METABOLIC EFFECTS OF CARTILAGE IN VIVO AND IN VITRO* BY

METABOLIC EFFECTS OF LATHYROGENIC AGENTS ON CARTILAGE IN VIVO AND IN VITRO*

BY MORRIS J. KARNOVSKY, M.B.B.CH., AND MANFRED L. KARNOVSKY, PH.D.

(From the Departments of Pathology and Biological Chemistry and the Biophysical ~ Laboratories, Harvard Medical School, Boston)

(Received for publication, August 31, 1960)

I t has been known for over twenty years that the feeding of sweet pea (Lathyrus odoratus) seeds to rats causes skeletal deformities (osteolathyrism) (1). Recently, Ponseti and co-workers (2) have revived interest in the experimental disease of osteolathyrism (or odoratism as it is sometimes called). The condition is characterised by severe and dramatic changes induced in the mesenchymal tissues of a variety of species by the administration of lathyrogenic agents. These include the seeds of L. odoratus (2), the toxic principle isolated from the seeds, /3-(N-7-L-glutamyl)aminopropionitrile (3), beta aminopropionitrilO (4) and various synthetic nitriles such as aminoacetoni- trilO (4), and methyleneaminoacetonitrile* (5). Beta mercapto-ethylamine x (6) and cystamine, as well as various carbonyl blocking reagents, such as semicarbazide (7) have also been implicated as lathyrogenic agents.

The experimental disease has been studied mostly in the rat, but the changes produced in various species (e.g. mice, rabbits, turkey pours, chick embryos, frogs and salamanders) are similar, in that essentially mesenchymal tissues are affected. To a greater or lesser extent, vascular, skeletal, and connective tissue lesions develop, including dissecting aneurysms of the aorta, degenerative changes in growth cartilages, marked subperiosteal new bone formation and bone deformities, hernias, and fragility and thinning of the skin (for review of the literature see references 8, 9). A character- istic feature is the extreme fragility of the connective tissues of lathyric animals (9).

Although it is generally agreed that a change in the state of organization of the connective tissues is involved in the pathogenesis of osteolathyrism, the literature is confusing on the nature of this change, probably owing in part to the large variety of techniques and experimental conditions used. On the basis of histological, histochemical, and radioactive tracer techniques, authors have variously ascribed the basic defect to a change in collagen, ground substance, elastin, or connective tissue cells.

* This investigation was supported by Grant A-2016 from the National Institutes of Health, United States Public Health Service, Training Grant No. 2G-113 from the United States Public Health Service, and the Eugene Higgins Trust through Harvard University.

1 The following abbreviations will be used: BAPN, beta aminopropionitrile; AAN, am|no- acetonitrile; MAAN, methyleneaminoacetonitrile; ME, beta mercaptoethylamine.

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382 METABOLIC EFFECTS O]? LATHYROGENS ON CARTILAGE

The literature regarding these changes in the components of connective tissue, more particularly collagen, has recently been surveyed by Levene and Gross (9). Although some workers have suggested decreased collagen synthesis, others have claimed that collagen is unaffected. The recent quantitative experiments of Levene and Gross (9, 10) clearly showed that there is a marked loss of tensile strength of connective tissue in osteolathyrism, which may be correlated with a marked increase in neutral salt- extractable collagen. On the basis of their extensive data, these authors suggest that one of the defects induced by lathyrogenic agents is an interference with the normal intermolecular cross-linking within the collagen molecule. Although evidence has been adduced by Follis and Tousimis (11) for normal synthesis of collagen, but failure to organize into fibers, van den Hooff, Levene, and Gross (10) believe that collagen is present in fibrillar form, but is loosely aggregated and is solubilized by extraction with cold saline. The mechanism of action whereby lathyrogenic agents induce these changes in collagen has not been elucidated.

In contrast to the evidence for changes in collagen, the evidence for changes in ground substance (acid mucopolysaccharides) is conflicting and less clear-cut. On the basis of increased histochemical staining for acid mucopolysaccharides several workers have claimed an increase in these components of connective tissues (12-16). Contrari- wise, the use of histochemical staining techniques has led others to propose either depolymerization of the ground substance (17, 18), defective formation, or excessive destruction (2, 8, 19-21).

Data obtained by the use of autoradiography utilizing sulfate-S 85 have also been contradictory. No change in incorporation of sulfate-S ~s into sulfated acid mucopolysaccharides has been reported (5). B61anger (22) found a normal or increased uptake of sulfate-S ~s into the cells of lathyric cartilage, but decreased binding of Ca 4s by lathyric cartilage matrix in vitro, suggesting increased turnover or utilization of sulfate. Irregular distribution of sulfate-S 35 in the hypertrophic zone, and delay in penetration into the matrix have recently been described (23). Increased uptake of sulfate-S 86 has been reported in lathyric femur shafts (24); this was attributed in part to enhanced accretion of cartilage and bone. Recently decreased uptake of sulfate-S ~ into fracture callus of BAPN-treated rats was reported (25).

Using hexosamine content as an index of mucopolysaccharide levels, several authors have reported no significant change (5, 26--28). However, Castellani and coworkers (29, 30) reported a marked decrease in the hexosamine content of lathyric rabbit epiphyscal cartilages, more specifically in the galactosamine level. A recent paper reported increased hexosamine and uronic acid levels in the aorta but not in other tissues (31).

In a preliminary report (32) we have suggested, on the basis of histochemical observations and biochemical studies on cartilage in vivo and in vitro, that lathyrogenic agents affect the metabolism of sulfated acid mucopolysaccharides. Further, studies on the inorganic composition of lathyric cartilage have lent indirect support to this view (33). This paper presents additional evidence that lathyrogenic agents have a marked effect on the metabolism of acid mucopolysaccharides of hyaline cartilage both in vivo and in vitro, and that other met-

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M. J. KARNOVSKY AND M. L. KARNOVSKY 383

abolic aspects of car t i lage are also affected. I t has p rev ious ly been shown t h a t

g lycogen m e t a b o l i s m is affected in os t eo la thy r i sm (34).

