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Problems in the analysis of urinary mucopolysaccharide excretion
BIOCHEMICAL
MEDICINE
2, 448-456
(1969)
Problems in the Analysis Mucopolysaccharide ALFRED Departments
LINKER,
LEIGH
R. EVANS,
of Biological Chemistry, University of Utah College Administration Hospital,
Received
of Urinary Excretion AND
JACK
A. MADSEN
Pathology, Neurology, and of Medicine and the Veterans Salt Lake City, Utah 84113 January
Pediatrics,
22 > 1969
The mucopolysaccharidoses are a group of heritable connective tissue disorders originally classified as gargoylism or Hurler’s syndrome. Patients with these diseases show severe abnormalities of the total connective tissue such as dwarI%m, deformities of skull and vertebrae, and pathological changes of the reticuloendothelial, cardiovascular, and central nervous systems. These disorders, though rare, represent a very interesting biochemical and genetic problem and appear to result from a basic defect in the metabolism of two (or three) acid mucopolysaccharides ( MPS ). Large amounts of these polymers are stored in a variety of tissues and excreted in the urine at levels of 10-25 times the normal amount. Recently, an attempt has been made to subdivide the mucopolysaccharidoses into six separate groups (1) which differ clinically and biochemically. As there is considerable overlap in clinical symptoms, the urinary MPS excretion pattern is of importance for the identification of a particular disease entity and also as background for studies involving fibroblast tissue cultures ( 2). A large number of assay methods have been used, most of them based on the original procedure by Di Ferrante (3) which uses precipitation of urinary MPS by cetyltrimethylammonium bromide (CTAB) , Considerable variation in methods exists from laboratory to laboratory and within the same laboratory at different times. Recently, we have noticed that small changes in procedure can lead to considerable error in total amount as well as pattern of MPS excreted. This report describes the effects of some variations in methodology on the recovery from urine of the two MPS most commonly associated with these disorders. namely, chondroitin sulfate B and heparitin sulfate. 448
URINARY
MUCOPOLYSACCHAFUDES
MATERIALS
449
AND METHODS
Analytical methods for uranic acid determination and paper chromatography of MPS were described previously (4). Electrophoresis was carried out on cellulose acetate (Sepraphore II) in 0.1 M Tris buffer pH 7.4, 0.05 N in LiCI. For estimation of total MPS 30-ml portions of 24-hour urine specimens were analyzed by the method of Teller et al. (5) and results expressed as milligrams of uranic acid (carbazole reaction) per gm creatinine (6). Total MPS were also isolated from large volumes of urine, fractionated, and identified by paper chromatography, ratio of uranic acid color reactions, glucosamine, and galactosamine content and paper electrophoresis (7). Standard MPS. Chondroitin sulfate B was prepared from pig skin (8) and heparitin sulfate purified from a commercial product (9). Patients. Three patients were used in this study. Patient D.W. (male) was described previously (7), had the typical features of Sanfillippo’s syndrome and excreted mainly heparitin sulfate. Patient J.F. (male), also described previously, represents either Hurler’s syndrome or a severe form of Hunter’s syndrome. He had been thought to excrete mainly chondroitin sulfate B (7). Patient D.K. (male) has the clinical features described for Hunter’s syndrome. His Y4PS excretion pattern will be described. RESULTS
Effect of Urine Concentration on Total UPS Recovery. While checking daily variations in urinary MPS excretion in patient D.W., we noticed that considerably less material seemed to be excreted when the creatinine values rose above 0.8 gm per liter, that is, when the urine became fairly concentrated. We, therefore, compared total MPS values obtained from the analysis of 30-ml urine samples prepared with and without dialysis prior to precipitation with CTAB. It can be seen in Table 1 that very large lossesof MPS occurred in the concentrated urines (shown by high creatinine values) when the samples were not dialyzed. As patient D.W. excreted almost entirely heparitin sulfate, losseswould be due to this compound. Therefore, another patient, J.F., previously shown to excrete mainly chondroitin sulfate B (about 80%of total) was investigated. As shown in Table 2, considerable lossesof MPS also occurred at high creatinine values without dialysis, though they were not comparable in magnitude to those in Table I. To identify the lost material, polysaccharide was isolated preparatively from two urine specimens (about 1200 ml each) with differing creatinine levels. Table 3 shows the effects of dialysis on this isolation. As can be seen the total
450
LINKER,EVANS,
AND
TABLE TOTAL
URINARY
MADSEN
1
MUCOPOLYSACCHARIDES
OF PATIENT
D.W.
