroblasts from alcoholic individual either with cirrhosis or without liver disease have comparable rates of collagen and noncollagen protein production. Acetaldehyde, in a concentration found in the liver during ethanol abuse, significantly increased collagen production by human skin fibroblasts (up to 140%), fetal fibroblasts (up to 240%) and hepatic fibroblasts (up to 70%) but the addition of ethanol had no significant effect on basal collagen production. The effect of acetaldehyde was dose-related and affected noncollagen protein production in a similar manner. Acetaldehyde did not cause changes in either proline transport or the specific activity of the proline precursor pool. This newly recognized stimulation of collagen production by acetaldehyde may be a possible mechanism of fibrogenesis in alcoholic individuals.

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Section: Nd 321mentioning

confidence: 68%

Section: Radiolabeling Of Cellsmentioning

confidence: 99%

Acetaldehyde Stimulates Collagen and Noncollagen Protein Production by Human Fibroblasts†

1984

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“…Fractions (0.7 ml) were collected at a flow rate of 0.35 ml/min; elution of proteins was monitored using dual label techniques (17)(18)(19)(20). SDS-polyacrylamide gel electrophoresis (PAGE) was used to assess protein purification (21).…”

Section: Introductionmentioning

confidence: 99%

Hepatocyte collagen production in vivo in normal rats.

Chojkier¹

1986

J. Clin. Invest.

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“…There were insignificant amounts of radioactivity in the region where 4-cis['4C]hydroxyproline would elute (33). To ascertain that neither isotope contaminants (34) nor ['4C]proline metabolites (32) would interfere with the analysis of radioactive hydroxyproline, bone sections were incubated in the presence of 1 mM a,a'-dipyridyl to inhibit hydroxyproline formation, and the low molecular weight fraction was analyzed, as suggested elsewhere (32). Amino acid analysis of hydrolyzates from these samples did not reveal any significant amount of radioactivity eluting in the hydroxyproline region.…”

mentioning

confidence: 99%

Specifically decreased collagen biosynthesis in scurvy dissociated from an effect on proline hydroxylation and correlated with body weight loss. In vitro studies in guinea pig calvarial bones.

Chojkier¹,

Spanheimer²,

Peterkofsky³

1983

J. Clin. Invest.

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A B S T R A C T The question whether ascorbate regulates collagen production solely through its direct role in proline hydroxylation was investigated. Proteins in calvarial bones from control and scorbutic weanling guinea pigs were labeled in short-term cultures with radioactive proline. Proteins were digested with purified bacterial collagenase to distinguish between effects on collagen polypeptide production and hydroxyproline formation. There was a preferential decrease in the absolute rate of collagen biosynthesis beginning after 2 wk of ascorbate deficiency, and this effect was temporally dissociated from decreased proline hydroxylation. There were no significant changes in the absolute rates of collagen degradation or noncollagen protein production. In vitro inhibition of proline hydroxylation in normal bone with a,a'-dipyridyl did not affect the relative rate of collagen synthesis, further dissociating these functions. Ascorbate added to scorbutic bone cultures reversed defective proline hydroxylation but not defective collagen synthesis, suggesting that the latter was an indirect effect of scurvy. There was a linear correlation between the extent of body weight lost during the 3rd and 4th wk of scurvy and the rate of collagen synthesis in scorbutic bone. This correlation also applied to control animals receiving ascorbate, but with weight loss induced by food restriction. These studies establish for the first time that ascorbate deficiency in guinea pigs leads to a specific decrease in collagen polypeptide synthesis and suggest that this decrease results from the reduced food intake and/or weight-loss characteristic of scurvy.

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Formation of proline metabolites in chick embryo bone: Interference with the measurement of free hydroxyproline by ion-exchange chromatography

Cited by 18 publications

References 23 publications

Acetaldehyde Stimulates Collagen and Noncollagen Protein Production by Human Fibroblasts†

Acetaldehyde Stimulates Collagen and Noncollagen Protein Production by Human Fibroblasts†

Hepatocyte collagen production in vivo in normal rats.

Specifically decreased collagen biosynthesis in scurvy dissociated from an effect on proline hydroxylation and correlated with body weight loss. In vitro studies in guinea pig calvarial bones.

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