Material and Methods

I . Experiments in V ivo .~

(a) General Procedures: Weanling male rats of the Sprague-Dawley strain, weighing between 40 and 50 gin. were used throughout these experiments. The rats were housed individually in plastic cages, and were fed on Purina Laboratory chow, with the restrictions on dietary intake specified in the Results section. Tap water was allowed ad libitum. The lathyrogenic agents, aminoacetonittile (AAN) and methyleneamlnoacetonittile (MAAN) were administered by stomach tube, or parenterally as noted later. The dally dose given in each experiment is specified in the Results section. The rats were killed by decapitation and the epiphyseal cartilages from the distal end of the femur and the proximal end of the tibia were dissected free of muscle, connective tissue, and bone (in so far as this was possible) under a dissecting microscope. In several experiments, samples from two or three animals were pooled.

(b) Analytical Methods: The samples were transferred to tared stoppered bottles and the wet weights obtained. They were then dried to constant weight in an oven at ll0°C. Samples were analyzed for hexosamine (35) and hydroxyproline (36) after hydrolysis in sealed tubes in 4.0 N HC1 in a boiling water bath for 6 hours. Recovery of added hexosamine was 75 to 82 per cent and of added hydroxyprollne 103 to 106 per cent. The precision for the hexosamine esti- mation was 3 per cent and for the hydroxyproline, 4 per cent.

Chondroitin sulfate extractions were performed according to Bollet et al. (37). The chondroitin sulfate was precipitated from the aliquots of the final dialysate as the protamine salt, and estimated as uronic acid (38). Hexosamine and uronic acid estimations on the isolated chondroitin sulfate gave molar ratios close to 1.0. Other aliquots of the dialysate were lyophilized and samples were examined in a Servall electrophoresis apparatus in 0.07 ~ phosphate buffer, pH 6.5, at 4°C. for 16 hours on Whatman paper No. 3 at 65 volts (2.5 ma.). The papers were dried and then stained with 0.06 per cent toluidine blue in 0.5 per cent acetic acid. Samples of rat epiphyseal cartilage chondroitin sulfate migrated at the same rate as commercial samples of chondroitin sulfate (Nutritional Biochemicals). No protein spots were detected.

(c) Histochemical Methods: Samples of epiphyseal cartilage were taken from representative animals and fixed in formol-alcohol, paraffin-embedded, sectioned and stained with hematoxy- lin-eosin, and for acid mucopolysaccharides with toluidine blue, alcian blue, and aldehyde- fuchsin. The periodic acid-Schiff reaction was also applied. The details of the histochemical techniques are to be found elsewhere (34).

(d) Administration of Sulfate-S~: was administered as sodium sulfate v/a the intraperi- toneal route. The doses used are given in the Results section. At the end of the experiments the epiphyseal cartilages were weighed, digested in a standard volume of hot 35 per cent KOH, neutralized, made to volume and suitable aliquots plated on planchets for counting.

H . Ezcperirnents in V i t ro .~

The ribs of freshly killed calves were obtained from the slaughter house, and were trans- ported to the laboratory in a thermos flask containing crushed ice. The ribs were dissected free of connective tissue, bone, and muscle and small cubes of cartilage (about 1 cm. s) were cut out with a scalpel. Slices about 600/z thick were made on a slicer (39) (mean thickness 619 4- 25/z). The slices were quickly weighed on a torsion balance and transferred to standard Warburg flasks containing the following medium: Krebs-Ringer bicarbonate buffer, 3.0 ml. total in main chamber, glucose (10.0/z~/ml.), 0.2 ml. 100 to 150 rag. of cartilage slices were placed in each flask. Lathyrogenic agents were included at a concentration of 2 #M/ml. final

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384 METABOLIC EFFECTS OF LATHYROGENS ON CARTILAGE

concentration in most experiments. In other experiments concentrations of 1.0, 3.0, and 5 #~/ml. final concentration were used. As MAAN is somewhat insoluble, it was first dis- solved in warm buffer and a suitable aliquot added to the flasks to give the requisite final concentration of MAAN.

In various experiments giucose-U-C 14 or glycine-l-C z4 were included in the incubation medium at levels of I to 2.5 #c. per ml. These materials were placed in the side-arm and added to the main chamber at the commencement of the experiment. Suitable control flasks containing 0.4 ml. of 0.4 per cent iodoacetate (neutralized with NaOH) were utilized as necessary. The gaseous phase was variously 95 per cent air-5 per cent COs, 95 per cent Or-5 per cent COs, 95 per cent N2-5 per cent CO2. The flasks were incubated at 37* and shaken. Mter equilibration the incubations were carried out for 2 hours, readings being made every 30 minutes. At the end of the incubation 0.4 ml. of 0.4 per cent iodoacetate was added to each flask. Results were determined as/zl. COs evolved/hour X 100 rag. wet weight of cartilage, and could be expressed as/zM lactate produced/hour X 100 rag. wet weight cartilage.

Other experiments were carried out in ~itro on a Dubnoff apparatus, under similar conditions of time, temperature, and gaseous phases. Cartilage slices (about 250 reg./flask) were placed in 25 ml. Erlenmeyer flasks containing 5 ml. Krebs-Ringer phosphate buffer, glucose (final concentration 10 #~t/ml.), and lathyrogenic agents at concentrations of 3.0 #g/ml . final concentration. Na2Sa60,, glucose-U-C 14 and giycine-1-C 14 were included in the media at levels of 1 /zc./ml. medium.

At the end of the incubations, the media were analyzed for lactic acid by the method of Barker and Summerson (40). The incorporation of the radioactive precursors into the slices was measured by a method based on that of Bostr6m et al. (41) in which counts were determined directly on the tissue. The slices were first washed several times in cold isotonic saline, and then were equilibrated overnight in cold saturated solutions of glycine, glucose, or sodium sulfate (depending on what labeled substance had been included in the incubation medium). Mter washing again three times with distilled water, and several times with acetone followed by ether, the slices were allowed to dry at room temperature. They were then weighed, placed on planchets, and counted. The per cent error for repeated counts on the same slice was 4-6 per cent and for different slices from the same flask, 4- 7 per cent. Because of the possible variation in factors such as age of the calves, the data acquired within each experiment were usually expressed as per cent of the control values, thus making comparison from experiment to experiment possible.

