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Studies on serum and urinary glycopeptides and glycosaminoglycans in aspartylglucosaminuria
CLINICA CHIMICA ACTA
431
Cc.4 4744
STUDIES
ON SERUM
AMINOGLYCANS
J. PALO
AND
URINARY
GLYCOPEPTIDES
AND
GLYCOS-
H. SAVOLAINEN
Department of Neurology, (Received
AND
IN ASPARTYLGLUCOSAMINURIA
August
University of Helsinki, Helsinki 29 (Finland)
3, 1971)
SUMMARY
Two-dimensional thin-layer chromatography of the urine samples of aspartylglucosaminuria patients showed the presence of large amounts of aspartylglucosamine that was not detected in their serum. An hitherto unidentified acidic carbohydrate containing aspartylglucosamine, N-acetylneuraminic acid, a hexosamine, and one or two uranic acid residues was also discovered in two-dimensional thin-layer chromatography. A distinct although weak glycosaminoglycan spot was present when urine samples were analysed with the aid of Sepraphore electrophoresis but it could not be detected with one-dimensional glass and serum and urine /3-glucuronidase Both aspartylglucosamine and probably derived from the extrusion
paper chromatography. Serum hyaluronidase, activities were increased. the newly discovered acidic carbohydrate are of lysosomes into the kidney tubules.
Aspartylglucosaminuria (AGU) is a hereditary disorder of metabolism characterized by the excretion of large amounts of z-acetamido-r-(P’-L-aspartamido)-r,zdideoxy-P-D-glucose (AADG) in the urine I. The clinical signs include mental retardation and various gargoyl-like structural abnormalities which tend to become worse with increasing age, lymphocyte vacuoles, occasional periods of diarrhoea and restlessness, and recurrent infections during childhood2J. The mode of inheritance is autosomal recessive. Thirty-four patients are known from Finland (Dr. S. Autio, personal communication) and two from England’. AADG is hydrolysed by a glycopeptide hydrolase (aspartylglucosylamine amido hydrolase, AADGase) associated with the lysosomal cell fraction. The AGU patients have a very low activity of AADGase in plasma and seminal fluid but the plasma AADGase activity is normal in their relatives 2,4. A marked decrease of AADGase activity to about one-fifth to one-third was recently observed in brain and liver biopsies taken from AGU patients5. Large amounts of AADG are excreted in the urine but only traces are found in the plasma and cerebrospinal fluid6. Traces of other glycoproteins seen in the urine have also been found in the cerebrospinal fluid. There have been three substances (and traces of others) of higher molecular weight than AADG that seem to contain Clin.
Chit-n. Acta, 36 (1972)
431-437
432
PALO, SAVOLAINEN
additional neutral carbohydrate residues; however, they have not been further characterized. No detailed analysis on urinary glycosaminoglycans has been performed, and there are no data on the AADG-linked urinary enzymes. It was therefore considered necessary to carry out a careful investigation on the various glycopeptides, glycosaminoglycans and enzymes in the serum and urine of AGU patients. MATERIALS
AND
METHODS
Amino acid analysis AADG can be cetected in desalted urine specimens with one- or two-dimensional amino acid paper chromatography3, and it can be isolated with column-chromatographic techniques4y7. Untreated urine can be analysed with high-voltage amino acid electrophoresis* or with one-dimensional thin-layer chromatographyg. These methods are suitable for screening purposes but the confirmation of the presence of AADG requires heating of the chromatogram at IIO' for 2-3 min. This procedure changes the colour of the AADG spot from brown to greenish-blue which is very characteristic and stays for several weeks. The following two-dimensional procedure is sufficient for clinical purposes and was used in the present study: Two microliters of untreated urine were pipetted on prepared cellulose layer on aluminium foil (E. Merck, Darmstadt, Germany), and the chromatogram was developed with n-butanol-pyridine-acetic acid-water-ethyl acetate (5 : I :2 :2 :2)l" in the first dimension and with n-butanol-acetone-acetic acid-water (35 : 35 : IO: 20)~ in the second dimension. The staining was according to the method of Ersser and Seakins9 with ninhydrin (0.5% in acetone) and with heating the plate at IIO' for 2-5 min. This method again produced the typical blue colour of AADG. proteinized serum were analysed with the same system.