Uranic acid (me) per creatinine Sample”
Creatinine (gm/liter)
Dialyzed*
1 2 3 4 5
1.25 1.7Q 2.00 1.35 0.28
111 121 1lQ 86 x0
(pm)
Non-dialyzed 68 43 13 15 50
a Samples were obtained on different days. Analyses were carried out on 30-ml ahquote obtained from the same urine specimen with and without dialysis. b Samples were dialyzed against 10 volumes of distilled water for 18 hours.
MPS yield in the more dilute specimen was about the same with and without dialysis while the more concentrated urine showed a loss of about 30% when dialysis was not used. The analytical data show an interesting pattern in the carbazole to orcinol ratio. This ratio in the undialyzed urine (0.70, 0.75) agrees with our earlier data (7) and indicates a large preponderance of chondroitin sulfate B (as only hepartin sulfate and chondroitin sulfate B are usually excreted in large amounts in the patients, the carbazole to orcinol ratio serves as a fairly reliable measure of the relative amounts of these two MPS). However, the much higher ratio of the material obtained from dialyzed urine indicates the presence of considerable amounts of heparitin sulfate in this sample. Paper chromatography and analysis for the presence of glucosamme (7) supported this observation.
Effect of Urine
Concentration
on Heparitin
Sulfate
Recovery.
The
following experiment was carried out to verify the loss of heparitin sulfate in the absence of dialysis. CTAB was added directly to a urine specimen (210 ml) of patient J.F., with a creatinine value of 1.3 gm/ TABLE TOTAL
URINARY
MUCOPOLYSACCHARIDES
2 OF
PATIENT
J.F.
Uranic acid (mg) per creatinine Sample”
Creatinine (gm/liter)
Dialyzed
1 2 3 4
0.72 1.20 1.34 0.92
90 119 120 111
aSee Table 1.
Non-dialyzed 88 84 59 86
(pm)
IBUNARY
TABLE ISOLATION
451
M.UcopoLYSACCHARLD~
OF MUCOPOLY~A~CHARIDE~
3
FROM
THE
UBINE
MPS Yield Carbazoleb Orcinol” Carbazole Orcinol
Non-dialyzed
95 mg 28 22 1.28
J.F.
Specimen 2a (1.30 gm Creatinine/liter)
Specimen la (0.85 gm Creatinine/liter) Dialyzed
OF PATIENT
Dialyzed 92 mg 31 24 1.28
115 mg 17 25 0.68
Nondialyzed 60 mg 14 20 0.70
a Urine specimens (about 1200 ml each) were divided in half and one-half dialyzed for 18 hours, the other precipitated directly. b Uranic acid content in percentage as determined by the color reaction indicated using glucuronic acid as standard.
liter after adjusting the pH to 5.0. The precipitate was collected and the MPS isolated as above. The supemate was then dialyzed for 16 hours and additional CTAB added. The precipitate which formed was collected and the material isolated. Results are shown in Table 4. The first precipitate consists mostly of chondroitin sulfate B by the criteria mentioned under methods and by paper electrophoresis as shown in Fig. 1. The second precipitate was fractionated with ethanol and found not to contain any material insoluble in 20% ethanol (chondroitin sulfate B is usually precipitated at this alcohol concentration). The higher ethanol fractions (7) contained mainly heparitin sulfate. Therefore, most of the heparitin sulfate would have been lost from concentrated urine without the dialysis step. It therefore seemsthat dialysis should be used as an obligatory step in the isolation procedures. However, one additional point needs clarification, a large difference in carbazole:orcinol ratio can be observed between dialyzed and non-dialyzed specimens (Table 3) TABLE IDENTIFICATION
First precipitate” Second precipitatea
OF “LOST”
4 MUCOPOLYSACCHAEIDE
Yield
Carbazole*
50 mg 60 mg
19% 27%
Orcinol” 32%
22%
--Carbazole Orcinol 0.59 1.23
s MPS waz precipitated from urine directly by CTAB and the supernate dialyzed. This waz followed by addition of CTAB to the retentate yielding the second precipitate. b Same az in Table 3.