I lL Isotopic Counting Techniques.--

The counting equipment used was the gas-flow counter described by Robinson (42), which operates in the proportional range. Where necessary, self-absorption factors were determined and other details were as given by Karnovsky el a/. (43).

RESULTS

Experiments in Viw.--

Effect of A A N on Dry Weight, ttexosamine, and ttydroxypyroline Content of Rat Epiphyseal Cartilage:

Two sets of experiments were run in this group. In the first experiment, (Experiment 1 in ~{vo, Table I) male weanling rats were given 40 rag. AAN daily by mouth for 3 days and were killed on the 4th day. The control animals were pair-fed to match the food intake of their experimental partners.

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Histological examination of the epiphyseal cartilages of the experimental group showed well developed lesions of osteolathyrism (34). The epiphyseal plates of the lathyric rats were considerably widened, irregular, gelatinous, and fragile. The cells were swollen and distributed in a random fashion in a copious matrix. The capsules of the cells were thickened, and the voluminous intercellular matr ix was intensely metachromatic , Alcian blue, and aldehyde- fuchsia-positive. The reaction to the periodic acid-schiff procedure was slight. Small splits containing metachromatic , Alcian blue, and aldehyde-fuchsia- positive material were noted in the matrix. Glycogen was characteristically

TABLE I Effect of A A N on Eplphyseal Cartilage of Weanling Rats in Vivo*

Experi- 1 AAN meat

mg.

1 0 (control) 4O Per cent

change Significance

2 0 (control) 10 Per cent

change Significance

Period

days

3

- - 5

10 5

N o . o f rats

10 10

Per cent dry weight

32.3 4- 0.9 29.2 4- 0.6

- - 9.5

P < 0.01

I34.8 4- 0.5 25.6 4- 1.0

- 26.5

P <0 .01

i

Hexosamine

reg.~gin, dry wt.

11.3 4- 0.4 10.4 4- 0.4

- - 5.5

P > 0.05

10.7 4- 0.5 12.2 4- 0.7

+ 14

0.05 <

P < 0 . 1

Hydroxyproline

m g./gm, dry t~.

20.0 4- 0.6 20.0 4- 0.6

0

19.6 4- 0.7 19.9 4- 0.5

+ 1.6

P > 0.05

Average weight:[: gain

10 3

* Specified amounts of AAN were given daily for the periods stated. :~ As per cent of mean original weight of 47 4- 2 gin.

absent from the cells (34). In contrast, the normal cartilage cells of the control were arranged in orderly columns; glycogen was present in considerable amount , and only the capsules stained intensely metachromat ical ly and with Alcian blue. The intercellular matr ix was slightly periodic acid-Schiff positive.

The results of the analytical determinations are shown in Table I. There were no significant changes in hexosamine and hydroxypyroline content expressed on a unit dry weight basis. However, the decrease in the percentage dry weight found in the experimental group is significant, and agrees with a water increase of 16 per cent found previously in a separate but similar experiment (33).

In the second experiment, (Experiment 2 in vivo, Table I) weanling male rats were s i m i -

l a r l y treated with 10 rag. AAN daily by mouth for 10 days and were killed on the l l th day. The results were similar to those found in the first experiment, there being no significant

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386 ~ETABOLIC EFFECTS OF LATHYROGEIqS ON CARTILAGE

change in hexosamine or hydroxypyroline concentration. The percentage dry weight in the lathyric rats showed an even greater decrease of 26.5 per cent (P < 0.01).

Eflext of A A N on Chondroitin Sulfate Content of Epipkyseal Cartilage:

In this experiment six pairs of rats were used, the controls being pair-fed to their experimental partners. The experimental group was given 40 mg. AAN daily by mouth for 3 days and the rats were killed on the 4th day. All animals had an initial weight of 50 gin. -4- 3 gin. The control animals lost an average of 8 per cent of their original weight, the experimental group, 14 per cent. Chondroitin sulfate of epiphyseal cartilage was determined by the method of Bollet et aL (37); i.e., it was isolated as the protamine salt and measured as uronic acid.

The control group gave a value of 1040 -4- 121 #g./100 nag. dry weight, the experimental group 950 4- 74 ttg./100 mg. dry weight (P < 0.02). In the con-

TABLE II

Effect of M A A N o n SulfateS ~ Incorporation into Epiphyseal Cartilage in Vivo*

E~perlmen_______~t Contro_______~l

S 36 entry~: . . . . . . . . . f I00 S 35 exit§ . . . . . . . . . . . I 100

Experimental No. of rats

54 4- 5 3 pairs 77 4- 5 4 pairs

P

<0.01 <0.02

Effect

Decrease Increase (?)

* Rats pair-fed. Results calculated as counts/minute X milligram dry weight of cartilage, and expressed as per cent of control values.

:~ 40 rag. MAAN given daily for 2 days. Then S ~5 and MAAN given on 3rd day. Rats killed on 5th day. Controls not given MAAN (see Results section).

§ Sulfate-S 85 given daily for 2 days, then MAAN 40 rag. given daily for 3 days. Controls not given MAAN (see Results section).

trol group this value of uronic acid corresponds to 960 /~g. hexosamine/100 rag. dry weight if calculated on an equimolar basis. The value of hexosamine actually found was 1130 /~g./100 mg. dry weight. The values for the experimental group are 690 #g. hexosamine/100 rag. dry weight (calculated) as against 1040/~g./100 mg. dry weight (measured value). Not all the hexosamine present in cartilage is that of chondroitin sulfate; other mucopolysaccharides may make a significant contribution 04). Our results are in conformity with this observation, and suggest that lathyrogenic agents cause an increase in the ratio of hexosamine to uronic acid.

