ASCORBIC-2-SULFATE METABOLISM BY HUMAN FIBROBLASTS * A. D. Bond Division of Science and Mathernatics Columbus College Columbus, Georgia 31 907
Introduction
Ascorbic-2-sulfate was first synthesized and studied in connection with its sulfate transferring properties.' I Its discovery in brine shrimp cysts and later in a variety of animal tissues suggested such a role in vivo. While the function of ascorbate is still incompletely understood, it has long been known to play a role in the sulfation of connective tissues. On the assumption that ascorbic-2-sulfate participated directly in the biosynthesis of mucopolysaccharides, ascorbi~-2-["~S]s~1Ifate was synthesized and its metabolism studied in ascorbutic guinea pigs. Results were inconclusive because of its rapid excretion from these animals.? Fibroblasts in culture are known to synthesize both collagen and mucopolysaccharides. In addition, ascorbic sulfate in the culture medium can not be excreted. Consequently, we set out to study metabolism of ascorbic-2-sulfate in human fibroblasts in vitro and to relate this to the function of ascorbate in human metabolism. Methods and Procedure
Human skin fibroblasts were subcultured on Eagle's minimum essential medium with 15% fetal calf serum at 3 7 ° C and, usually, with 250 pg/ml ascorbate. Medium was changed daily after the cells had reached approximately -7/4 of confluency. Because the effect of ascorbic sulfate on cells and its mode of metabolism were unknown, several experimental regimes were used. Cells were first cultured in a medium containing a supplement of 1 m M diammonium ascorbic-2-sulfate and their rates of growth were compared to cells receiving ascorbate. Representative cultures were treated with 0.25 % trypsin and shaken loose, and the number of cells per flask were counted. The medium was supplemented at various stages of growth and at various periods after confluency by ascorbi~-2-[:~~SS]suIfate, prepared as previously described.8 Permeability of cells to ascorbic sulfate was determined by separating cells from the medium, washing briefly, then counting aliquots of medium and of homogenized cells in BBOT-Toluene in a Packard Tricarb liquid scintillation counter.
* This work was jointly sponsored by the Biology Division of Oak Ridge National Laboratories and USPHS, NIH, Grants IF03 AMS1218 and 2F03 AM 51218-02. A travel contract from Oak Ridge Associated Universities also assisted these studies. .I- Unpublished observations. 307
308
Annals New York Academy of Sciences
Sodium [5S]lsulfate (New England Nuclear) was added to the medium at various stages of cell growth and after confluency, in the presence and absence of ascorbate. The total incorporation and types of molecules into which sulfate was incorporated were both examined. Some cultures were supplemented with 0.05 mCi l-[lC]ascorbate (New England Nuclear) with a total ascorbate content of 175 pg/ml. These cultures were then examined in the same way as those receiving ascorbic sulfate or inorganic sulfate supplements. The medium was removed and an aliquot centrifuged at 100,000 X ,q for 5 hours in a 5-20% sucrose buffer containing 0.05 M Tris buffer, pH 8.0. The gradient was dripped onto paper disks and counted in BBOT-Toluene. Combined medium and homogenized cells were filtered through a UM-10 ultrafilter (DiaflowB) , followed by washing until filtrate counted background. The retentate was concentrated, counted for total activity and electrophoresed on Whatman No. 1 paper at 200 V for 90 minutes. Migration was determined by cutting thin strips and counting them in BBOT-Toluene, using heparin and chondroitin sulfate B as standards. Duplicates were stained for mucopolysaccharides or protein. Attempts were made to precipitate mucopolysaccharidcs from the retentate with tetraalkyl ammonium ions and to coprecipitate them with chondroitin sulfate B. Aliquots were additionally fractionated on G-200 resin (Sephadexe’) and the fractions assayed for radioactivity and protein. Low-molecular-weight compounds were fractionated either from the UM-10 ultrafiltrate