452
LINKER,
1. Electrophoresis of MPS FIG sulfate standard; B, 20% ethanol standard.
EVANS,
AND
MADSEN
on Sepraphore II. fraction of patient
66V/cm, $5 hour. J.F.: C, chondroitin
A, heparitin sulfate B
even when little difference in total MPS yield is apparent, indicating a possible compensatory loss occurring with dialysis. Effect of Dialysis on Chondroitin Sdfate B Recovery. Total MPS was, therefore, isolated from a urine specimen of patient J.F. (creatinine 0.85 gm/liter, Table 3). The sample was divided in half (600 ml each) and MPS isolated with and without dialysis. The material obtained was fractionated with ethanol and the results are shown in Table 5. It is apparent that the dialyzed urine sample contains hardly any material precipitating with 20% ethanol and that, therefore, much of the chondroitin sulfate B had been lost in this case. As the dialyzate when checked did not contain significant amounts of MPS this loss was not due to dialyzability of the polysaccharide. Further investigation indicated that part of the chondroitin sulfate B present in the CTAB precipitate of the dialyzed urine had remained insoluble in the calcium acetate solution used for redissolving the precipitate (7) but could be recovered by extraction with 0.1 N NaOH. This was not the case with
URINARY
TABLE ETHANOL
FRACTIONATION ISOLATED
20’;
453
MUCOPOLYSACCHARJDES
WITH
5 OF RIuc~PoLY~AccH.~RIDE~
AND
WITHOUT
Ethanola
DIALYSIS
407~ Et,hanok
Carbazolec
SOY0 Ethanola
Carbazolec
Isolation procedure
Yield”
Orcinol
Yieldb
Orcinol
Dialyzed Non-dialyzed
‘) 3.1
1.3 0.4
4x 3’9
1.6 0.9
n MPS precipitated at this ethanol concentration from its solution 2.55& calcium acetate. b Percentage yield of total. c Ratio of the two calorimetric reactions for uranic arid.
Carbazolec Yieldb 50 39
Orcinol 1 .:z 1.3
(at 10 mg/ml’j
in
the same precipitate from nondialyzed urine. The other part of the chondroitin sulfate loss seems due to incomplete precipitation from dialyzed urine. Another patient, D.K., also excreting a mixture of heparitin sulfate and chondroitin sulfate B, was investigated. The urine specimen used had a creatinine value of 0.67 gm/liter and l/s of the heparitin sulfate was lost without dialysis and 1/ of the chondroitin sulfate with dialysis. The losses, therefore, were less severe than in patient J.F., probably due to the less concentrated urine. By applying the method described above of precipitating first, followed by dialysis and reprecipitation, patient D.K. was found to excrete equal amounts of heparitin sulfate and chondroitin sulfate B. DISCUSSION
Studies of the heritable disorders of connective tissue involving mucopolysaccharide metabolism have shown promise to lead to a better understanding of the biological function of these rather elusive polymers. A reliable method for the analysis of urinary MPS is of considerable importance for identification of patients with mucopolysaccharidoses. Many of them are difficult to separate on a clinical basis alone from a large number of other skeletal abnormalities which they resemble but which do not involve the metabolism of MPS. In addition, the urine analysis is valuable for classification of subgroups of these disorders, for evaluating the effects of experimental treatment, and is essential as a background for tissue culture studies. Fibroblast tissue cultures have recently been shown (2, 10) to be useful for the identi&ation of the mucopolysaccharidoses. They have the advantage of being closer to the basic genetic defect and seem to allow the determination of heterozygote
4754
LINKER,
EVANS,
AND
MADSEN
carriers of the diseases. The cultures have, however, certain drawbacks: ( 1) Space and personnel are not always available; (2) considerable time is required before resuits are obtained; (3) several disorders show the same MPS pattern in the skin fibroblasts and metachromasia of granules alone is not characteristic of connective tissue disorders per se (11). The urinary MPS excretion pattern which is more easily determined, though obviously somewhat removed from the basic defect, should still reflect the primary disease process. Normal urine has been shown to contain chondroitin 4-sulfate, chondroitin g-sulfate, chondroitin sulfate B, chondroitin, heparitin sulfate, keratosulfate, hyaluronic acid, and some chondroitin sulfate polypeptide ( 4. E-14). Chondroitin 4 and 6 sulfate are the major components and considerable variation in sulfate content was noted by some of the authors. Patients with mucopolysaccharidoses have been found to excrete mainly chondroitin sulfate B and heparitin sulfate (15, 16). As these two MPS are present in amounts of 10-20 times the tom1 normal MPS levels they completely overshadow the other constituents. It is, therefore, mainly the excretion pattern of heparitin sulfate and chondroitin sulfate 13 which appears to be characteristic for this group of diseases (Marquio’s syndrome, which is still fairly controversial, is not included here). At present, however, as different laboratories use similar but varying methodology the results obtained are difficult to interpret. As shown here, large amounts of heparitin sulfate can be lost as it does not precipitate well from concentrated urines. This difficulty is obscured when recovery from urine is checked by using tissue-derived heparitin sulfate preparations as standards. At urine concentrations usually encountered precipitation