Incorporation of Sulfate-S 35 into Epipkyseal Cartilage in Vivo.-- Weanling male rats (mean weight 48 ± 3 gin.) were given 40 rag. MAAN daily by mouth

for 3 days. On the 3rd day sulfate-S u (40 #c. in 0.1 ml. saline) was administered intraperitoneally. After 48 hours without further treatment the rats were killed and the epiphyseal cartilages (distal, femoral, and proximal tibial) were dissected out, weighed, and hydrolyzed in hot 35 per cent KOH. The hydrolysates were then neutralized, brought to volume and aliquots plated. Throughout the experiment, (Expriment 3, in vivo, Table II) the control rats were pair-fed to their experimental partners.

In a similar experiment, (Experiment 4, in vlvo, Table II) sulfate-S ~ was given intra-

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peritoneally daily for 2 days, and then MAAN was given daily for 3 days. This experiment was designed to show the effect of MAAN on the "exit" of sulfate-S u from epiphyseal cartilage.

The results of both experiments are shown in Table I I . I t can be seen (Ex- per iment 3, in vivo) t ha t the incorporat ion (entry) of sulfate-S a5 was marked ly depressed in the la thyr ic group, being about half of the value obta ined in the control group. I t should also be noted tha t the experimental group lost 7 per cent of i ts original weight (mean 40 4- 2 gm.) whereas the control group gained an average of 6 per cent over the original weight (mean 48 4- 3 gm.).

TABLE III Effect of A A N on Sulfate-S 36 Imor wration into Rat Epiphyseal Cartilage in Vivo*

Counts/rain. X 100 rag. wet weight cartilage

Control Experimental

Control Experimental

Wt. gain as per cent of original weight:~

Hours after administration of sulfate-S u

(a): (b)

900 1520 410 390

33 17 5 16

8

(a) (b)

77O 6OO 44O 3OO

4O 33 5 20

12

(a) (b)

L30 820 ~50 430

40 43 i 40 20

'It.

24 48

(al (b) (~) (b)

:720 370 380 53( 220 200 3 2 0 25(

5 32 24 2~ 13 32 16 14

~r Those pairs of control and experimental rats in which the weight loss or gain was the same, or where the experimental rats gained more than their respective controls.

* 20 rag. AAN given daily intraperitoneally for 2 days, then 5 mg. daily for 2 days. I0/~c. sulfate-S 35 per rat given intraperitoneally on the 3rd day. Pairs (a) and (b) killed at each time interval.

Mean original weight 40 4- 2 gm.

In the fourth sulfate-S 35 experiment (exit), the experimental group showed a decreased incorporat ion of sulfate-S 86 a t the end of the experiment, namely 77 per cent of the control value. Both groups gained an average of 13 per cent of their original weight (mean 48 4- 3 gm). The results of this experiment are indicat ive of an increased ra te of turnover or loss of sulfate-S 8s in the la thyr ic group: nevertheless the possible inhibi tory effect of M A A N on reincorporat ion of sulfate-S 3~ released from chondrot in sulfate turnover in the period following the commencement of the la thyrogenic t r ea tment cannot be excluded.

In another experiment, (Table III) weanling rats were given 20 rag. AAN intraperitoneally for 2 days and were then given 10 ~uc. sulfate-S a6 intraperitoneally on the 3rd day. Two pairs of rats from each group were then killed at intervals of 2, 5, 8, 12, 24, and 48 hours thereafter. During the final 48 hours of the experiment the experimental rats were given 5 mg. AAN intraperitoneally daily. The controls were paired by weight gain or loss to their experimental partners in so far as this was possible.

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388 ~[ETABOLIC EFFECTS O]F LATFIYROGENS ON CARTILAGE

The results are shown in Table I I I . I t can readily be seen that where the weight gain of the control rat and its experimental partner were the same, the incorporation of sulfate-S 85 at all time intervals was lower in the experimental animals (starred columns in Table I I I ) . In those cases in which the control rat gained weight at a greater rate than its experimental partner, this difference in sulfate-S 85 uptake was even more exaggerated. I t should also be noted that both groups showed a progressive decrease in growth rate, and actual failure to gain in the latter part of the experiment: thus, the incorporation of sulfate-S 8s does not increase up to 48 hours postinjection of S 85, as might be expected in rats showing a progressive weight gain. The failure to gain weight is reflected in the time curve for the uptake of sulfate-S 35. The weight changes in Table I I I are shown on an exaggerated scale, as a percentage of the original weight of each rat. The mean original weight was 40 4- 2 gin.

I t is apparent from the above results that a more rapid rate of growth in the control rats, as compared with the experimental rats, would exaggerate the degree of inhibition of sulfate-S 35 uptake in the lathyric rats.

Effect of A A N on Incorporation of Sulfate-S a5 into Chondroitin Sulfate in Vivo.--The above experiments relate to the incorporation of sulfate-S 3~ into the whole cartilage, the results being expressed on a unit wet or dry weight basis. The assumption was made, from data in the literature (41), that nearly all sulfate-S 35 incorporated into cartilage is in fact incorporated into chondroitin sulfate. The present experiment (Table IV) deals with the specific activity of sulfate-S ~5 incorporated into isolated chondroitin sulfate. In order to obviate as far as possible the effects of differences in growth rates between experimental and control groups, the controls in this experiment were paired by weight gain or loss to their respective experimental partners, in so far as this was possible in the short period of time over which the experiment was run. The mean weight of the rats at the beginning of the experiment was 45 :k 4 gm.

Experimental rats were given a total of 20 mg. AAN subcutaneously daily in two divided doses of 10 mg. in 0.25 ml. water, morning and evening. The controls received an equivalent amount of water. On each day, after the 1st day, 360 /zc. sulfate-S ~ (0.1 mg. SO4) in 0.25 ml. isotonic saline was given to four rats from each group. Two pairs were killed at 6 and 24 hours after the sulfate-S ~ administration, respectively. The proximal epiphyses of the tibiae and the distal epiphyses of the femurs from each pair of rats were pooled, dried to constant weight, and chondroitin sulfate isolated as the protamine salt (37). The specific activities are expressed in terms of uronic acid in the final samples obtained. (The results are shown in Table IV).

