INTRACELLULAR SYNTHESIS OF CHONDROITIN SULFATE

In autoradiograms of slices of costal cartilage, incubated for 4 hours in a salt solution containing S35-sulfate and then washed extensively and dehydrated, about 85 per cent of the radioactivity was assignable to the chondrocytes. From alkaline extracts of similarly prepared slices of cartilage, 64 to 83 per cent of the total sulfur-35 in the slices was isolated as chondroitin sulfate by chromatography on an anion-exchange resin. In view of the estimate that only about 15 per cent of the radioactivity was in the matrix, the isolation of 64 to 83 per cent of the total sulfur-35 as chondroitin sulfate is a strong argument that the chondrocytes are the loci in which chondroitin sulfate(s) is synthesized

TURNOVER OF S35-SULFATE IN THE MUCOSA OF THE GASTROINTESTINAL TRACT OF RATS AS SEEN IN AUTORADIOGRAMS

Segments of the gastrointestinal tract removed from rats after intervals of time following injection of S35-sulfate were fixed in aqueous formalin and then washed in water. Contact and coated autoradiograms were prepared. The suggestion made by others that more of the labelled sulfate is fixed by the mucosa than by the underlying coats of the gastrointestinal tract is confirmed. In addition it was found that the isotope is fixed to a greater extent in the lower intestine than in the middle or upper portions of it. Coated autoradiograms revealed that 6 hours after administration of S35-sulfate more of the label was present in the goblet cells lying deep in the crypts of the mucosa than in those adjacent to the intestinal lumen. By the 24th hour the concentration of the isotope was strikingly higher and more uniform from cell to cell. The mucus in the intestinal lumen was also highly radioactive. At the end of 48 hours very little of the sulfur-35 remained in the intestinal wall or could be made out in the mucus of the lumen: the autoradiographic reaction was faint and diffuse as contrasted with the punctiform and intense reaction given by the specimens removed at the end of shorter intervals of time

EFFECT OF AGE ON SOME ASPECTS OF SULFATE METABOLISM IN THE RAT

S35-labelled sodium sulfate was administered to rats 10, 30, and 300 days old in an intraperitoneal dose of 0.3 µc. per gm. of body weight. Representative animals of each age were sacrificed 12, 24, 48, and 96 hours after injection. The concentration of sulfur-35 in the pooled sera of the 10-day-old rats was found to be strikingly higher than the level in the sera of the 30-day-old and the 300-day-old rats, while the levels of sulfur-35 in the sera of rats in the latter two age groups were similar. The difference was not explained by the differences in binding of sulfate by serum proteins. Although no binding could be detected when sulfate was added to serum in vitro, a substantial fraction, up to 80 per cent by the 96th hour, was observed to be bound after injection into the living rat. The 10-day-old rats differed from the older ones in having lower levels of serum proteins and lesser amounts of bound sulfate. The non-dialyzable sulfur-35 was associated to the largest extent with the albumin component in the sera. The age of the rats found expression in the specific activities of the sulfate-sulfur of mucopolysaccharides isolated from the skeletons, pelts, and viscera. The highest specific activities were observed in the mucopolysaccharides isolated from the tissues of the youngest rats; the lowest in those from the oldest rats. Though the maximum concentration was rapidly attained in the mucopolysaccharides from the various tissues in each of the age groups, the subsequent decreases in concentration were slow. Radiochemical analyses for sulfur-35 in ends and shafts of femurs and radioautographs of humeri supported the assumption that the labelled sulfate had been incorporated into the chondroitin sulfate of growing cartilage