or from the medium following deproteinization with HCIO,,. The fractionation procedure was the same used to purify ascorbic-2-sulfate from synthetic preparations, as previously described.:’ Particular interest was directed to detection of labeled ascorbic-2-sulfate from preparations containing [Y3] or [“C].Because specific activities were high and anticipated yields low, a marker of unlabeled ascorbic sulfate was added just prior to fractionation. Fractions containing unknown labeled compounds were separated and purified and characterization was begun. In particular, a fraction labled “Y” was chromatographed, hydrolysed, and subjected to qualitative analyses, and some derivatives were prepared. A trimethylsilylated derivative was prepared and fractionatcd by GLC, and the volatile fractions were submitted to analysis by mass spcctroscopy. R esii Its cind Discussion
FIGURE1 represents the growth curves for cells receiving respectively ascorbate and ascorbic sulfate. The time necessary to reach confluence was always a little longer in the presence of ascorbic sulfate but did not appear to be significantly so. Ascorbic-Z-[ ’5SS]sulfateseems passively permeable; the ratio of corints in the washed cell layer to those in the medium was not significantly different from the ratio of cell volume to media volume. Metabolism of ascorbic sulfate in young, rapidly growing cultures is limited. With cells approximately % grown, only about 5% of ascorbi~-2-(:’~S]sulfate was changed even after 36 hours. At this stage of growth, little [ : T I sulfate was taken up either and no detectable synthesis of ascorbi~-2-[:~~S]sulfate occurred. Earlier observations of Upton ’’ indicated that in live animals little sulfate uptake occurred during wound healing until several days after wound closure. Therefore, we maintained fibroblasts for 5 days postconfluence before supplementing with the isotopic tracer. Twenty to twenty-five percent of a
Bond : Ascorbic-2-Sulfate Metabolism
309
FIGURE I . Growth of fibroblasts on MEM (EaIOC gle's) with 15% fetal calf serum. Cells grown P in medium supplemented with 250 gg/ml sodium X ascorbate represented by (-) and those in 1 m M diamnionium ascorhic-7- V
e
1
sulfate, by (- - -).
to
/
7
a ' e ' e ' Ib
TIME AFTER PLANTING (days) 1 mM ascorbic sulfate solution was then altered within 24 hours. Also, 1-["C]ascorbatc disappeared rapidly from these fibroblasts, but, as anticipated, it was destroycd in sterile medium as well. However, a small amount of ['Clascorbic2-sulfate was detected in these cells. Similarly, when these aged fibroblasts were was detected. Only given [:'fiS]sulfate, a small amount of [:43S]ascorbic-2-s~ilfate very small concentrations of ascorbic sulfatc were formed. It was interesting that I-['"C]ascorbate of fairly low specific activity gave higher counts in a (TABLE1 ) . A logical sulfated derivative than high-specific-activity [:+~SS]sulfate explanation would be that the sulfate is predominantly derived from a nonsulfate source. McCulley has reported a similar observation in an ascorbate-requiring process of cells having a n inborn error in sulfur-amino-acid metabolism. TABLEI INCORI'ORATION
Source I -["C]Ascorbate ["S]Sodium sulfate
OF
["CI
FROM l-["C]ASCORBATE ["S1SLJl.PATE XNTO C O M P O U N D
AND OF
["s] F R O M
"Y"
Approximate Specific Activity
Counts per Minute in Compound "Y"
0.3 mCi/mmole
3 00 34
3 rnCi/rnmole
310
Annals New York Academy of Sciences TARIE2
[“s]
INCORPORATION OF FROM SULFATE A N D FROM ASCORBIC-2-SULFATE INTO MUCOPOL.YSACCHARIDES ( MPS) BY FIBROBLASTS
Specific activity Total counts MPS in medium MPS in cell layer Percent total counts incorporated
[:“slSUl fa te
Ascorbic-?i-[”S] Sulfate
3.3 mCi/mmole 2 6 . 6 10’cpm ~ 3 1,700 cpm 13,300 cpm
0.34 mCi/mmole 1 3 . 2 10“ ~ cpm 3,500 cpm 1,080 cpm
0.2%
0.03 %