Four
microliters
of de-
Analysis 0-f acidic carbohydrates ad oligosaccharides Twenty microliters of untreated urine were pipetted on prepared Silica gel layer (E. Merck, Darmstadt, Germany) and developed with n-butanollacetic acid-water (50 : 25 : 25) in one dimensionli. The plate was dried and sprayed with a diphenylamine reagent prepared by dissolving I mg diphenylamine in I ml of aniline and by adding this mixture to 90 ml of acetone with IO ml of o-phosphoric acid12. The plate was then heated for zo min at roo’.Ten microliters of both untreated and deproteinized serum were also analysed. Analysis of &cosaminoglycans Isolation and electrophoresis of urinary glycosaminoglycans was performed as described by Wolfe et a1.13. Cetylpyridine chloride (z"/b)was used for the fractionation of the various glycosaminoglycans. A better resolution between chondroitin 4- and 6-sulphate and heparin was obtained by thin-layer chromatography on silicateimpregnated glass paper developed with varying concentrations of ethanol and calcium acetate and stained with 1% toluidine blue solution in 7076 ethanol-acetic acid (95:5,
v/v) for 3 minr”.
Enzyme determinations The activities of serum and urinary hyaluronidase, phatase and amino acid arylamidase (LAP) were assayed mercial methods15. Clin.
Chim.
Acta,
36 (1972)
z+j1-.+37
,&glucuronidase, arylsulaccording to routine com-
SERUM
AND
URINE
Fig. 1. Two-dimensional spot of I\XDG (arrow),
IN ASPARTYLGLUCOSAMINURIA
thin-layer chromatograms B, Control.
433
of urinary
amino acids. ,4, AGU
with a dis tinct
RESULTS
AADG
and amino acids Large amounts of AADG were found in the urine but not in the plasn ia and serum of AGU patients or, if present, the amounts were small enough to avoid dc?tecClin. Chirn. Acta, 36 (1972) 431-437
PALO,
434
SAVOLAINEN
tion with the present analytical methods (Fig. I). This finding was in agreement with the results of our earlier investigation where we were unable to detect AADG with the aid of a column-chromatographic system7. All other plasma amino acids were normal. Acidic carbohydrates and oligosaccharides Thin-layer chromatographic analysis of urine samples produced an unidentified spot with an RF value of 0.13 (Fig. 2). It was not detected in 20 urine samples obtained from adult, non-neurological patients but it was present in all AGU samples. It was excreted in rather constant amounts while the excretion of AADG showed wide variations. Its colour was brownish when stained with diphenylamine reagent. It was not present
in the AGU serum samples.
Fig. 2. One-dimensional thin-layer chromatograms of urinary acidic carbohydrates. From left to right: AGI:; control; neutral sugar standard mi\-ture; glucoce standard; ACU; control. The unknown compound is the lowermost band in the AGU urine samples. Note its equal intensity in the two different patients (the band jnst above it is caused by the solvent system).
The compound was isolated further analysis. It was found to N-acetyl neuraminic acid (NANA) the area of uranic acid residues. investigation. Clin.
Chim.
A4cta, 36
(1972)
131-437
from the silica gel and subjected to hydrolysis and contain AADG as one of its constituents, and also and hexosamine in addition to one or two bands in Its precise structure is at present under a closer
435
SERUM AND URINE IN ASPARTYLGLUCOSAMINURIA
Glycosaminoglycans Analysis of urinary glycosaminoglycans showed a rather weak although distinct spot in the electropherograms of AGU samples (Fig. 3). It was most distinct in the cetylpyridine chloride (CPC)-precipitated fraction but may have been present also in the CPC-soluble group. It was too weak or overlapped by the other compounds in order to be visible in the glass paper chromatograms, cr it may have remained at the origin. Because the chondroitin sulphates and heparin, which come out as a single spot in the Sepraphore electropherogram, can be easily separated on the glass paper, it could be concluded that the spot was none of these. Enzymes Hyaluronidase activity was increased in the serum, and it was normal or slightly decreased in the urine of AGU patients (Table I). The activity of P-glucuronidase was increased both in the serum and urine. The LAP activity was above the upper normal limit in the urine but not in the serum. No arylsulphatase activity was detected in the control and AGU samples.