would be complete. However, the polysaccharide when excreted in the urine is of much lower molecular weight and varies considerably in sulfate content ( 17). Both factors can prevent precipitation at much lower salt concentration than that affecting standard preparations ( 18). The same problem also applies to urinary MPS fractionation procedures whether based on ethanol precipitation (4), solubility of cetylpyridinium complexes ( 19) or ion exchange chromatography ( 13, 20). Identification is generally based on undegraded standards obtained from connective tissue sources while the urinary MPS investigated appear considerably degraded ( 13, 17). The MPS loss in a patient excreting mainly heparitin sulfate would express itself in a lowered total excretion value, but in a patient also excreting excessive chondroitin sulfate B the proportion between the two polysaccharides would be affected. In our original study of patient J.F. (7) when d’la ly sis was not used we found a ratio of chondroitm sulfate B to heparitin sulfate of 4 to 1 and at the present, using dialysis, a proportion of 1 to 1. The differences with and without dialysis are not
URINARY
MUCOPOLYSACCHARIDES
4.55
as great in patient D.K. mainly because his urine has been generally less concentrated. In other words, differences in excretion pattern ascribed to a difference in disease entity may frequently be merely due to variations in urine concentration. In addition, as shown here, when dialysis of urine is routinely used, as done by some laboratories, considerable amounts of chondroitin sulfate B may be lost. The simplest procedure, therefore, would appear to be dilution, or alternatively concentration of urines to an optimum ionic strength. However, our data indicate that it would be diEcult to find optimum conditions for both polysaccharides as heparitin sulfate precipitates best from dialyzed urine and chondroitin sulfate B from a somewhat concentrated urine. In addition, salt concentration per se is not the only determining factor as the precipitation of MPS by CTAB seems also influenced by the level of urinary “mucoids” (21). At low levels recovery seems incomplete. The best method would seem to be direct precipitation with CTAB from urine followed by dialysis of the supematant and additional CTAB precipitation. We obtained good recoveries by this method. However, it should perhaps be stressed that no best generally applicable method can be recommended. Di Ferrante (21) has shown that considerable heparitin sulfate can be lost with, rather than without, dialysis as this MPS may be dialyzable. This is in contrast to the data reported here but different patients may very well excrete MPS of different molecular weights and the dialysis conditions used by that author were somewhat more vigorous than those used here. Data presented here show that chondroitin sulfate B may be lost from very dilute urines and therefore concentration may be necessary as a first step. In general, therefore, care should be used in analyzing the excretion in each patient and possible losses should be checked rather than relying on a standard procedure. If this is done the urinary excretion pattern should be a valuable diagnostic tool and also should reflect at least to some extent the basic disease process. The confusion about some .of the mucopolysaccharidoses showing similar excretion pattern but quite different clinical features may also be resolved ( 1). SUMMARY
In a study of polysaccharide excretion in patients with mucopolysaccharidoses large differences in yield as well as pattern, depending on the assay procedure used, were observed. When urines with high creatmine values were not dialyzed prior to polysaccharide precipitation with CTAB, large amounts of heparitin sulfate remained in solution and were therefore lost to analysis. On the other hand, when urines were routinely dialyzed considerable lossesof chondroitin sulfate B appeared to occur. Both factors can lead to serious errors in mucopolysaccharide analysis
456
LINKER,
EVANS,
AND
MADSEX
and significantly affect any attempt to correlate disease classification with urinary excretion pattern. An improved method has been proposed and investigators are advised to check for possible losses in the urine of individual patients rather than to rely on a general method. ACKNOWLEDGMENT This 08080.
work
was
supported
in part
by
U.
S. Public
Health
Service
Grant
No.
HE
REFERENCES 1. MCKWICK, V. A., “Heritable Disorders of Connective Tissue” (3rd ed.) Mosby, Saint Louis, Missouri, 1966. 2. DANES, B. S., AND BEARN, A. G., Lancer I, 241 ( 1967). 3. DI FERRANTE, N. M., AND RICH, C., C&n. Chim. Acta 1, 519 (1956). 4. LINKER, A., AND TERRY, K. D., PTOG. Sot. Exptl. Biol. Med. 113, 743 (1963). 5. TELLER, W. M., BURKE, E. C., ROSEVEAR, J. W., AND MCKENZLE, B. F., J. Lab. Clin. Med. 59, 95 ( 1962). 6. BONSNES, R. W., AND TAUSSKY, H. H., J. Biol. Chem. 158, 581 (1945). 7. TERRY, K. D., AND LINKER, A., Proc. Sot. Exptl. Biol. Med. 115, 394 (1964). 8. MEYER, K., DAVIDSON, E., LINKER, A., AND HOFF&IAN, P., Biochim. Biophys. Acta 21, 506 (1956). 9. CIFONELLI, J. A., AND DORFMAN, A., J. BioZ. Chem. 235, 3283 (1960). 10. MATALON, R., AND DORFIMAN, A., Proc. Natl. Acad. Sci. 60, 179 (1968). 11. DANES, B. S., AND BEARN, A. G., Science 161, 1347 (1968). 12. BERENSON, G. S., AND DALFERES, E. R., JR., Biochim. Biophys. Acta 101, 183
(1965). 13. VARADI,
D.