The number of sulfated molccules retaincd by ultrafiltration did increase rapidly as the cells matured in the presence of ascorbate. Similar increases were seen using ascorbic-2-[:~‘S]sulfate. The incorporation pattern suggested that 2). That ascorbic sulfate was used at least as well as inorganic sulfate (TABLE is, sulfate of 10 times greater activity was incorporated at less than 10 times the rate of ascorbic sulfate and. of course, only about 20% of the latter was hydrolysed. It was observed that in the absence of ascorbate the amount of inorganic sulfate incorporated in polymer did not increase as rapidly. Furthermore, the labeled material did not migrate with the mucopolysaccharides, heparin, or chondroitin sulfate B, nor were they coprecipitated with it. Fractionation on G-200 Sephadex showed that the principle material formed in the absence of ascorbate was proteinaceous. In the presence of ascorbate, radioactivity was 2). not associated with the protein fraction (FIGURE Fractionation of low-molecular-weight components by the procedure used to purify ascorbic sulfate allowed us to assay for this important compound 2 significantly while isolating other metabolites. From ascorbi~-2-[~~~SS]sulfate, labeled species were detected. One, fraction B, was absorbed in acidic charcoal but washed free in water, whereas ascorbic sulfate required an NH,OH-ethanol eluting solvent. This molecule had no uv absorption but behaved chromatographically like ascorbic sulfate. A second species behaved, in the isolation procedure, like inorganic sulfate but was not coprecipitated with BaCI, and H,SO,. Identification of these was postponed with attention diverted to an unknown compound, “Y,” which appeared together with labeled ascorbic sulfate in media supplemented with [35S]sulfate or 1-[‘‘C]ascorbate. In addition, “Y” was found to be formed in cultures treated with labeled ascorbic sulfate. From labeled ascorbate or sulfate, “Y” bore more counts than the ascorbic sulfate detected. This molecule is difficult to distinguish from ascorbic sulfate in many ways, except that it does not absorb uv light significantly. Unfortunately, the quantity of this substance is very small, making its identification difficult. However, its ubiquitous distribution with greater label than ascorbic sulfate suggests that it could provide an important clue to ascorbate metabolism. Compound Y is readily hydrolysed in dilute HCI. When it is derived from [Y3]sulfatc, all radioactivity appears in the precipitate formed by treating the hydrolysate with BaC1, and H,SO,. We could detect the hydrolysed compound by I, on chromatographic sheets. Qualitative tests suggested a carbohydrate-like molecule.
Bond : Ascorbic-2-Sulfatc Metabolism
31 1
Conversion of the hydrolysed Y to volatile trimethylsilylated derivatives allowed separation of 4 major components. Mass spectroscopy of these indicated they represented thc parent molecule increasingly trimethylsilylated. Comparison of the tetra-substituted derivative's disintegration patterns with those of ascorbic sulfate and ascorbic acid derivatives was made, Both ascorbic acid and ascorbic sulfate produced the same fragmentation pattern. Sulfate is apparently rapidly cleaved in the trimethylsilylating medium. The pattern from the TMS 3 ) has a mass exceeding that of the correderivative of compound Y (FIGURE sponding ascorbic acid derivative by 2 units. This is just the mass increase
FRACTION NUMBER
50
FIGURE 2. [%]Sulfate containing fractions from G-200 (Sephadex). Counts per minute, CPM, represented by (-) and concentration of protein (- - - -) from cells receiving no ascorbate supplement. Cells receiving ascorbate had CPM represented by (---) and concentration of protein (. . . . . ).
Annals New York Academy of Sciences
312
expected if ascorbic sulfate had had its double bond reduced. The use of deuterated TMS derivatives produced evidence that there were 4 -OH groups silylated as in ascorbate. However, the heavier-mass fragments represented primarily the loss of CH,- or TMS-groups typical of these types of derivatives, and we were unable to decipher an identifiable pattern. Ascorbate derivatives have previously been observed to undergo various internal cyclizations or rearrangements,ll, la which could explain a loss of uv absorption, and we
I
374
419 464
I , .)I.
