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Radiochemical Purity of [14C]-Proline: Implications for Measurement of Intracellular Collagen Degradation
Collagen ReI. Res. Vol. 411984, pp. 195-200
Radiochemical Purity of [ 14 C]-Proline:Implications for Measurement of Intracellular Collagen Degradation ROBERT S. BIENKOWSKI Pulmonary Division, Department of Pediatrics, Albert Einstein College of Medicine, Bronx, New York 10461 USA. Address after 1. July 1984: see page 200.
Abstract Measurement of intracellular degradation of newly synthesized collagen requires use of radiochemically pure [14C]-proline. It is particularly important that levels of contaminating hydroxy-[14C]-proline or other radioactive species that migrate with hydroxyproline on cation-exchange resins be very low. The objective of this study was to determine how far the levels of these impurities could be reduced. Commercially available [14C]-proline was subjected to preparative ion-exchange chromatography. The procedure was capable of adequately separating authentic hydroxyproline from proline, however, upon rechromatography of the "purified" isotope on an analytic resin it was found that background radioactivity in the hydroxyproline region could not be reduced below approximately 500 parts per 10 6 parts of [14C]-proline. The contaminants appear to be generated during the procedure itself, suggesting that further efforts to purify the material would be unproductive. The background level can be a limiting factor in attempting to measure degradation in systems that synthesize very small amounts of collagen. Key words: collagen, hydroxyproline, intracellular degradation, proline, radiochemical purity.
Introduction Intracellular degradation of newly synthesized collagen is usually quantitated by incubating cells or tissue with (14C]-proline and measuring the hydroxy[14C]-proline in a low molecular weight fraction relative to the total hydroxy(14C]-proline produced by the system (Bienkowski and Engels, 1981). Radiochemical purity of [14C]-proline is an important consideration in these experiments, since the presence of hydroxy-(14C]-proline as a contaminant can artefactually increase the measured level of degradation. Until recently it was not unusual for commercial preparations of [14C]-proline to contain as much as 10f0
196
R. S. Bienkowski
hydroxy-(14C]-proline, or another radioactive species that migrates with hydroxyproline on an analytic cation-exchange resin, and it was necessary to purify the isotope before it could be used for degradation studies. However, (14C]-proline currently available from commercial suppliers is very pure, and the question arises whether even lower levels of impurity can be achieved. The issue is especially important when attempting to quantitate degradation in cell systems that produce very small amounts of hydroxyproline. Results presented in this communication show that there is a lower limit to the level of radiochemical impurity than can be achieved for [14C]-proline, and that further attempts at purification would be unproductive. Materials and Methods
Radioactive Isotopes L-[U- 14 C]-Proline, specific activity 280 mCilmol (approx), was purchased from either New England Nuclear, Boston, MA, or Amersham, Arlington Heights, IL; the stated radiochemical purity of each lot as :> 98 Ofo. Trans-4-Hydroxy-L-[G3H]-proline, spec. act. 5.4 Ci/mol, was purchased from New England Nuclear and purified as described previously (Bienkowski and Engels, 1981). Chromatography Systems Radioactive hydroxyproline and other species that co migrate with it were quantitated using a 0.9 X 28 cm column of DC6A resin (Dionex) eluted with citrate buffer (Pierce, Pico-Buffer IIA, adjusted to pH 2.65) at a flow rate of 66 mllh; the column temperature was 37 DC. Two ml fractions of effluent were collected and mixed with 18 ml of scintillation fluid; radioactivity in each fraction was measured using automatic external standardization and double label counting techniques when necessary. Carbon-14 was counted with an efficiency of 26 Ofo and tritium was counted with an efficiency of 34 Ofo. [14C]-Proline was separated from radioactive contaminants by chromatography on 1 X 30 cm columns of AG50-X8 resin (Na+ form) eluted with 0.1 N citrate buffer, pH 2.85. Fractions eluted from the AG50 columns were desalted during 2 X 5 cm columns of AG2-X8 resin (OH- form); each column was washed with 1 I of H 2 0 and the bound radioactive species were eluted with 0.5 M acetic acid. Complete details for operating these columns were published previously (Bienkowski and Engels, 1981). Results Radiochemical purity of [14C]-proline was assessed by chromatographing a sample of isotope, usually 1 oder 2 ,uCi, on an analytic cation-exchange resin (DC6A). The relative amount of radioactive impurity in each fraction was quantitated using the following equation: Percent Impurity in Fraction N = 100 X (Radioactivity in Fraction N) -7(Total Radioactivity Applied to Column)