Fig. 3. One-dimensional Sepraphore electrophoresis of urinary glycosaminoglycans. From left to right: AGU, CPC-non-precipitated fraction; AGU, CPC-precipitated fraction (the extra spot marked with arrow) ; bovine chondroitin sulfate (standard) ; whale chondroitin sulfate (standard) ; heparin (standard) ; control, CPC-non-precipitated fraction; control, CPC-precipitated fraction; whale chondroitin sulfate: bovine chondroitin sulfate. TABLE
I
ACTIVITIES OF HYALURO~IDASE, B-GLUCUROSIDASE
AGU
SERUM ANDURINEOFCONTROLAND
AND
AMIKO
ACID
(L_kPI IS THE
ARYLAMIDASE
PATIENTS
The activities for urinary enzymes are calculated per standard creatinine concentration moles/l. Each figure is the mean of two determinations.
of IO m-
LAP* Sevum Controls:
AGU
I 2 3
0.070
0.146 0.462
0.126
0.117
* Normal range: serum S-z?. mu/ml,
0.000 0.008
0.006 0.000 0.012
10.8 10.8
L'rzm?
1.9 3.3 5.6
urine 0.6-4.7 U/115. Clin. Chim. Acta,
36
(1972)
431-437
I’ALO,
436
SAVOLAINEN
DISCUSSION
AADG residues occur in nature linking carbohydrate
groups to protein chains.
The protein chain is continued on by peptide bonds on either side of the asparagine moiety in the usual way, while the carbohydrate chain is attached to the N-acetylglucosamine residue4. Decreased a.ctivity of AADGase leads into an abnormal urinary excretion of AADG and into aspartylglucosaminuria. AADG has been the main target of the screening techniques aimed at the detectian of AGU3. On the basis of the present investigation it seems as if the new acidic carbohydrate would be equally suitable for screening purposes. It is excreted in rather constant amounts while the excretion of AADG varies from one patient to the other. However, its role in the pathogenesis of AGU is even more obscure than that of AADG. It is definitely different from the glucose, galactose and hydroxylysine-containing glycopeptide observed in the urine of some neuroblastoma patients16; interestingly enough, this peptide was thought to be present in the glomerular basement membrane and it was expected to be found in patients suffering from kidney or collagen disease. Electron microscopic data have suggested that the cause of the urinary excretion of AADG in aspartylglucosaminuria may be the extrusion of lysosomes into the kidney tubules (Arstila et al., submitted for publication). It is possible that the other compound is excreted by the same mechanism. In any case, both of them are metabolites of the catabolic pathway of glycoproteins and have been found thus far only in the patients with AGU. Despite the several gargoyl-like clinical features enough bicchemical evidence has been gathered to delineate AGU as an independent metabolic disorder, especially different from the mucopolysaccharidose.9. The significance of the extra spot observed in the glycosaminoglycan analysis is at present unknown although it may well reflect the generalised connective tissue disorder. There is thus by far no evidence to support the inclusion of AGU into the group of mucolipidoses17. The lysosomal nature of AGU is now well established5. Increased activity of fi-D-glucuronidase in liver, kidney and brain has been reported, as has also been the case with a number cf other acid hydrolases. The increased activity of this enzyme in urine is in good agreement with these results and fits well into the concept of a generalised lysosomal disorder. However, the increased LAP activity supports more the theory of a tubular leakage as mentioned above. On the other hand, these two theories do not necessarily exclude each other but may reflect the multidimensional nature of the disease. The very large lysosomes observed in AGU biopsy specimens contain some unidentified storage material which is at present under investigation (Palo and Savolainen, to be published). The fact that it appears electron-lucent in liver and kidney and more electron-dense in brain5 may indicate differences in its biochemical composition in various tissues. Its relation to AADG and to the newly discovered acidic carbohydrate compound remains to be determined. ACKNOWLEDGEMEiXTS
This investigation was supported by grants from the National for Medical Sciences and from Kehitysvammaliitto r.y., Finland. Clin. Chim.