The pairing by weight gain or loss was not entirely successful owing to the short duration of the experiment and the rapid changes in weight in the experimental group. There is marked inhibition of incorporation of sulfate-S 85 into chondroitin sulfate in the experimental rats at 6 hours after sulfate-S 35 administration, the weight changes between the control and experimental groups being relatively slight. This inhibition is especially marked on days 2 and 3 of

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M. J . K A R N O V S K Y A N D M. L . K A R N O V S K Y 389

the experiment being less obvious on day 4. On day 2, at 24 hours after sulfate- S 8" was given, incorporation is also decreased in the lathyric rats. The difference between control and experimental animals is no longer seen on days 3 and 4 in regard to incorporation at 24 hours after sulfate-S 8~ administration. I t should be noted that the net incorporation of sulfate-S 3s also fell in both groups as the experiment proceeded in time, this owing to a virtual cessation of growth in both control and experimental groups.

The 24 hour incorporation levels were higher in each instance than the 6 hour levels, the steepest rise being on day 2 when growth was most rapid in both

Incorporation of S~

Day . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Specific activity~; Control (C) . . . . . . . . . . . Experiment (E) . . . . . . . . . Ratio E/C . . . . . . . . . . . . .

Per cent weight change§ Control . . . . . . . . . . . . . . . . Experiment . . . . . . . . . . . .

TABLE IV ~ate-S 8s into Ctwndroitin Sulfate o] AAN-Treated Rats*

6 hrs 24 hrs.

79 160~2 38 0.48 0.62

0 --8 --5 --11

6hrs.

48 20 0.41

+2 --2

24 hrs.

41 37 0.90

--1 --2

6 hrs.

44 35 0.79

+10 +6

/,4 hrs.

47 46

1.00

+6 +2

* AAN 20 rag. subcutaneously given daily. Sulfate-S ~ given on days 2, 3, and 4 and rats killed at 6 and 24 hours after sulfate-S 85 administration.

Specific activity of sulfate-SSa-labeled chondroitin sulfate as per cent of 24 hour control value, day 2.

§ Mean original weight 45 4- 4 gin.

groups. Thereafter the 24 hour levels were not as high as the initial (day 2) value, and gradually dropped to only slightly above the respective 6 hour levels. In one instance (24 hour control, day 3) the 24 hour level is slightly lower than the 6 hour level: this was probably owing to a precipitous drop in weight which occurred in the final 24 hours (55 to 45 gm. average). The apparent absence of any significant difference of sulfate-S 35 incorporation between control and experimental animals in the 24 hour values on days 3 and 4 is thought to be due to virtual cessation of growth and this thus becomes a limiting factor.

Experiments in Vitro.--

L Effect of Lathyrogenic Agents on Organic Acid Production by Cartilage Slices (Qorga, ~c ,c~a) :

In preliminary experiments it was found that calf costal cartilage slices had

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390 METABOLIC EFFECTS OF LATHYROGENS ON CARTILAGE

an extremely low oxygen consumption of about 0.5/~l./hour X rag. wet weight of cartilage. The production of organic acid, however, was considerable, equivalent to 0.63 #1. CO2/hour X mg. dry weight which agrees with reported values for QI~o~** of 0.65 for calf hyaline cartilage (45). The effects of lathyrogenic agents on organic acid (mainly lactic acid) production by calf costal cartilage are shown in Fig. 1. There is a tendency for a decreased production of organic acid under aerobic conditions, in the presence of lathyrogenic agents, which is more

z ~ I 0 0 " IJJ 0

~ 5o

AEROBIC ANAE RO BIC

N M A B B N M A B B O A A A A 0 A A A A R A N P P R A N p p M N N N M N N N A X5 A X5 L L

0 I0 9 3 3 I I 9 3 3

NUMBER OF OBSE RVATIONS

M E

3

FIo. 1. Effect of lathyrogenic agents on organic acid production (Qorganlc aoid) by calf cartilage slices. Final concentration of lathyrogens was 2 pM/ml. (BAPN × 5 = 10/z,~/ml.). The results are expressed as per cent of normal aerobic control values. The increment in acid production by normal cartilage (in the absence of lathyrogens) for anaerobic conditions as compared with aerobic conditions is significant at the 0.01 level. Where columns are labeled S, a significant difference (P < 0.01) exists between the values for cartilage slices treated with lathyrogens and their respective aerobic or anerobic normal, untreated controls.

apparent under anaerobic conditions. It should be noted that there is also a significant increase in acid production, in the absence of lathyrogenic agents, under anaerobic as compared with aerobic conditions (Pasteur effect).

Chemical estimations for lactic acid gave somewhat unusual results in that the chemical values were frequently higher than the manometric values. This was found to be the case even when both the chemical and manometric values were corrected for values obtained from control slices killed with iodoacetate at the beginning of each experiment; these control values give a correction for the amount of acid already present in the tissues and leaching into the medium before the manometric readings were commenced. Another factor which complicated the chemical analysis for lactate was the observation that some of the lathyrogenic agents (especially MAAN) gave strong interfering colors with the

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~ . J. KA.RNOVSK¥ AND ~I. L. KARNOVSKY 391

colorimetric reagents, thus making it impossible to obtain chemical values when these agents were used. Nevertheless, it is of some interest that despite the fact that the chemical method gave higher values than expected, probably owing to some interfering substance, the invremcttt of acid production by normal control cartilage found under anaerobic conditions was confirmed by both the chemical and manometric estimations, and can therefore be largely attributed to an increase in lactate production per se. In a representative experiment the

AEROBIC ANAEROBIC (~ M B M N M A

N P P R N M N N N M N A X15 A L L

~. 150

,~ 100

_u u_ 50 o Ld O. ¢0

0

S_ m S

S

- 5 7 4 3 2 3

NUMBER OF OBSERVATIONS FIG. 2. Effect of lathyrogenic agents on sulfate-S s6 incorporation by calf cartilage slices

l~mal concentration of lathyrogens was 2 p~x/ml. (BAPN X 15 = 30 gu/ml.). The results are expressed as per cent of normal aerobic control values. The decreased value for normal slices (in the absence of lathyrogens) under anerobic conditions is significant at the 0.01 level. S above a column represents a significant difference (P < 0.01) when the values from cartilage slices treated with lathyrogens are compared with their respective aerobic or anerobie normal untreated controls, s above a column represents significance at the P < 0.05 level.

increment in acid production by normal cartilage under anaerobic as opposed to aerobic conditions measured manometrically was 0.44/zM/100 rag. cartilage (fresh weight) per hour, whereas the chemical value obtained was 0.34 pM lactate.