(1)
Radiochemical purity of [14C]-proline
197
(This equation is applicable only for those regions of the elution profile that do not contain proline.) Impurity levels are very low, typically in the range 0.01-0.02 percent in each fraction, in the region of the elution profile where authentic trans-4-hydroxyproline is known to appear ("OHProline region"); since the OHProline region comprises 3 fractions, the total percent impurity is 0.03-0.06, or 300-600 parts per million (ppm). Several attempts were made to remove the impurities by chromatographing the isotope on preparative ionexchange columns of AG50 resin_ Subsequent analysis on DC6A resin showed that the impurity levels in the OHProline region were not significantly lower than in the original isotope. A separate experiment showed that this could not be attributed to use of preparative chromatography procedures. A sample of [14C]-proline was chroma to graphed on DC6A resin and then an aliquot of the major peak of radioactivity was immediately rechromatographed on the same column; there was no reduction in the impurity level in the OHProline region of the elution profile (not shown). Frequent analysis of the isotope showed that the impurity level did not increase appreciably with time in storage (in 2 Ofo ethanol as per manufacturer's specifications). Another set of experiments was carried out to investigate the problem further. A mixture of 15 ,uCi [14C]-proline and 1.2 ,uCi trans-4-hydroxy-[3H]-proline was divided into 6 equal samples. Three samples were chroma to graphed directly on DC6A resin. The (14C]-radioactivity was expressed as percent impurity using Equation 1 and the chromatograms are shown in Figure 1 A; the trans-4-hydroxy[3H]-proline eluted between 36 and 40 ml and this is labeled "OHProline region" in Figure 1 A. The three other samples were chromatographed on AG50 columns (Fig. 1 B); the fractions containing significant amounts of either hydroxy-[3H]proline or [14C]-proline were pooled and desalted on AG2 columns, and these were then rechromatographed on DC6A resin. Percent impurity for the [14C]proline-containing samples was calculated using Equation 1, and the elution profiles are the curves labeled "Proline" in Figure 1 C. The [14C]-radioactivity that comigrated with hydroxy-[3H]-proline on AG50 was expressed in terms of an equivalent percent impurity: Equivalent Percent Impurity in Fraction N RN X ([3H]/[14C]}orig
=
(2)
where RN is the ratio of [14C]-radioactivity in fraction N to the total hydroxy[3H]-proline in the rechromatographed samples; and ([3H]/[14C])orig is the isotope ratio of the original mixture. The elution profiles for these samples are the curves labeled "OHProline" in Figure 1 C. Comparison of the upper family of curves in Figure 1 C ("Proline") with the elution profiles in Figure 1 A shows that percent impurity in the OHProline region of rechromatographed [14C]-proline is either equal to, or slightly greater than, percent impurity of the original isotope. Interestingly, however, comparison of the lower family of curves in Figure 1 C ("OHProline") with the elution profiles in Figure 1 A shows that the equivalent percent impurity of the (14C]radioactivity that comigrated with hydroxy-[3H]-proline on AG50 is approximately 1/5 the percent impurity of the original isotope. The purified [14C]-proline contained less than 2 % of the hydroxy-[3H]-proline present initially (not shown); this demonstrates that AG50 chromatography illustrated in Figure 1 B efficiently separates proline and hydroxyproline. However, when the (14C]-radioactivity
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Fig. 1. Analysis of the radiochemical purity of [14C]-proline. A mixture of [14C]-proline and trans-4-hydroxy-[3H]-proline was divided into 6 equal samples. A. Three samples were chromatographed on a column of DC6A analytic cation-exchange resin. In this system, trans-4-hydroxyproline elutes between 36 and 40 ml (OHProline region) and proline elutes between 70 and 80 ml (not shown). [14C]-Radioactivity was expressed as percent impurity using Equation 1. B. Three samples of the original mixture were chromatographed on preparative columns of AG50 resin (for clarity the elution profile of only one sample is shown). Fractions containing significant amounts of either hydroxy-[3H]-proline or [14C]-proline were pooled and desalted. C. Samples containing either [14C]-proline or hydroxy-[3H]-proline that had been separated on AG50 resin (see panel B) were rechromatographed on DC6A resin. Percent impurity for the [14C]-proline-containing samples was calculated using Equation 1 ("Proline" curves). Equation 2 was used to calculate equivalent percent impurity for the [14C]radioactivity that chromatographed with hydroxy-[3H]-proline on AG50 ("OHProline" curves).