SULFATE-SULFUR METABOLISM IN THE RAT FETUS AS INDICATED BY SULFUR-35

Twenty-four hours after the intraperitoneal injection of sodium sulfate-S35 into pregnant rats, sulfur-35 was found in the embryos. The amount of the sulfur-35 retained by the embryos was directly related to their degree of development in utero. A large fraction of the sulfur-35 found in the embryos was insoluble in 5 per cent trichloroacetic acid. At the 9th to 10th day of development, about 40 per cent of the sulfur-35 was present in this fraction. In 20-day-old embryos this fraction accounted for nearly 90 per cent of the total. Radioautographs of sections of embryos fixed in a solution of formaldehyde revealed that the sulfur-35 was most highly concentrated in the cartilaginous portion of the skeleton. All other tissues gave much weaker autographic reactions, comparable with the over-all reaction obtained when sections from embryos fixed in a solution of formaldehyde saturated with barium hydroxide were used. By analysis for the sulfur-35 content of individual tissues the concentration of the sulfur-35 in humeri from 20-day-old embryos was found to be about 30 times that in the maternal sternum. The concentration of the isotope in the skeletal muscle, brain, heart, and skin of the same embryos was also higher than in the corresponding maternal tissues. On the other hand, the concentration of the sulfur-35 in the maternal gastrointestinal tract plus contents was higher than in the gastrointestinal tract and contents of the embryos

RADIOAUTOGRAPHIC VISUALIZATION OF SULFUR-35 DISPOSITION IN THE ARTICULAR CARTILAGE AND BONE OF SUCKLING RATS FOLLOWING INJECTION OF LABELED SODIUM SULFATE

The localization of sulfur-35 administered intraperitoneally as sodium sulfate to 7-day-old rats was determined by radioautography of sections of humeri and tibia-knee-femur combinations removed 24, 48, 96, 216, and 290 hours after injection of this isotope. Radioautography of sections of bone and cartilage that had been fixed in formalin indicated that the tracer isotope was present throughout the entire epiphysis. Its concentration therein was highest initially at the epiphyseal-diaphyseal junction where the more mature cartilage cells were present. By the 96th hour post injection the sulfur-35 had become more uniformly distributed in the epiphyses and an even distribution of it throughout the epiphyseal cartilage was almost attained by the 216th hour post injection. As centers of secondary ossification arose in the epiphyseal cartilage, the sulfur-35 appeared to diminish in concentration and disappear from these loci. However, radioautographs of cartilage fixed in formalin saturated with barium hydroxide, instead of in formalin only, disclosed the fact that the tracer isotope was still present in these loci. When bone and bone marrow were fixed in formalin the autographs indicated the presence of sulfur-35 primarily in the periosteum. Only a negligible amount appeared to be present in the bone shaft and marrow. However, when these tissues were fixed in formalin saturated with barium hydroxide it was possible to demonstrate the presence of the tracer isotope in both bone and bone marrow

VITAMIN A AND ENDOCHONDRAL OSSIFICATION IN THE RAT AS INDICATED BY THE USE OF SULFUR-35 AND PHOSPHORUS-32

The administration of vitamin A to vitamin A-deficient rats resulted in a decreased concentration of inorganic sulfate-sulfur in the serum from a value of 2.5 mg. per cent to 1.8 mg. per cent, the latter being close to the value of 2.0 mg. per cent found in normal rats of the same age. The uptake of sulfate and phosphate by femurs and tibiae of vitamin A-deficient rats was less than that in normal rats of the same age. An increased uptake followed the administration of vitamin A: radioautography indicated that in the case of sulfate, its uptake was particularly increased in the epiphyseal cartilage; an increased uptake of phosphate was particularly evident in the diaphysis immediately adjacent to the epiphyseal cartilage plate. The specific activity of the sulfate-sulfur in the chondroitin sulfate samples isolated from the skeletons of vitamin A-deficient rats fell progressively as the deficiency continued. Following administration of vitamin A, the specific activity approached and exceeded the value given by the sample from the skeletons of normal rats of the same age. A substantial increase was found in the value of the specific activity of the sulfate-sulfur of sulfomucopolysaccharides isolated from skins of vitamin A-deficient rats that had been given vitamin A. Following administration of vitamin A to rats deficient in this vitamin, an increased accumulation of some sulfur-containing material was found in regions of active calcification