P.,
CIFONELLI,
J. A.,
AND
DORFMAN,
A., Biochim.
Biophys.
Acta
141, 103 (1967). AND HA~KSWORTH, J., J. Clin. Puthol. 21, 339 (1968). A. E., Proc. Natl. Acad. Sci. 43, 443 ( 1957). MEYER, K., GRIJMBACH, M. M., LINKER, A., AND HOFFMAN, P., Proc. Sot. Exptl. Biol. Med. 97, 275 ( 1958). KNECHT, J., CIFONELLI, J. A., AND D~RFMAN, A., J. Biol. Chem. 242, 4652 (1967). SCOTT, J. E., Methods B&hem. Anal. 8, 145 (1960). SVEJCAR, J., AND ROBERTSON, W. VAN B., Anal. Biochem. 18, 333 ( 1967). TELLER, W., AND ZIEMANN, A., K&n. Wochschr. 44, 1142 (1966). DI FERRANTE, N. M., Anal. Biochem. 21, 98 (1967).
14. MANLEY,
15. DORFMAN, 16. 17. 18. 19. 20. 21.
G.,
SEVERN,
M.,
A., AND LORINCZ,
MEDICINE
2, 448-456
(1969)
Problems in the Analysis Mucopolysaccharide ALFRED Departments
LINKER,
LEIGH
R. EVANS,
of Biological Chemistry, University of Utah College Administration Hospital,
Received
of Urinary Excretion AND
JACK
A. MADSEN
Pathology, Neurology, and of Medicine and the Veterans Salt Lake City, Utah 84113 January
Pediatrics,
22 > 1969
The mucopolysaccharidoses are a group of heritable connective tissue disorders originally classified as gargoylism or Hurler’s syndrome. Patients with these diseases show severe abnormalities of the total connective tissue such as dwarI%m, deformities of skull and vertebrae, and pathological changes of the reticuloendothelial, cardiovascular, and central nervous systems. These disorders, though rare, represent a very interesting biochemical and genetic problem and appear to result from a basic defect in the metabolism of two (or three) acid mucopolysaccharides ( MPS ). Large amounts of these polymers are stored in a variety of tissues and excreted in the urine at levels of 10-25 times the normal amount. Recently, an attempt has been made to subdivide the mucopolysaccharidoses into six separate groups (1) which differ clinically and biochemically. As there is considerable overlap in clinical symptoms, the urinary MPS excretion pattern is of importance for the identification of a particular disease entity and also as background for studies involving fibroblast tissue cultures ( 2). A large number of assay methods have been used, most of them based on the original procedure by Di Ferrante (3) which uses precipitation of urinary MPS by cetyltrimethylammonium bromide (CTAB) , Considerable variation in methods exists from laboratory to laboratory and within the same laboratory at different times. Recently, we have noticed that small changes in procedure can lead to considerable error in total amount as well as pattern of MPS excreted. This report describes the effects of some variations in methodology on the recovery from urine of the two MPS most commonly associated with these disorders. namely, chondroitin sulfate B and heparitin sulfate. 448
URINARY
MUCOPOLYSACCHAFUDES
MATERIALS
449
AND METHODS
Analytical methods for uranic acid determination and paper chromatography of MPS were described previously (4). Electrophoresis was carried out on cellulose acetate (Sepraphore II) in 0.1 M Tris buffer pH 7.4, 0.05 N in LiCI. For estimation of total MPS 30-ml portions of 24-hour urine specimens were analyzed by the method of Teller et al. (5) and results expressed as milligrams of uranic acid (carbazole reaction) per gm creatinine (6). Total MPS were also isolated from large volumes of urine, fractionated, and identified by paper chromatography, ratio of uranic acid color reactions, glucosamine, and galactosamine content and paper electrophoresis (7). Standard MPS. Chondroitin sulfate B was prepared from pig skin (8) and heparitin sulfate purified from a commercial product (9). Patients. Three patients were used in this study. Patient D.W. (male) was described previously (7), had the typical features of Sanfillippo’s syndrome and excreted mainly heparitin sulfate. Patient J.F. (male), also described previously, represents either Hurler’s syndrome or a severe form of Hunter’s syndrome. He had been thought to excrete mainly chondroitin sulfate B (7). Patient D.K. (male) has the clinical features described for Hunter’s syndrome. His Y4PS excretion pattern will be described. RESULTS
Effect of Urine Concentration on Total UPS Recovery. While checking daily variations in urinary MPS excretion in patient D.W., we noticed that considerably less material seemed to be excreted when the creatinine values rose above 0.8 gm per liter, that is, when the urine became fairly concentrated. We, therefore, compared total MPS values obtained from the analysis of 30-ml urine samples prepared with and without dialysis prior to precipitation with CTAB. It can be seen in Table 1 that very large lossesof MPS occurred in the concentrated urines (shown by high creatinine values) when the samples were not dialyzed. As patient D.W. excreted almost entirely heparitin sulfate, losseswould be due to this compound. Therefore, another patient, J.F., previously shown to excrete mainly chondroitin sulfate B (about 80%of total) was investigated. As shown in Table 2, considerable lossesof MPS also occurred at high creatinine values without dialysis, though they were not comparable in magnitude to those in Table I. To identify the lost material, polysaccharide was isolated preparatively from two urine specimens (about 1200 ml each) with differing creatinine levels. Table 3 shows the effects of dialysis on this isolation. As can be seen the total
450
LINKER,EVANS,
AND
TABLE TOTAL
URINARY
MADSEN
1
MUCOPOLYSACCHARIDES
OF PATIENT
D.W.