Z. Incorporatiott of Sulfate-S .6 into Cartilage Slices: The incorporation of sulfate-S 35 into cartilage slices under anaerobic conditions was about 50 per cent of that obtained under aerobic conditions. This is in accordance with results obtained by others in chick embryo cartilage where there was inhibition of incorporation into chondroitin sulfate under anaerobic conditions (46). The lathyrogenic agents used significantly depress sulfate-S 85 incorporation under aerobic conditions (Fig. 2). Only AAN was tested under anaerobic conditions, with a similar depressant effect being found.

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392 METABOLIC EI~FECTS OF LATHYROGENS ON CARTILAGE

3. Glucose-U-C 14 Incorporation into Cartilage Slices: Lathyrogenic agents depressed the incorporation of glucose-U-C 14 into cartilage slices; once again the effect was more obvious under anaerobic conditions (Fig. 3).

4. Incorporation of Glycine-l-O 4 into Cartilage Slices: The effect of lathyrogenic agents on glycine-l-C 14 incorporation into cartilage slices is shown in Fig. 4. MAAN had an inhibitory effect on incorporation which was more marked under anaerobic conditions. ME had a marked effect under aerobic conditions, but AAN had no apparent effect in the concentrations tested under both aerobic and anaerobic conditions.

> I,- <

u_

laJ n or)

150

AEROBIC ANAEROBIC

N M A B B N M A B B 0 A A A A O A A A A R A N P P R A N P P M N N N M N N N A X5 A X5 L L

I00

50

0 6 .5 4 .5 6 3 4 3

NUMBER OF OBSERVATIONS FI6. 3. Effect of lathyrogenic agents on glucose-U-C 14 incorporation by calf cartilage slices.

Final concentration of lathyrogens was 2 #~/ml . (BAPN X 5 = 10 #M/ml.). The results are expressed as per cent of normal aerobic control values. S and s have the same meaning as in Fig. 2.

5. Dose-Response Curve: Effect of A A N on Sulfate-S 35 Incorporation into Calf Cartilage Slices: A typical dose-response curve for the incorporation of sulfate-S 35 into cartilage slices in the presence of varying concentration of AAN is shown in Fig. 5.

6. Effect of Glutamine on M A A N Inhibition of Incorporation of Sulfate-S 8s Glycine-l-C 14, and Gtucose-U-O 4 into Cartilage and Organic Acid Production: The effect of glutamine on the inhibition of sulfate-S 35 incorporation by MAAN is shown in Fig. 6. The incorporation of sulfate-S 35 by normal cartilage was stimulated in the presence of glutamine, confirming the results of others (50). The inhibitory effect of MAAN (2 t~M/ml.) on sulfate-S as incorporation was reversed by glutamine at a concentration of 5 #g./ml. (0.03 ~ / m l . ) .

Similar results were obtained in regard to glycine-l-C 14 incorporation (Fig.

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AEROBIC

N M A M

2001 L

1 5 0

r ,oo I I

0 6 6 4

ANAEROBIC

N M A 0 A A

t

NUMBER OF OBSERVATIONS FIO. 4. Effect of lathyrogenic agents on glycine-l-C 14 incorporation by calf cartilage slices.

Final concentration of lathyrogens was 2/z~/ml. The results are expressed as per cent of normal aerobic control values. S has the meaning assigned in Fig. 2.

>.- I--- > t--- (J

LL (.) ILl Q.. 03

J 0 r,,'

0 (..) LL 0

,,=, 0.1.

130

120

I10

I00 •

90

80

70

50

40

30

20

I I I I I I I I I I I I I I I I00 50 20 I0 5.0 2.0 1.0 0.5 0 2 0.1 0.05 0.02 0.01 0.005 0.002 0.001

LOG AAN ( MG. / ML.) FIG. S. Dose response curve: effect of AAN on sulfate-S 85 incorporation by calf cartilage

slices under aerobic conditions. Results expressed as per cent of normal untreated control values. Each point is the mean of two observations.

393

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2 0 0

>- t - t50 I - -

I 0 0 _o 14. 0 " ' 5 0 U')

O ....... 0 5 2O 2O0

GLUTA MINE (.,ug./ml.)

FIO. 6. Effect of glutamine on sulfate-S 86 incorporation by calf cartilage slices in the presence of MAAN under air-CO2. Final concentration of MAAN was 2 #x*/ml. The results are expressed as per cent of values from normal control slices untreated with MAAN or glutamine. S refers to a significance of P < 0.01 when the values for cartilage slices treated with MAAN in the absence of glutamine are compared with normal untreated control slices. Solid columns: values obtained in the presence of MAAN; open columns: values obtained in absence of MAAN. Each bar is the mean of six observations.

3 0 0

25O

I . - 200 I - - c

o '~ 150 N T

I i O

100 13_ I f )

50

0 I0 5 0 200

GLUTAMINE (.pg./rnl.) FIG. 7. Effect of glutamine on glycine-l-C 14 incorporation by cartilage slices in the presence

of MAAN under air-CO2. Final concentration of MAAN was 3 #M/ml. The results are expressed as per cent of values from normal control slices untreated with MAAN or glutamine. S has the meaning ascribed in Fig. 6. Solid columns: values obtained in the presence of MAAN; open columns: values obtained in absence of MAAN. Each bar is the mean of six observations.

394

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M. J. KARNOVSKY AND ~I. L. KARNOVSKY 395

7). Again glutamine had a marked stimulating effect on glycine-l-C 1. incorporation by normal cartilage. The inhibitory effect of MAKN (3/zM/ml.) on glycine-l-C 14 incorporation was overcome by glutamine at a concentration of 10/~g./ml. (0.06/z~r/ml.).