RADIOAUTOGRAPHIC STUDIES OF SULFATE-SULFUR (S35) METABOLISM IN THE ARTICULAR CARTILAGE AND BONE OF SUCKLING RATS

Fifteen minutes after intraperitoneal injection of sulfur-35 as sodium sulfate to 7-day-old rats the concentration of the isotope was highest in the cartilage at the epiphyseal-diaphyseal junction of long bones but was demonstrable throughout the entire epiphysis. Up to the 24th hour the pattern of deposition did not change as the concentration continued to increase. As centers of secondary ossification developed, there occurred in them an increased concentration of some form of sulfur, insoluble as the barium salt. This sulfur was probably derived from the cartilage which the center of secondary ossification replaced. Up to about the 30th minute after injection the sulfur-35 was deposited transitorily in a high concentration in discrete loci of the bone marrow. Excluding this transitory deposition, the highest concentration of the radioisotope in the bone marrow was seen in the specimens removed 24 hours after administration. In the bone shaft the sulfur-35, in a form which was insoluble in an alkaline solution of barium ions, was deposited diffusely up to the 24th hour after its administration. Thereafter, the radioisotope decreased in concentration in the middle portion of the bone shaft. However, the concentration of a similarly characterized sulfur-35-bearing component in the ends of the diaphysis continued to increase up to at least the 96th hour after injection

SYNTHESIS OF SULFOMUCOPOLYSACCHARIDES IN THYROIDECTOMIZED RATS

The thyroid glands of rats were made non-functional by a single dose of iodine-131 on the 1st day after birth or by surgical removal on the 28th day. The incorporation of sulfate-sulfur into the sulfomucopolysaccharides of skeletons and pelts was found to be significantly depressed by thyroidectomy. Daily supplements of 5 or 10 γ of L-thyroxine, started on the 28th day, increased the uptake of sulfur-35, although it did not reach normal in the 2 weeks of treatment. Autoradiograms of sections of tibiae and pelts confirmed the analytical data. The findings suggest that the synthesis of sulfomucopolysaccharides is depressed in thyroidectomized rats

TURNOVER OF THE ORGANIC MATRIX OF CARTILAGE AND BONE AS VISUALIZED BY AUTORADIOGRAPHY

Tibiae and humeri were removed from suckling rats at intervals of time after intraperitoneal injection of C14-L-phenylalanine, C14-L-leucine, S35-sulfate, or Ca45 Cl2. Autoradiograms of sections of the bones were prepared. Ca45 was removed from sections treated with dilute acetic acid; neither the concentration of S35 nor that of C14 was thereby markedly decreased. The S35 was removed from the demineralized sections on incubation in a solution of testicular hyaluronidase; the C14 was not. These results are interpreted as indicating that most of the S35 was present in the bones as chondroitin sulfate and that most of the C14 in the bones was present as protein. In the epiphyses, the C14 was initially concentrated in the proliferaing and hypertrophic chondrocytes, as was the S35. Secretion of S35- and C14-labeled materials into the matrix followed. Thereafter, however, although the S35-labeled material (chondroitin sulfate) persisted in the matrix, albeit at a diminished concentration, and was incorporated into metaphyseal bone, the C14-labeled material (protein) was almost completely removed from the matrix. When rats were given repeated doses of 17-β-estradiol benzoate so as to inhibit resorption of their metaphyses, repeated doses of S35-sulfate were discerned as strata of S35 in their metaphyses. This was not the case if the rats received repeated doses of C14-L-phenylalanine or C14-L-leucine. On the basis of the results in these experiments it is suggested that although a portion of the chondroitin sulfate produced by the chondrocytes of the epiphyseal plate is retained and becomes part of the cores of metaphyseal spicules of bone, the protein of the proteinpolysaccharide is somehow removed before calcification of the cartilage ensues

A CONSIDERATION OF THE PERMEABILITY OF CARTILAGE TO INORGANIC SULFATE

On the basis of an examination of autoradiograms of knee-joints fixed so as to remove chondroitin sulfate or inorganic sulfate, or to minimize the loss of both, it is suggested that the cartilage is permeable to inorganic sulfate in vivo and in vitro. In vivo and in vitro, almost as rapidly as it enters the cartilage, inorganic sulfate is utilized by the cells in the synthesis of chondroitin sulfate. The net result is a continuing low concentration of inorganic sulfate in the cartilage