Ah,
36 (1972)
431-437
Research
Council
SERUM
AND
URINE
437
IN ASPARTYLGLUCOSAMINURIA
We wish to express for technical assistance.
our appreciation
to Miss K. Lindberg
and Mr. P. MIBrGnen
REFERENCES F. R. J. R. J. R.
A. JENNER AND R. J. POLLITT, Biochem. J., 103 (1967) 48P. J. POLLITT, F. A. JENNER AND H. MERSKEY, Luncet, ii (1968) 253. PALO AI\‘D K. MATTSSON, J. Mental Deficiency Res., 14 (1970) 168. J. POLLITT AND F. A. JENNER, Clin. Chim. Acta, 25 (1969) 413. PALO, P. RIEKKINEN, A. ARSTILA AND S. AUTIO, Neurology, in Press. J. POLLITT AND F. A. JENNER, in J. D. ALLAN AEII D. N. RAINE (Eds.), Some lnhevited Disorders of Brain and Mznscle, E. & S. Livingstone, Edinburgh-London, 1969, p. 80. J. PALO AND K. MATTSSON, J. Chvomatog., 50 (1970) 534. J. PALO, J. K. VISAKORPI, J. PERHEENTUPA .%NDT. LOUHIMO, Stand. J. Clin. Invest., 25 (1970) 61. 9 R. S. ERSSERAPI‘D J. W. T. SEAKINS, Natzlve, 223 (1969) 1388. 10 M. BRENXER, A. NIEDERUTIESER AND G. PATAKI, in E. STAHL (Ed.), Diinnschichtchvomatographic, ein LaboratoriulnshandbzIch, 2. Ed., SPringer-Verlag. Berlin-Heidelberg-New York, 1967, P. 720. B. A. LEWIS AND F. SMITH, ibid., p. 795. K. LAUNIALA, J. PERHEEP~TUPA, J. VISAKORPI AND N. HALLMAN, Pedia!Yics, 34 (1964) 615. 13 L. S. WOLFE, J. CALLAHAN, J. S. FAWCETT, F. ANDERMIINN AND C. R. SCRIVER, Neurology, 20 (1970) 23. ‘4 L. LIPPIELLO AND H. J. MANKIN, Avuzl. Biochem., 39 (1971) 54, 1970, 15 Biochemica Katalog. Boehringer Mannheim GmbH, Mannheim, Germany, 16 H. SHIMIZU AND E. H. LABROSSE. Clin. Chim. Acta, 22 (1968) 623. 17 J. W. SPRAXGI~R AND H.-R. WIEDERMANN, Humangenetik, 9 (1970) 113. II I2
Cl&. Chim. Acta, 36 (1972) 431-437
431
Cc.4 4744
STUDIES
ON SERUM
AMINOGLYCANS
J. PALO
AND
URINARY
GLYCOPEPTIDES
AND
GLYCOS-
H. SAVOLAINEN
Department of Neurology, (Received
AND
IN ASPARTYLGLUCOSAMINURIA
August
University of Helsinki, Helsinki 29 (Finland)
3, 1971)
SUMMARY
Two-dimensional thin-layer chromatography of the urine samples of aspartylglucosaminuria patients showed the presence of large amounts of aspartylglucosamine that was not detected in their serum. An hitherto unidentified acidic carbohydrate containing aspartylglucosamine, N-acetylneuraminic acid, a hexosamine, and one or two uranic acid residues was also discovered in two-dimensional thin-layer chromatography. A distinct although weak glycosaminoglycan spot was present when urine samples were analysed with the aid of Sepraphore electrophoresis but it could not be detected with one-dimensional glass and serum and urine /3-glucuronidase Both aspartylglucosamine and probably derived from the extrusion
paper chromatography. Serum hyaluronidase, activities were increased. the newly discovered acidic carbohydrate are of lysosomes into the kidney tubules.