Uranic acid (me) per creatinine Sample”
Creatinine (gm/liter)
Dialyzed*
1 2 3 4 5
1.25 1.7Q 2.00 1.35 0.28
111 121 1lQ 86 x0
(pm)
Non-dialyzed 68 43 13 15 50
a Samples were obtained on different days. Analyses were carried out on 30-ml ahquote obtained from the same urine specimen with and without dialysis. b Samples were dialyzed against 10 volumes of distilled water for 18 hours.
MPS yield in the more dilute specimen was about the same with and without dialysis while the more concentrated urine showed a loss of about 30% when dialysis was not used. The analytical data show an interesting pattern in the carbazole to orcinol ratio. This ratio in the undialyzed urine (0.70, 0.75) agrees with our earlier data (7) and indicates a large preponderance of chondroitin sulfate B (as only hepartin sulfate and chondroitin sulfate B are usually excreted in large amounts in the patients, the carbazole to orcinol ratio serves as a fairly reliable measure of the relative amounts of these two MPS). However, the much higher ratio of the material obtained from dialyzed urine indicates the presence of considerable amounts of heparitin sulfate in this sample. Paper chromatography and analysis for the presence of glucosamme (7) supported this observation.
Effect of Urine
Concentration
on Heparitin
Sulfate
Recovery.
The
following experiment was carried out to verify the loss of heparitin sulfate in the absence of dialysis. CTAB was added directly to a urine specimen (210 ml) of patient J.F., with a creatinine value of 1.3 gm/ TABLE TOTAL
URINARY
MUCOPOLYSACCHARIDES
2 OF
PATIENT
J.F.
Uranic acid (mg) per creatinine Sample”
Creatinine (gm/liter)
Dialyzed
1 2 3 4
0.72 1.20 1.34 0.92
90 119 120 111
aSee Table 1.
Non-dialyzed 88 84 59 86
(pm)
IBUNARY
TABLE ISOLATION
451
M.UcopoLYSACCHARLD~
OF MUCOPOLY~A~CHARIDE~
3
FROM
THE
UBINE
MPS Yield Carbazoleb Orcinol” Carbazole Orcinol
Non-dialyzed
95 mg 28 22 1.28
J.F.
Specimen 2a (1.30 gm Creatinine/liter)
Specimen la (0.85 gm Creatinine/liter) Dialyzed
OF PATIENT
Dialyzed 92 mg 31 24 1.28
115 mg 17 25 0.68
Nondialyzed 60 mg 14 20 0.70
a Urine specimens (about 1200 ml each) were divided in half and one-half dialyzed for 18 hours, the other precipitated directly. b Uranic acid content in percentage as determined by the color reaction indicated using glucuronic acid as standard.
liter after adjusting the pH to 5.0. The precipitate was collected and the MPS isolated as above. The supemate was then dialyzed for 16 hours and additional CTAB added. The precipitate which formed was collected and the material isolated. Results are shown in Table 4. The first precipitate consists mostly of chondroitin sulfate B by the criteria mentioned under methods and by paper electrophoresis as shown in Fig. 1. The second precipitate was fractionated with ethanol and found not to contain any material insoluble in 20% ethanol (chondroitin sulfate B is usually precipitated at this alcohol concentration). The higher ethanol fractions (7) contained mainly heparitin sulfate. Therefore, most of the heparitin sulfate would have been lost from concentrated urine without the dialysis step. It therefore seemsthat dialysis should be used as an obligatory step in the isolation procedures. However, one additional point needs clarification, a large difference in carbazole:orcinol ratio can be observed between dialyzed and non-dialyzed specimens (Table 3) TABLE IDENTIFICATION
First precipitate” Second precipitatea
OF “LOST”
4 MUCOPOLYSACCHAEIDE
Yield
Carbazole*
50 mg 60 mg
19% 27%
Orcinol” 32%
22%
--Carbazole Orcinol 0.59 1.23
s MPS waz precipitated from urine directly by CTAB and the supernate dialyzed. This waz followed by addition of CTAB to the retentate yielding the second precipitate. b Same az in Table 3.