I
Ascorbic
d8
.d
.a
259
332
Acid- 4TMS
_.
FIGURE 3. Mass spectrum of volatile TMS derivatives of ascorbic acid, ascorbic-2-sulfate and compound Y. The portion of the spectrum reproduced is, in each case, for the tetra-substituted derivative.
Ascorbic -2- sulfate - 4TMS
Compound Y-
1 31:1
1 502
473
388
Compound Y-4TMScH)
..
A
.
r
.
P
..
3
1..
.-,
cannot rule out ascormc-3-suirate as one 01 rne orner meraooiites. w e nave searched diligently in both natural and synthetic reaction mixtures for it without success. We conclude that fibroblasts utilize and synthesize ascorbic-2-sulf ate at distinct periods in their development, at times utilizing ascorbic sulfate at least as readily as inorganic sulfate. The prominent occurrence of other sulfated ascorbate metabolites and evidence of reduction suggest that sulfate transfer is only one role of this compound.
Bond: Ascorbic-2-Sulfate Metabolism
31 3
Acknowledgments The cooperation and guidance of F. J. Finamore and J. Regan, in whose laboratories the work was done, and the assistance of W. Rainey with mass spectroscopy is gratefully acknowledged. Ref erencrs
1 . FORD,E. A. & P. M. RUOFF. 1965. Chem. Comrnun. : 630. 2. MUMMA,R. 0. 1968. Biochem. Biophys. Acta 165: 571. 1972. 3. BOND,A. D., B. W. MCCLELLAND, J. R . EINSTEIN& F. J. FINAMORE. Arch. Biochem. Biophys. 153: 207. 4. CHU, T. M. & W. R. SLAUNWHJTE, JR. 1968. Steroids 12: 309. 5. MEAD,C. G. & F. J. FINAMORE.1969. Biochemistry 8: 2652. 6. MUMMA,R. 0. & A. I. VERLANGIERI. 1971. Fed. Proc. 30: 370. B. M., D. J. ISHERWOOD, R. W. ATCHELY & E. M. BAKER.1971. Fed. 7. TOLBERT, Proc. 30: 529. 8. BAKER,E. M., 111, D. C. HAMMER, S. C. MARCH,B. M. TOLBERT & J. E. CANHAM. 1971. Science 173: 826. 9. UPTON, A. C. & T. T. ODELL,JR. 1956. Arch. Path. 62: 194. K . S. 1972. Amer. I. Path. 66: 83. 10. MCCULLY, W. N., E. L. HIRST& J. K. N . JONES. 1937. J. Chem. SOC.: 549. 1 1 . HAWORTH, 12. JACKSON, K. G. A. & J. K. N. JONES. 1965. Can. J. Chem. 43: 450.
DISCUSSION (Hoflrnann-La Roche, Nutley, N.J.) : Did you subject the DR. S. SHAPIRO sulfate of the macromolecule to proteolytic digestion? DR. BOND: Yes we did. We then attempted to precipitate chondroitin sulfate, but were unable to. DR.SHAPIRO:Well, you mentioned it resembled a sulfate with protein. DR.BOND: In the absence of ascorbate you have a material that looks like a sulfated protein. In the presence of ascorbate or ascorbic sulfate, you have a material that appears to be a mucopolysaccharide. DR.SHAPIRO: What was the basis for this conclusion? DR. BOND: They stain histologically as protein or mucopolysaccharide. DR. J. GROSS: Are you sure your compound Y is not a high molecular weight compound? DR. BOND: It should not be because it passes through the filter readily and acts very much like ascorbate sulfate. The mass spectrum and so forth supports this. DR.B. M . TOLBERT:I was rather curious, regarding your last slide, whether there was any possibility your trimethylsilyl derivative of the ascorbate sulfate might have desulfated and actually been a trimethylsilyl derivative of ascorbic acid. The similarity between patterns were certainly remarkable. DR.BOND: They are identical.