Radiochemical purity of [14C]-proline
199
that comigrated with hydroxy-[3H]-proline was rechromatographed on DC6A, a significant amount (46 0/0) eluted in the Proline region and only 5 % eluted in the OHProline region; the rest was distributed fairly uniformly among the other fractions of efluent (not shown). A simple interpretation of these data is that when "purified" [14C]-proline is rechromatographed on DC6A, the [14C]radioactivity that elutes in the OHProline region was generated spontaneously from the proline during the purification procedure. Discussion The results presented here strongly suggest that radioactive "impurities" are generated spontaneously and rapidly from [14C]-proline. The various radioactive species appear to be in equilibrium since the amount of impurity does not increase with time. Neither the nature of the reactions nor the identity of the impurities is known; however, the practical significance of this work is that attempts to reduce the impurity level in the hydroxyproline region below approximately 500 parts per 106 parts of [14C]-proline would be unproductive. Another point of methodologic significance is that when making a correction for background radioactivity in a sample containing biologically-generated hydroxyproline as well as a large amount of (14C]-proline, the amount to be subtracted is not the amount contained in the original isotope, which is a function of percent impurity, but rather the amount in a processed sample, which is a function of equivalent percent impurity; thus, the correction factor is 100 ppm rather than 500 ppm. It should be noted that separation of hydroxyproline and proline on AG50 followed by desalting on AG2, and then final analysis on DC6A is the procedure used to quantitate collagen degradation (Bienkowski and Engels, 1981). It is interesting to consider when the background might become a limiting factor in measuring degradation. For example, if 10 .uCi [14C]-proline is used to label a cell culture, then the background adds approximately 2500 dpm to the level of hydroxy-[14C]-proline in the low molecular weight fraction, and analysis of replicate samples shows that the uncertainty in this measurement is approximately ± 500 dpm (unpublished observation). In a cell system that produces a large amount of collagen and in which degradation is appreciable, such as cultured fibroblasts, the background contribution is relatively minor; however, it can be substantial when attempting to measure degradation in a system that synthesizes a very small amount of collagen or collagenous molecules such as liver cells (Bienkowski et ai., 1984) or macrophages (Myllyla and Seppa, 1979). In these cases, the uncertainty in the background may be comparable to the amount of biologically produced hydroxy-[14C]-proline in the low molecular weight fraction, and quantitation of degradation would be subjected to very large uncertainty. Acknowledgement This work was supported by NIH Grant HL 22729. The assistance of JoAnn Maestri in preparing this manuscript is greatly appreciated.
200
R. S. Bienkowski
References Bienkowski, R. S. and Engels, c.: Measurement of intracellular collagen degradation. Anal. Biochem. 116: 414-424, 1981. Bienkowski, R. S., Wu, C. H. and Wu, G. Y.: Intracellular degradation of newly synthesized collagen in liver. Submitted for publication (1984). Myllyla, R. and Seppa, H.: Studies on enzymes of collagen biosynthesis and the synthesis of hydroxyproline in macrophages and mast cells. Biochem. J. 182: 311-316, 1979. Dr. Robert S. Bienkowski, Pulmonary Division, Department of Pediatrics, Room 426, Van Etten, Albert Einstein College of Medicine, Bronx, New York 10461 USA.