452
LINKER,
1. Electrophoresis of MPS FIG sulfate standard; B, 20% ethanol standard.
EVANS,
AND
MADSEN
on Sepraphore II. fraction of patient
66V/cm, $5 hour. J.F.: C, chondroitin
A, heparitin sulfate B
even when little difference in total MPS yield is apparent, indicating a possible compensatory loss occurring with dialysis. Effect of Dialysis on Chondroitin Sdfate B Recovery. Total MPS was, therefore, isolated from a urine specimen of patient J.F. (creatinine 0.85 gm/liter, Table 3). The sample was divided in half (600 ml each) and MPS isolated with and without dialysis. The material obtained was fractionated with ethanol and the results are shown in Table 5. It is apparent that the dialyzed urine sample contains hardly any material precipitating with 20% ethanol and that, therefore, much of the chondroitin sulfate B had been lost in this case. As the dialyzate when checked did not contain significant amounts of MPS this loss was not due to dialyzability of the polysaccharide. Further investigation indicated that part of the chondroitin sulfate B present in the CTAB precipitate of the dialyzed urine had remained insoluble in the calcium acetate solution used for redissolving the precipitate (7) but could be recovered by extraction with 0.1 N NaOH. This was not the case with
URINARY
TABLE ETHANOL
FRACTIONATION ISOLATED
20’;
453
MUCOPOLYSACCHARJDES
WITH
5 OF RIuc~PoLY~AccH.~RIDE~
AND
WITHOUT
Ethanola
DIALYSIS
407~ Et,hanok
Carbazolec
SOY0 Ethanola
Carbazolec
Isolation procedure
Yield”
Orcinol
Yieldb
Orcinol
Dialyzed Non-dialyzed
‘) 3.1
1.3 0.4
4x 3’9
1.6 0.9
n MPS precipitated at this ethanol concentration from its solution 2.55& calcium acetate. b Percentage yield of total. c Ratio of the two calorimetric reactions for uranic arid.
Carbazolec Yieldb 50 39
Orcinol 1 .:z 1.3
(at 10 mg/ml’j
in
the same precipitate from nondialyzed urine. The other part of the chondroitin sulfate loss seems due to incomplete precipitation from dialyzed urine. Another patient, D.K., also excreting a mixture of heparitin sulfate and chondroitin sulfate B, was investigated. The urine specimen used had a creatinine value of 0.67 gm/liter and l/s of the heparitin sulfate was lost without dialysis and 1/ of the chondroitin sulfate with dialysis. The losses, therefore, were less severe than in patient J.F., probably due to the less concentrated urine. By applying the method described above of precipitating first, followed by dialysis and reprecipitation, patient D.K. was found to excrete equal amounts of heparitin sulfate and chondroitin sulfate B. DISCUSSION
Studies of the heritable disorders of connective tissue involving mucopolysaccharide metabolism have shown promise to lead to a better understanding of the biological function of these rather elusive polymers. A reliable method for the analysis of urinary MPS is of considerable importance for identification of patients with mucopolysaccharidoses. Many of them are difficult to separate on a clinical basis alone from a large number of other skeletal abnormalities which they resemble but which do not involve the metabolism of MPS. In addition, the urine analysis is valuable for classification of subgroups of these disorders, for evaluating the effects of experimental treatment, and is essential as a background for tissue culture studies. Fibroblast tissue cultures have recently been shown (2, 10) to be useful for the identi&ation of the mucopolysaccharidoses. They have the advantage of being closer to the basic genetic defect and seem to allow the determination of heterozygote
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carriers of the diseases. The cultures have, however, certain drawbacks: ( 1) Space and personnel are not always available; (2) considerable time is required before resuits are obtained; (3) several disorders show the same MPS pattern in the skin fibroblasts and metachromasia of granules alone is not characteristic of connective tissue disorders per se (11). The urinary MPS excretion pattern which is more easily determined, though obviously somewhat removed from the basic defect, should still reflect the primary disease process. Normal urine has been shown to contain chondroitin 4-sulfate, chondroitin g-sulfate, chondroitin sulfate B, chondroitin, heparitin sulfate, keratosulfate, hyaluronic acid, and some chondroitin sulfate polypeptide ( 4. E-14). Chondroitin 4 and 6 sulfate are the major components and considerable variation in sulfate content was noted by some of the authors. Patients with mucopolysaccharidoses have been found to excrete mainly chondroitin sulfate B and heparitin sulfate (15, 16). As these two MPS are present in amounts of 10-20 times the tom1 normal MPS levels they completely overshadow the other constituents. It is, therefore, mainly the excretion pattern of heparitin sulfate and chondroitin sulfate 13 which appears to be characteristic for this group of diseases (Marquio’s syndrome, which is still fairly controversial, is