Introduction
Ascorbic-2-sulfate was first synthesized and studied in connection with its sulfate transferring properties.' I Its discovery in brine shrimp cysts and later in a variety of animal tissues suggested such a role in vivo. While the function of ascorbate is still incompletely understood, it has long been known to play a role in the sulfation of connective tissues. On the assumption that ascorbic-2-sulfate participated directly in the biosynthesis of mucopolysaccharides, ascorbi~-2-["~S]s~1Ifate was synthesized and its metabolism studied in ascorbutic guinea pigs. Results were inconclusive because of its rapid excretion from these animals.? Fibroblasts in culture are known to synthesize both collagen and mucopolysaccharides. In addition, ascorbic sulfate in the culture medium can not be excreted. Consequently, we set out to study metabolism of ascorbic-2-sulfate in human fibroblasts in vitro and to relate this to the function of ascorbate in human metabolism. Methods and Procedure
Human skin fibroblasts were subcultured on Eagle's minimum essential medium with 15% fetal calf serum at 3 7 ° C and, usually, with 250 pg/ml ascorbate. Medium was changed daily after the cells had reached approximately -7/4 of confluency. Because the effect of ascorbic sulfate on cells and its mode of metabolism were unknown, several experimental regimes were used. Cells were first cultured in a medium containing a supplement of 1 m M diammonium ascorbic-2-sulfate and their rates of growth were compared to cells receiving ascorbate. Representative cultures were treated with 0.25 % trypsin and shaken loose, and the number of cells per flask were counted. The medium was supplemented at various stages of growth and at various periods after confluency by ascorbi~-2-[:~~SS]suIfate, prepared as previously described.8 Permeability of cells to ascorbic sulfate was determined by separating cells from the medium, washing briefly, then counting aliquots of medium and of homogenized cells in BBOT-Toluene in a Packard Tricarb liquid scintillation counter.
* This work was jointly sponsored by the Biology Division of Oak Ridge National Laboratories and USPHS, NIH, Grants IF03 AMS1218 and 2F03 AM 51218-02. A travel contract from Oak Ridge Associated Universities also assisted these studies. .I- Unpublished observations. 307
308
Annals New York Academy of Sciences
Sodium [5S]lsulfate (New England Nuclear) was added to the medium at various stages of cell growth and after confluency, in the presence and absence of ascorbate. The total incorporation and types of molecules into which sulfate was incorporated were both examined. Some cultures were supplemented with 0.05 mCi l-[lC]ascorbate (New England Nuclear) with a total ascorbate content of 175 pg/ml. These cultures were then examined in the same way as those receiving ascorbic sulfate or inorganic sulfate supplements. The medium was removed and an aliquot centrifuged at 100,000 X ,q for 5 hours in a 5-20% sucrose buffer containing 0.05 M Tris buffer, pH 8.0. The gradient was dripped onto paper disks and counted in BBOT-Toluene. Combined medium and homogenized cells were filtered through a UM-10 ultrafilter (DiaflowB) , followed by washing until filtrate counted background. The retentate was concentrated, counted for total activity and electrophoresed on Whatman No. 1 paper at 200 V for 90 minutes. Migration was determined by cutting thin strips and counting them in BBOT-Toluene, using heparin and chondroitin sulfate B as standards. Duplicates were stained for mucopolysaccharides or protein. Attempts were made to precipitate mucopolysaccharidcs from the retentate with tetraalkyl ammonium ions and to coprecipitate them with chondroitin sulfate B. Aliquots were additionally fractionated on G-200 resin (Sephadexe’) and the fractions assayed for radioactivity and protein. Low-molecular-weight compounds were fractionated either from the UM-10 ultrafiltrate or from the medium following deproteinization with HCIO,,. The fractionation procedure was