The effect of glutamine on the incorporation of glucose-U-C 14 in the presence of MAAN (3 #M/ml.) was similar: once again there was marked stimulation of incorporation by normal cartilage, and reversal of the depressant action of MAAN, but not quite as completely as in the previous instances (Fig. 8).

2 5 0

>.. 2 0 0 I-. >_ P 150 o

o ,T I 0 0 o bJ 12. oo 5 0

0

G LUTAMINE (,ug./ml.)

Fio. 8. Effect of glutamine on incorporation of glucose-U-C l* by cartilage slices in the presence of MAAN under air-CO,. Final concentration of MAKN was 3 #•/ml. The results are expressed as per cent of values from normal control slices untreated with MAAN or glutamine. S has the meaning ascribed in Fig. 6. Solid columns: values obtained in presence of MAAN'; open columns: values obtained in absence of MAAN. Each bar is the mean of six observations.

The effect of glutamine on glycine-l-C 14 incorporation in the presence of ME is shown in Fig. 9. ME (0.7 /z~c/ml.) had a marked inhibitory effect on glycine-l-C 14 incorporation, which was not overcome by glutamine at a concentration of 0 . 6 / ~ / m l . In this respect, as well as others, ME appeared to be the most potent of the lathyrogenic agents studied in vitro.

The effect of glutamine on organic acid production in the presence of MAAN and ME respectively is shown in Fig. 10. Glutamine stimulated the production of acid by normal cartilage: this stimulatory effect was greater under anaerobic conditions. There was a tendency for glutamine to reverse the inhibitory effects of MAAN (3 jz~/ml.) and ME (3 /zM/ml.), but the reversal was not as complete as was noted above for sulfate-S 35 and glycine-l-C I* incorporations.

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:550

300

25O >- I-- 200-

0 ,7" 150

8 ~ I00

5O

0

8

oR L

0 20

GLUTAMINE

80

(.pg./mO Fro. 9. Effect of glutamine on incorporation of glycine-l-C 14 by calf cartilage slices in the

presence of ME under air CO2. Final concentrations of ME were 0.7/z~/ml. and 3.5 #M/ml. The results are expressed as per cent of values from normal control slices untreated with ME or glutamine. S has the meaning ascribed in Fig. 6. Hatched columns: values obtained in presence of ME (0.7/zM/ml.); cross-hatched: values obtained in presence of ME (3.5/z~/ml.); open columns: values obtained in absence of ME. Each bar is the mean of six observations.

ANAEROBIC AEROBIC

- 200 _1

z ° c o ~ 0

~ N

~ o ° T to D ~ R 1:3o 0 '~(n ~C~ ,.,.n"~Oa'"'~c" _-I00~

0 2 5 200 0

GLUTAMINE Cug./ml.)

I 2 5

FIG. 10. Effect of glutamine on organic acid production by cartilage slices in presence of MAAN and ME under aerobic and anaerobic conditions. Final concentrations of lathyrogens were 3 #M/ml, The results are expressed as per cent of values from normal control slices untreated with lathyrogen or glutamine. S as in Fig. 6. Hatched columns: values obtained in presence of MAAN; cross-hatched columns: values obtained in presence of ME; open columns: values obtained in absence of lathyrogens. Each bar is the mean of two observations (MAAN) and three observations (ME).

396

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M. J. KARNOVSKY AND ~[. L. KARNOVSKY 397

Chemical analysis showed that the increment in acid production induced by glutamine was largely due to increased lactate production. In a representative experiment the inclusion of 1.5/z~ of glutamine per ml. of medium induced an increment of 0.37 #~ of acid per 100 rag. cartilage (fresh weight) per hour as measured manometrically, and of 0.34/z~r of lactate as measured chemically: the incubation was performed anaerobically.

DISCUSSION

Lathyrogenic agents were found to induce profound effects on the metabolism of cartilage cells. In particular, the biosynthesis of sulfated acid mucopolysaccharides appeared to be affected, and effects were obtained with these agents in experiments both in vivo and in vitro.

We chose to work with rats suffering from acute lathyrism so that the disease could be analyzed in as "pure" a form as possible. This eliminated the complications of resistance and hyperadrenalism which are known to develop (8). Reactive changes such as marked subperiosteal bone formation and healing, which would have complicated analysis of the lesions, were also avoided in this way. The weight changes which occur in lathyrism were controlled as far as was possible. Results obtained under our experimental conditions are not directly comparable with those of other workers who failed, in longer term studies, to find changes in sulfated acid mucopolysaccharide metabolism.

The hexosamine levels of cartilage were unchanged in our experiments, as in other studies (5, 26-28). However, the concentration of hexosamine is not a specific measure of the galactosamine of chondroitin sulfate, since considerable amounts of other mucopolysaccharides containing hexosamine (e.g. glucosamine of keratosulfate) may also be present in cartilage (44). A marked decrease in the galactosamine content of lathyric rabbit cartilage has, indeed, been reported (29, 30). This observation and the decrease in uronic acid levels obtained in our work indicate that the sulfated acid mucopolysaccharide (chondroitin sulfate) content of lathyric cartilage is diminished. An increased water content of lathyric tissues has previously been reported (9, 27, 33), and a possible mechanism for the markedly increased water content of epiphyseal cartilage in lathyrism has previously been suggested (33). The absence of any change in hydroxyproline content (as an index of total collagen) is in agree- ment with others (9, 11, 29).

Our histochemical observations showing increased metachromasia, alcian blue, and aldehyde-fuchsin staining, and the similar findings of others, must be interpreted with caution because of our failure to demonstrate chemically a quantitative increase in the mucopolysaccharide content of the lathyric tissues. The increase in staining intensity probably reflects merely an increase in the number of anionic groups available for binding the dyes. The increased staining for acid mucopolysaccharides (12-17) suggests that lathyrism produces Some, as yet undefined, steric change in these components of connective tissue.

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398 METABOLIC EFFECTS OF LATHYROGENS O N CARTILAGE

The pitfalls in interpreting data obtained on the incorporation of sulfate-S a5 into cartilage in in vivo experiments and the requirements for estimating rates of synthesis and turnover have recently been discussed (47). Among the modi- fying factors which may cause difficulty in the study of pathological conditions are differences between the sulfate pools in control and experimental groups and factors which modify the growth rate of cartilage cells, such as differences in dietary intake between control and experimental groups (48). Depression of growth causes a non-specific decrease in the sulfate-S 36 incorporation into cartilage (48).