Address after 1 July 1984: Schneider Children's Hospital, Long Island Jewish-Hillside Medical Center, New Hyde Park, NY 11042 USA.
Radiochemical Purity of [ 14 C]-Proline:Implications for Measurement of Intracellular Collagen Degradation ROBERT S. BIENKOWSKI Pulmonary Division, Department of Pediatrics, Albert Einstein College of Medicine, Bronx, New York 10461 USA. Address after 1. July 1984: see page 200.
Abstract Measurement of intracellular degradation of newly synthesized collagen requires use of radiochemically pure [14C]-proline. It is particularly important that levels of contaminating hydroxy-[14C]-proline or other radioactive species that migrate with hydroxyproline on cation-exchange resins be very low. The objective of this study was to determine how far the levels of these impurities could be reduced. Commercially available [14C]-proline was subjected to preparative ion-exchange chromatography. The procedure was capable of adequately separating authentic hydroxyproline from proline, however, upon rechromatography of the "purified" isotope on an analytic resin it was found that background radioactivity in the hydroxyproline region could not be reduced below approximately 500 parts per 10 6 parts of [14C]-proline. The contaminants appear to be generated during the procedure itself, suggesting that further efforts to purify the material would be unproductive. The background level can be a limiting factor in attempting to measure degradation in systems that synthesize very small amounts of collagen. Key words: collagen, hydroxyproline, intracellular degradation, proline, radiochemical purity.
Introduction Intracellular degradation of newly synthesized collagen is usually quantitated by incubating cells or tissue with (14C]-proline and measuring the hydroxy[14C]-proline in a low molecular weight fraction relative to the total hydroxy(14C]-proline produced by the system (Bienkowski and Engels, 1981). Radiochemical purity of [14C]-proline is an important consideration in these experiments, since the presence of hydroxy-(14C]-proline as a contaminant can artefactually increase the measured level of degradation. Until recently it was not unusual for commercial preparations of [14C]-proline to contain as much as 10f0
196
R. S. Bienkowski
hydroxy-(14C]-proline, or another radioactive species that migrates with hydroxyproline on an analytic cation-exchange resin, and it was necessary to purify the isotope before it could be used for degradation studies. However, (14C]-proline currently available from commercial suppliers is very pure, and the question arises whether even lower levels of impurity can be achieved. The issue is especially important when attempting to quantitate degradation in cell systems that produce very small amounts of hydroxyproline. Results presented in this communication show that there is a lower limit to the level of radiochemical impurity than can be achieved for [14C]-proline, and that further attempts at purification would be unproductive. Materials and Methods
Radioactive Isotopes L-[U- 14 C]-Proline, specific activity 280 mCilmol (approx), was purchased from either New England Nuclear, Boston, MA, or Amersham, Arlington Heights, IL; the stated radiochemical purity of each lot as :> 98 Ofo. Trans-4-Hydroxy-L-[G3H]-proline, spec. act. 5.4 Ci/mol, was purchased from New England Nuclear and purified as described previously (Bienkowski and Engels, 1981). Chromatography Systems Radioactive hydroxyproline and other species that co migrate with it were quantitated using a 0.9 X 28 cm column of DC6A resin (Dionex) eluted with citrate buffer (Pierce, Pico-Buffer IIA, adjusted to pH 2.65) at a flow rate of 66 mllh; the column temperature was 37 DC. Two ml fractions of effluent were collected and mixed with 18 ml of scintillation fluid; radioactivity in each fraction was measured using automatic external standardization and double label counting techniques when necessary. Carbon-14 was counted with an efficiency of 26 Ofo and tritium was counted with an efficiency of 34 Ofo. [14C]-Proline was separated from radioactive contaminants by chromatography on 1 X 30 cm columns of AG50-X8 resin (Na+ form) eluted with 0.1 N citrate buffer, pH 2.85. Fractions eluted from the AG50 columns were desalted during 2 X 5 cm columns of AG2-X8 resin (OH- form); each column was washed with 1 I of H 2 0 and the bound radioactive species were eluted with 0.5 M acetic acid. Complete details for operating these columns were published previously (Bienkowski and Engels, 1981). Results Radiochemical purity of [14C]-proline was assessed by chromatographing a sample of isotope, usually 1 oder 2 ,uCi, on an analytic cation-exchange resin (DC6A). The relative amount of radioactive impurity in each fraction was quantitated using the following equation: Percent Impurity in Fraction N = 100 X (Radioactivity in Fraction N) -7(Total Radioactivity Applied to Column)