Aspartylglucosaminuria (AGU) is a hereditary disorder of metabolism characterized by the excretion of large amounts of z-acetamido-r-(P’-L-aspartamido)-r,zdideoxy-P-D-glucose (AADG) in the urine I. The clinical signs include mental retardation and various gargoyl-like structural abnormalities which tend to become worse with increasing age, lymphocyte vacuoles, occasional periods of diarrhoea and restlessness, and recurrent infections during childhood2J. The mode of inheritance is autosomal recessive. Thirty-four patients are known from Finland (Dr. S. Autio, personal communication) and two from England’. AADG is hydrolysed by a glycopeptide hydrolase (aspartylglucosylamine amido hydrolase, AADGase) associated with the lysosomal cell fraction. The AGU patients have a very low activity of AADGase in plasma and seminal fluid but the plasma AADGase activity is normal in their relatives 2,4. A marked decrease of AADGase activity to about one-fifth to one-third was recently observed in brain and liver biopsies taken from AGU patients5. Large amounts of AADG are excreted in the urine but only traces are found in the plasma and cerebrospinal fluid6. Traces of other glycoproteins seen in the urine have also been found in the cerebrospinal fluid. There have been three substances (and traces of others) of higher molecular weight than AADG that seem to contain Clin.
Chit-n. Acta, 36 (1972)
431-437
432
PALO, SAVOLAINEN
additional neutral carbohydrate residues; however, they have not been further characterized. No detailed analysis on urinary glycosaminoglycans has been performed, and there are no data on the AADG-linked urinary enzymes. It was therefore considered necessary to carry out a careful investigation on the various glycopeptides, glycosaminoglycans and enzymes in the serum and urine of AGU patients. MATERIALS
AND
METHODS
Amino acid analysis AADG can be cetected in desalted urine specimens with one- or two-dimensional amino acid paper chromatography3, and it can be isolated with column-chromatographic techniques4y7. Untreated urine can be analysed with high-voltage amino acid electrophoresis* or with one-dimensional thin-layer chromatographyg. These methods are suitable for screening purposes but the confirmation of the presence of AADG requires heating of the chromatogram at IIO' for 2-3 min. This procedure changes the colour of the AADG spot from brown to greenish-blue which is very characteristic and stays for several weeks. The following two-dimensional procedure is sufficient for clinical purposes and was used in the present study: Two microliters of untreated urine were pipetted on prepared cellulose layer on aluminium foil (E. Merck, Darmstadt, Germany), and the chromatogram was developed with n-butanol-pyridine-acetic acid-water-ethyl acetate (5 : I :2 :2 :2)l" in the first dimension and with n-butanol-acetone-acetic acid-water (35 : 35 : IO: 20)~ in the second dimension. The staining was according to the method of Ersser and Seakins9 with ninhydrin (0.5% in acetone) and with heating the plate at IIO' for 2-5 min. This method again produced the typical blue colour of AADG. proteinized serum were analysed with the same system.