Under our experimental conditions, lathyric rats lost weight at a greater rate than their pair-fed controls, but the weight differences were small (c/. reference 48). Further, in our experiments, both when the controls lost less weight than the lathyric rats, and when the controls were paired by weight loss to their experimental partners, a decreased incorporation of sulfate-S 35 into whole cartilage and into chondroitin sulfate was obtained. Eventual failure to grow did become a limiting factor in our experiments and at that stage both lathyric and control rats showed a low incorporation of sulfate-S .5 without any observable difference between the two groups.

On the basis of autoradiographic evidence, an increased rate of turnover of sulfated mucopolysaccharides in lathyric epiphyseal cartilage has been suggested (22). Whether decreased incorporation of sulfate-S 35 reflects a decreased rate of synthesis or a changed rate of turnover of sulfated mucopolysaccharides cannot be deduced from the experiments in vivo. Further, chondroitin sulfate may possibly be lost from the cartilage matrix by diffusion, due to decreased binding of chondroitin sulfate to the protein moiety of chondromucoprotein, or due to changes occurring in other components of the matrix, such as collagen. It has been suggested that loss of chondroitin sulfate from the matrix occurs owing to depolymerization, and this has been associated with increased mucoprotein and hexosamine levels in the blood of lathyric rabbits (17). The difficulty in interpreting the results with respect to the rate of synthesis of sulfated mucopolysaccharides is extreme in the case of the epiphyseal cartilage of weanling rats, a tissue which is not in a state of equilibrium, but has a rapid growth rate and a turnover period of the cells of two days (49).

The use of an in vitro system obviates many complicating factors. The incorporation of sulfate-S .5 into sulfated mucopolysaccharides was again shown to be decreased in the presence of lathyrogenic agents. The incorporation of glucose-U-C z4 was similarly decreased. Rod6n et al. have shown that counting directly on the whole slice is a reasonable reflection of the incorporation of both sulfate-S 85 and glucose-U-C 14 into chondroitin sulfate (41, 50, 51). Our glycine-l-C z4 studies probably reflect, at least in part, the biosynthesis of the protein moiety of chondromucoprotein, and of other proteins as well. I t is probable, therefore, that the inhibitory effects of lathyrogenic agents observed on the incorporation of labeled precursors reflect a decrease in the synthesis of

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~. ~. KARNOVSKYANDM. L. KARNOVSKY 399

the whole molecule of chondromucoprotein. Decreased incorporation of glycine- 1-C x4 into proteins has also been observed in vivo in lathyric rats: it was suggested that the lathyrogen primarily affects amino acid incorporation into collagen or ground substance, rather than into cellular proteins (52).

Recently glutamine has been shown to stimulate sulfate-S 35, glucose-U-C 14, and C14-acetate incorporation into chondroitin sulfate of cartilage slices (41, 50, 51). Partial protection in vivo against lathyrogenic agents was found when glutamine was included in the diet (53). In addition, the enzymatic synthesis of hexosamine from glutamine and glucose-6-phosphate in epiphyseal plates of lathyric rabbits was markedly impaired (54).

We found marked stimulation of all the aspects of cartilage metabolism which we have investigated in vitro, in the presence of glutamine. The concentration of glutamine which produced the maximal stimulation of sulfate-S a5 and glucose-U-C 14 incorporation into the whole slice was approximately the same as that found by others (50, 51). The stimulatory effects on organic acid production and glycine-l-C 14 incorporation have hitherto not been reported to our knowledge. Our results showed that in the presence of glutamine the inhibitory effects of lathyrogenic agents were overcome to some extent, in that the metabolic levels were restored to approximately those obtained with normal cartilage. However, these levels fell below the degree of stimulation caused by glutamine in cartilage not treated with lathyrogens.

The relationship of the changes in the metabolism of acid mucopolysaccharides to changes in the state of molecular aggregation of collagen reported by others (9-10) is at present a matter of speculation. The role of acid mucopolysaccharides in the formation of collagen fibers and in maintaining the stability of the formed fibers has not been conclusively demonstrated in vivo, but there has been much suggestive evidence obtained in systems in vitro (55-58). An understanding of the relationship of the changes in mucopolysaccharides to the changes in collagen which occur in osteolathyrism requires further elucida- tion of the role, if any, of acid mucopolysaccharides in collagen fiber formation and stability.

SUMMARY

The effects of lathyrogenic agents in dvo and in ~tro are described, in respect to some biochemical indices of cartilage metabolism. Lathyrogenie agents in vivo inhibited the incorporation of radiosulfate into rat epiphyseal cartilage and the isolated chondroitin sulfate. No significant changes in hydroxyproline or hexosamine content of epiphyseal cartilage were found, but there was a marked increase in water content. The content of chondroitin sulfate, measured as uronic acid, was decreased. The importance of taking growth rate differences between control and experimental rats into account in assessing the effects of lathyrogenic agents in vivo is emphasized.

In an in vitro system, utilizing fresh calf costal cartilage slices, the presence

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400 MIETABOLIC EFFECTS OF LATHYROGENS ON CARTILAGE

of low concentrations of lathyrogenic agents markedly affected various metabolic events. The incorporation into cartilage slices of sulfate-S 85, glucose- U-C 14, and glycine-l-C 14 was significantly depressed, as was the production of organic acids, including lactic acid. In general, these effects were more severe under anaerobic conditions. Glutamine restored the activities of the slices treated with lathyrogenic agents to control values obtained in the absence of either lathyrogen or glutamine.

The authors are grateful to Mrs. Peggy Shepard and Mrs. Rosanne Pruneau for skilled technical assistance.

Aminoacetonitrile was kindly supplied by Dr. George H. Berryman, Abbott Laboratories, North Chicago.

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4O2 METABOLIC EFFECTS OF LATHYROGENS ON CARTILAGE

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M. J. KAR~OVSKY AND M. L. KARNOVSKY 403

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