(1)
Radiochemical purity of [14C]-proline
197
(This equation is applicable only for those regions of the elution profile that do not contain proline.) Impurity levels are very low, typically in the range 0.01-0.02 percent in each fraction, in the region of the elution profile where authentic trans-4-hydroxyproline is known to appear ("OHProline region"); since the OHProline region comprises 3 fractions, the total percent impurity is 0.03-0.06, or 300-600 parts per million (ppm). Several attempts were made to remove the impurities by chromatographing the isotope on preparative ionexchange columns of AG50 resin_ Subsequent analysis on DC6A resin showed that the impurity levels in the OHProline region were not significantly lower than in the original isotope. A separate experiment showed that this could not be attributed to use of preparative chromatography procedures. A sample of [14C]-proline was chroma to graphed on DC6A resin and then an aliquot of the major peak of radioactivity was immediately rechromatographed on the same column; there was no reduction in the impurity level in the OHProline region of the elution profile (not shown). Frequent analysis of the isotope showed that the impurity level did not increase appreciably with time in storage (in 2 Ofo ethanol as per manufacturer's specifications). Another set of experiments was carried out to investigate the problem further. A mixture of 15 ,uCi [14C]-proline and 1.2 ,uCi trans-4-hydroxy-[3H]-proline was divided into 6 equal samples. Three samples were chroma to graphed directly on DC6A resin. The (14C]-radioactivity was expressed as percent impurity using Equation 1 and the chromatograms are shown in Figure 1 A; the trans-4-hydroxy[3H]-proline eluted between 36 and 40 ml and this is labeled "OHProline region" in Figure 1 A. The three other samples were chromatographed on AG50 columns (Fig. 1 B); the fractions containing significant amounts of either hydroxy-[3H]proline or [14C]-proline were pooled and desalted on AG2 columns, and these were then rechromatographed on DC6A resin. Percent impurity for the [14C]proline-containing samples was calculated using Equation 1, and the elution profiles are the curves labeled "Proline" in Figure 1 C. The [14C]-radioactivity that comigrated with hydroxy-[3H]-proline on AG50 was expressed in terms of an equivalent percent impurity: Equivalent Percent Impurity in Fraction N RN X ([3H]/[14C]}orig
=
(2)
where RN is the ratio of [14C]-radioactivity in fraction N to the total hydroxy[3H]-proline in the rechromatographed samples; and ([3H]/[14C])orig is the isotope ratio of the original mixture. The elution profiles for these samples are the curves labeled "OHProline" in Figure 1 C. Comparison of the upper family of curves in Figure 1 C ("Proline") with the elution profiles in Figure 1 A shows that percent impurity in the OHProline region of rechromatographed [14C]-proline is either equal to, or slightly greater than, percent impurity of the original isotope. Interestingly, however, comparison of the lower family of curves in Figure 1 C ("OHProline") with the elution profiles in Figure 1 A shows that the equivalent percent impurity of the (14C]radioactivity that comigrated with hydroxy-[3H]-proline on AG50 is approximately 1/5 the percent impurity of the original isotope. The purified [14C]-proline contained less than 2 % of the hydroxy-[3H]-proline present initially (not shown); this demonstrates that AG50 chromatography illustrated in Figure 1 B efficiently separates proline and hydroxyproline. However, when the (14C]-radioactivity
198
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Fig. 1. Analysis of the radiochemical purity of [14C]-proline. A mixture of [14C]-proline and trans-4-hydroxy-[3H]-proline was divided into 6 equal samples. A. Three samples were chromatographed on a column of DC6A analytic cation-exchange resin. In this system, trans-4-hydroxyproline elutes between 36 and 40 ml (OHProline region) and proline elutes between 70 and 80 ml (not shown). [14C]-Radioactivity was expressed as percent impurity using Equation 1. B. Three samples of the original mixture were chromatographed on preparative columns of AG50 resin (for clarity the elution profile of only one sample is shown). Fractions containing significant amounts of either hydroxy-[3H]-proline or [14C]-proline were pooled and desalted. C. Samples containing either [14C]-proline or hydroxy-[3H]-proline that had been separated on AG50 resin (see panel B) were rechromatographed on DC6A resin. Percent impurity for the [14C]-proline-containing samples was calculated using Equation 1 ("Proline" curves). Equation 2 was used to calculate equivalent percent impurity for the [14C]radioactivity that chromatographed with hydroxy-[3H]-proline on AG50 ("OHProline" curves).