not included here). At present, however, as different laboratories use similar but varying methodology the results obtained are difficult to interpret. As shown here, large amounts of heparitin sulfate can be lost as it does not precipitate well from concentrated urines. This difficulty is obscured when recovery from urine is checked by using tissue-derived heparitin sulfate preparations as standards. At urine concentrations usually encountered precipitation would be complete. However, the polysaccharide when excreted in the urine is of much lower molecular weight and varies considerably in sulfate content ( 17). Both factors can prevent precipitation at much lower salt concentration than that affecting standard preparations ( 18). The same problem also applies to urinary MPS fractionation procedures whether based on ethanol precipitation (4), solubility of cetylpyridinium complexes ( 19) or ion exchange chromatography ( 13, 20). Identification is generally based on undegraded standards obtained from connective tissue sources while the urinary MPS investigated appear considerably degraded ( 13, 17). The MPS loss in a patient excreting mainly heparitin sulfate would express itself in a lowered total excretion value, but in a patient also excreting excessive chondroitin sulfate B the proportion between the two polysaccharides would be affected. In our original study of patient J.F. (7) when d’la ly sis was not used we found a ratio of chondroitm sulfate B to heparitin sulfate of 4 to 1 and at the present, using dialysis, a proportion of 1 to 1. The differences with and without dialysis are not
URINARY
MUCOPOLYSACCHARIDES
4.55
as great in patient D.K. mainly because his urine has been generally less concentrated. In other words, differences in excretion pattern ascribed to a difference in disease entity may frequently be merely due to variations in urine concentration. In addition, as shown here, when dialysis of urine is routinely used, as done by some laboratories, considerable amounts of chondroitin sulfate B may be lost. The simplest procedure, therefore, would appear to be dilution, or alternatively concentration of urines to an optimum ionic strength. However, our data indicate that it would be diEcult to find optimum conditions for both polysaccharides as heparitin sulfate precipitates best from dialyzed urine and chondroitin sulfate B from a somewhat concentrated urine. In addition, salt concentration per se is not the only determining factor as the precipitation of MPS by CTAB seems also influenced by the level of urinary “mucoids” (21). At low levels recovery seems incomplete. The best method would seem to be direct precipitation with CTAB from urine followed by dialysis of the supematant and additional CTAB precipitation. We obtained good recoveries by this method. However, it should perhaps be stressed that no best generally applicable method can be recommended. Di Ferrante (21) has shown that considerable heparitin sulfate can be lost with, rather than without, dialysis as this MPS may be dialyzable. This is in contrast to the data reported here but different patients may very well excrete MPS of different molecular weights and the dialysis conditions used by that author were somewhat more vigorous than those used here. Data presented here show that chondroitin sulfate B may be lost from very dilute urines and therefore concentration may be necessary as a first step. In general, therefore, care should be used in analyzing the excretion in each patient and possible losses should be checked rather than relying on a standard procedure. If this is done the urinary excretion pattern should be a valuable diagnostic tool and also should reflect at least to some extent the basic disease process. The confusion about some .of the mucopolysaccharidoses showing similar excretion pattern but quite different clinical features may also be resolved ( 1). SUMMARY
In a study of polysaccharide excretion in patients with mucopolysaccharidoses large differences in yield as well as pattern, depending on the assay procedure used, were observed. When urines with high creatmine values were not dialyzed prior to polysaccharide precipitation with CTAB, large amounts of heparitin sulfate remained in solution and were therefore lost to analysis. On the other hand, when urines were routinely dialyzed considerable lossesof chondroitin sulfate B appeared to occur. Both factors can lead to serious errors in mucopolysaccharide analysis
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and significantly affect any attempt to correlate disease classification with urinary excretion pattern. An improved method has been proposed and investigators are advised to check for possible losses in the urine of individual patients rather than to rely on a general method. ACKNOWLEDGMENT This 08080.
work
was
supported
in part
by
U.
S. Public
Health
Service
Grant
No.
HE
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