the same used to purify ascorbic-2-sulfate from synthetic preparations, as previously described.:’ Particular interest was directed to detection of labeled ascorbic-2-sulfate from preparations containing [Y3] or [“C].Because specific activities were high and anticipated yields low, a marker of unlabeled ascorbic sulfate was added just prior to fractionation. Fractions containing unknown labeled compounds were separated and purified and characterization was begun. In particular, a fraction labled “Y” was chromatographed, hydrolysed, and subjected to qualitative analyses, and some derivatives were prepared. A trimethylsilylated derivative was prepared and fractionatcd by GLC, and the volatile fractions were submitted to analysis by mass spcctroscopy. R esii Its cind Discussion
FIGURE1 represents the growth curves for cells receiving respectively ascorbate and ascorbic sulfate. The time necessary to reach confluence was always a little longer in the presence of ascorbic sulfate but did not appear to be significantly so. Ascorbic-Z-[ ’5SS]sulfateseems passively permeable; the ratio of corints in the washed cell layer to those in the medium was not significantly different from the ratio of cell volume to media volume. Metabolism of ascorbic sulfate in young, rapidly growing cultures is limited. With cells approximately % grown, only about 5% of ascorbi~-2-(:’~S]sulfate was changed even after 36 hours. At this stage of growth, little [ : T I sulfate was taken up either and no detectable synthesis of ascorbi~-2-[:~~S]sulfate occurred. Earlier observations of Upton ’’ indicated that in live animals little sulfate uptake occurred during wound healing until several days after wound closure. Therefore, we maintained fibroblasts for 5 days postconfluence before supplementing with the isotopic tracer. Twenty to twenty-five percent of a
Bond : Ascorbic-2-Sulfate Metabolism
309
FIGURE I . Growth of fibroblasts on MEM (EaIOC gle's) with 15% fetal calf serum. Cells grown P in medium supplemented with 250 gg/ml sodium X ascorbate represented by (-) and those in 1 m M diamnionium ascorhic-7- V
e
1
sulfate, by (- - -).
to
/
7
a ' e ' e ' Ib
TIME AFTER PLANTING (days) 1 mM ascorbic sulfate solution was then altered within 24 hours. Also, 1-["C]ascorbatc disappeared rapidly from these fibroblasts, but, as anticipated, it was destroycd in sterile medium as well. However, a small amount of ['Clascorbic2-sulfate was detected in these cells. Similarly, when these aged fibroblasts were was detected. Only given [:'fiS]sulfate, a small amount of [:43S]ascorbic-2-s~ilfate very small concentrations of ascorbic sulfatc were formed. It was interesting that I-['"C]ascorbate of fairly low specific activity gave higher counts in a (TABLE1 ) . A logical sulfated derivative than high-specific-activity [:+~SS]sulfate explanation would be that the sulfate is predominantly derived from a nonsulfate source. McCulley has reported a similar observation in an ascorbate-requiring process of cells having a n inborn error in sulfur-amino-acid metabolism. TABLEI INCORI'ORATION
Source I -["C]Ascorbate ["S]Sodium sulfate
OF
["CI
FROM l-["C]ASCORBATE ["S1SLJl.PATE XNTO C O M P O U N D
AND OF
["s] F R O M
"Y"
Approximate Specific Activity
Counts per Minute in Compound "Y"
0.3 mCi/mmole
3 00 34
3 rnCi/rnmole
310
Annals New York Academy of Sciences TARIE2
[“s]
INCORPORATION OF FROM SULFATE A N D FROM ASCORBIC-2-SULFATE INTO MUCOPOL.YSACCHARIDES ( MPS) BY FIBROBLASTS
Specific activity Total counts MPS in medium MPS in cell layer Percent total counts incorporated
[:“slSUl fa te
Ascorbic-?i-[”S] Sulfate
3.3 mCi/mmole 2 6 . 6 10’cpm ~ 3 1,700 cpm 13,300 cpm
0.34 mCi/mmole 1 3 . 2 10“ ~ cpm 3,500 cpm 1,080 cpm
0.2%
0.03 %