Radiochemical purity of [14C]-proline
199
that comigrated with hydroxy-[3H]-proline was rechromatographed on DC6A, a significant amount (46 0/0) eluted in the Proline region and only 5 % eluted in the OHProline region; the rest was distributed fairly uniformly among the other fractions of efluent (not shown). A simple interpretation of these data is that when "purified" [14C]-proline is rechromatographed on DC6A, the [14C]radioactivity that elutes in the OHProline region was generated spontaneously from the proline during the purification procedure. Discussion The results presented here strongly suggest that radioactive "impurities" are generated spontaneously and rapidly from [14C]-proline. The various radioactive species appear to be in equilibrium since the amount of impurity does not increase with time. Neither the nature of the reactions nor the identity of the impurities is known; however, the practical significance of this work is that attempts to reduce the impurity level in the hydroxyproline region below approximately 500 parts per 106 parts of [14C]-proline would be unproductive. Another point of methodologic significance is that when making a correction for background radioactivity in a sample containing biologically-generated hydroxyproline as well as a large amount of (14C]-proline, the amount to be subtracted is not the amount contained in the original isotope, which is a function of percent impurity, but rather the amount in a processed sample, which is a function of equivalent percent impurity; thus, the correction factor is 100 ppm rather than 500 ppm. It should be noted that separation of hydroxyproline and proline on AG50 followed by desalting on AG2, and then final analysis on DC6A is the procedure used to quantitate collagen degradation (Bienkowski and Engels, 1981). It is interesting to consider when the background might become a limiting factor in measuring degradation. For example, if 10 .uCi [14C]-proline is used to label a cell culture, then the background adds approximately 2500 dpm to the level of hydroxy-[14C]-proline in the low molecular weight fraction, and analysis of replicate samples shows that the uncertainty in this measurement is approximately ± 500 dpm (unpublished observation). In a cell system that produces a large amount of collagen and in which degradation is appreciable, such as cultured fibroblasts, the background contribution is relatively minor; however, it can be substantial when attempting to measure degradation in a system that synthesizes a very small amount of collagen or collagenous molecules such as liver cells (Bienkowski et ai., 1984) or macrophages (Myllyla and Seppa, 1979). In these cases, the uncertainty in the background may be comparable to the amount of biologically produced hydroxy-[14C]-proline in the low molecular weight fraction, and quantitation of degradation would be subjected to very large uncertainty. Acknowledgement This work was supported by NIH Grant HL 22729. The assistance of JoAnn Maestri in preparing this manuscript is greatly appreciated.
200
R. S. Bienkowski
References Bienkowski, R. S. and Engels, c.: Measurement of intracellular collagen degradation. Anal. Biochem. 116: 414-424, 1981. Bienkowski, R. S., Wu, C. H. and Wu, G. Y.: Intracellular degradation of newly synthesized collagen in liver. Submitted for publication (1984). Myllyla, R. and Seppa, H.: Studies on enzymes of collagen biosynthesis and the synthesis of hydroxyproline in macrophages and mast cells. Biochem. J. 182: 311-316, 1979. Dr. Robert S. Bienkowski, Pulmonary Division, Department of Pediatrics, Room 426, Van Etten, Albert Einstein College of Medicine, Bronx, New York 10461 USA.
Address after 1 July 1984: Schneider Children's Hospital, Long Island Jewish-Hillside Medical Center, New Hyde Park, NY 11042 USA.