The number of sulfated molccules retaincd by ultrafiltration did increase rapidly as the cells matured in the presence of ascorbate. Similar increases were seen using ascorbic-2-[:~‘S]sulfate. The incorporation pattern suggested that 2). That ascorbic sulfate was used at least as well as inorganic sulfate (TABLE is, sulfate of 10 times greater activity was incorporated at less than 10 times the rate of ascorbic sulfate and. of course, only about 20% of the latter was hydrolysed. It was observed that in the absence of ascorbate the amount of inorganic sulfate incorporated in polymer did not increase as rapidly. Furthermore, the labeled material did not migrate with the mucopolysaccharides, heparin, or chondroitin sulfate B, nor were they coprecipitated with it. Fractionation on G-200 Sephadex showed that the principle material formed in the absence of ascorbate was proteinaceous. In the presence of ascorbate, radioactivity was 2). not associated with the protein fraction (FIGURE Fractionation of low-molecular-weight components by the procedure used to purify ascorbic sulfate allowed us to assay for this important compound 2 significantly while isolating other metabolites. From ascorbi~-2-[~~~SS]sulfate, labeled species were detected. One, fraction B, was absorbed in acidic charcoal but washed free in water, whereas ascorbic sulfate required an NH,OH-ethanol eluting solvent. This molecule had no uv absorption but behaved chromatographically like ascorbic sulfate. A second species behaved, in the isolation procedure, like inorganic sulfate but was not coprecipitated with BaCI, and H,SO,. Identification of these was postponed with attention diverted to an unknown compound, “Y,” which appeared together with labeled ascorbic sulfate in media supplemented with [35S]sulfate or 1-[‘‘C]ascorbate. In addition, “Y” was found to be formed in cultures treated with labeled ascorbic sulfate. From labeled ascorbate or sulfate, “Y” bore more counts than the ascorbic sulfate detected. This molecule is difficult to distinguish from ascorbic sulfate in many ways, except that it does not absorb uv light significantly. Unfortunately, the quantity of this substance is very small, making its identification difficult. However, its ubiquitous distribution with greater label than ascorbic sulfate suggests that it could provide an important clue to ascorbate metabolism. Compound Y is readily hydrolysed in dilute HCI. When it is derived from [Y3]sulfatc, all radioactivity appears in the precipitate formed by treating the hydrolysate with BaC1, and H,SO,. We could detect the hydrolysed compound by I, on chromatographic sheets. Qualitative tests suggested a carbohydrate-like molecule.
Bond : Ascorbic-2-Sulfatc Metabolism
31 1
Conversion of the hydrolysed Y to volatile trimethylsilylated derivatives allowed separation of 4 major components. Mass spectroscopy of these indicated they represented thc parent molecule increasingly trimethylsilylated. Comparison of the tetra-substituted derivative's disintegration patterns with those of ascorbic sulfate and ascorbic acid derivatives was made, Both ascorbic acid and ascorbic sulfate produced the same fragmentation pattern. Sulfate is apparently rapidly cleaved in the trimethylsilylating medium. The pattern from the TMS 3 ) has a mass exceeding that of the correderivative of compound Y (FIGURE sponding ascorbic acid derivative by 2 units. This is just the mass increase
FRACTION NUMBER
50
FIGURE 2. [%]Sulfate containing fractions from G-200 (Sephadex). Counts per minute, CPM, represented by (-) and concentration of protein (- - - -) from cells receiving no ascorbate supplement. Cells receiving ascorbate had CPM represented by (---) and concentration of protein (. . . . . ).
Annals New York Academy of Sciences
312
expected if ascorbic sulfate had had its double bond reduced. The use of deuterated TMS derivatives produced evidence that there were 4 -OH groups silylated as in ascorbate. However, the heavier-mass fragments represented primarily the loss of CH,- or TMS-groups typical of these types of derivatives, and we were unable to decipher an identifiable pattern. Ascorbate derivatives have previously been observed to undergo various internal cyclizations or rearrangements,ll, la which could explain a loss of uv absorption, and we
I
374
419 464
I , .)I.
I
Ascorbic
d8
.d
.a
259
332
Acid- 4TMS
_.
FIGURE 3. Mass spectrum of volatile TMS derivatives of ascorbic acid, ascorbic-2-sulfate and compound Y. The portion of the spectrum reproduced is, in each case, for the tetra-substituted derivative.
Ascorbic -2- sulfate - 4TMS
Compound Y-
1 31:1
1 502
473
388
Compound Y-4TMScH)
..
A
.
r
.
P
..
3
1..
.-,
cannot rule out ascormc-3-suirate as one 01 rne orner meraooiites. w e nave searched diligently in both natural and synthetic reaction mixtures for it without success. We conclude that fibroblasts utilize and synthesize ascorbic-2-sulf ate at distinct periods in their development, at times utilizing ascorbic sulfate at least as readily as inorganic sulfate. The prominent occurrence of other sulfated ascorbate metabolites and evidence of reduction suggest that sulfate transfer is only one role of this compound.
Bond: Ascorbic-2-Sulfate Metabolism
31 3
Acknowledgments The cooperation and guidance of F. J. Finamore and J. Regan, in whose laboratories the work was done, and the assistance of W. Rainey with mass spectroscopy is gratefully acknowledged. Ref erencrs
1 . FORD,E. A. & P. M. RUOFF. 1965. Chem. Comrnun. : 630. 2. MUMMA,R. 0. 1968. Biochem. Biophys. Acta 165: 571. 1972. 3. BOND,A. D., B. W. MCCLELLAND, J. R . EINSTEIN& F. J. FINAMORE. Arch. Biochem. Biophys. 153: 207. 4. CHU, T. M. & W. R. SLAUNWHJTE, JR. 1968. Steroids 12: 309. 5. MEAD,C. G. & F. J. FINAMORE.1969. Biochemistry 8: 2652. 6. MUMMA,R. 0. & A. I. VERLANGIERI. 1971. Fed. Proc. 30: 370. B. M., D. J. ISHERWOOD, R. W. ATCHELY & E. M. BAKER.1971. Fed. 7. TOLBERT, Proc. 30: 529. 8. BAKER,E. M., 111, D. C. HAMMER, S. C. MARCH,B. M. TOLBERT & J. E. CANHAM. 1971. Science 173: 826. 9. UPTON, A. C. & T. T. ODELL,JR. 1956. Arch. Path. 62: 194. K . S. 1972. Amer. I. Path. 66: 83. 10. MCCULLY, W. N., E. L. HIRST& J. K. N . JONES. 1937. J. Chem. SOC.: 549. 1 1 . HAWORTH, 12. JACKSON, K. G. A. & J. K. N. JONES. 1965. Can. J. Chem. 43: 450.
DISCUSSION (Hoflrnann-La Roche, Nutley, N.J.) : Did you subject the DR. S. SHAPIRO sulfate of the macromolecule to proteolytic digestion? DR. BOND: Yes we did. We then attempted to precipitate chondroitin sulfate, but were unable to. DR.SHAPIRO:Well, you mentioned it resembled a sulfate with protein. DR.BOND: In the absence of ascorbate you have a material that looks like a sulfated protein. In the presence of ascorbate or ascorbic sulfate, you have a material that appears to be a mucopolysaccharide. DR.SHAPIRO: What was the basis for this conclusion? DR. BOND: They stain histologically as protein or mucopolysaccharide. DR. J. GROSS: Are you sure your compound Y is not a high molecular weight compound? DR. BOND: It should not be because it passes through the filter readily and acts very much like ascorbate sulfate. The mass spectrum and so forth supports this. DR.B. M . TOLBERT:I was rather curious, regarding your last slide, whether there was any possibility your trimethylsilyl derivative of the ascorbate sulfate might have desulfated and actually been a trimethylsilyl derivative of ascorbic acid. The similarity between patterns were certainly remarkable. DR.BOND: They are identical.