Four
microliters
of de-
Analysis 0-f acidic carbohydrates ad oligosaccharides Twenty microliters of untreated urine were pipetted on prepared Silica gel layer (E. Merck, Darmstadt, Germany) and developed with n-butanollacetic acid-water (50 : 25 : 25) in one dimensionli. The plate was dried and sprayed with a diphenylamine reagent prepared by dissolving I mg diphenylamine in I ml of aniline and by adding this mixture to 90 ml of acetone with IO ml of o-phosphoric acid12. The plate was then heated for zo min at roo’.Ten microliters of both untreated and deproteinized serum were also analysed. Analysis of &cosaminoglycans Isolation and electrophoresis of urinary glycosaminoglycans was performed as described by Wolfe et a1.13. Cetylpyridine chloride (z"/b)was used for the fractionation of the various glycosaminoglycans. A better resolution between chondroitin 4- and 6-sulphate and heparin was obtained by thin-layer chromatography on silicateimpregnated glass paper developed with varying concentrations of ethanol and calcium acetate and stained with 1% toluidine blue solution in 7076 ethanol-acetic acid (95:5,
v/v) for 3 minr”.
Enzyme determinations The activities of serum and urinary hyaluronidase, phatase and amino acid arylamidase (LAP) were assayed mercial methods15. Clin.
Chim.
Acta,
36 (1972)
z+j1-.+37
,&glucuronidase, arylsulaccording to routine com-
SERUM
AND
URINE
Fig. 1. Two-dimensional spot of I\XDG (arrow),
IN ASPARTYLGLUCOSAMINURIA
thin-layer chromatograms B, Control.
433
of urinary
amino acids. ,4, AGU
with a dis tinct
RESULTS
AADG
and amino acids Large amounts of AADG were found in the urine but not in the plasn ia and serum of AGU patients or, if present, the amounts were small enough to avoid dc?tecClin. Chirn. Acta, 36 (1972) 431-437
PALO,
434
SAVOLAINEN
tion with the present analytical methods (Fig. I). This finding was in agreement with the results of our earlier investigation where we were unable to detect AADG with the aid of a column-chromatographic system7. All other plasma amino acids were normal. Acidic carbohydrates and oligosaccharides Thin-layer chromatographic analysis of urine samples produced an unidentified spot with an RF value of 0.13 (Fig. 2). It was not detected in 20 urine samples obtained from adult, non-neurological patients but it was present in all AGU samples. It was excreted in rather constant amounts while the excretion of AADG showed wide variations. Its colour was brownish when stained with diphenylamine reagent. It was not present
in the AGU serum samples.
Fig. 2. One-dimensional thin-layer chromatograms of urinary acidic carbohydrates. From left to right: AGI:; control; neutral sugar standard mi\-ture; glucoce standard; ACU; control. The unknown compound is the lowermost band in the AGU urine samples. Note its equal intensity in the two different patients (the band jnst above it is caused by the solvent system).
The compound was isolated further analysis. It was found to N-acetyl neuraminic acid (NANA) the area of uranic acid residues. investigation. Clin.
Chim.
A4cta, 36
(1972)
131-437
from the silica gel and subjected to hydrolysis and contain AADG as one of its constituents, and also and hexosamine in addition to one or two bands in Its precise structure is at present under a closer
435
SERUM AND URINE IN ASPARTYLGLUCOSAMINURIA
Glycosaminoglycans Analysis of urinary glycosaminoglycans showed a rather weak although distinct spot in the electropherograms of AGU samples (Fig. 3). It was most distinct in the cetylpyridine chloride (CPC)-precipitated fraction but may have been present also in the CPC-soluble group. It was too weak or overlapped by the other compounds in order to be visible in the glass paper chromatograms, cr it may have remained at the origin. Because the chondroitin sulphates and heparin, which come out as a single spot in the Sepraphore electropherogram, can be easily separated on the glass paper, it could be concluded that the spot was none of these. Enzymes Hyaluronidase activity was increased in the serum, and it was normal or slightly decreased in the urine of AGU patients (Table I). The activity of P-glucuronidase was increased both in the serum and urine. The LAP activity was above the upper normal limit in the urine but not in the serum. No arylsulphatase activity was detected in the control and AGU samples.
Fig. 3. One-dimensional Sepraphore electrophoresis of urinary glycosaminoglycans. From left to right: AGU, CPC-non-precipitated fraction; AGU, CPC-precipitated fraction (the extra spot marked with arrow) ; bovine chondroitin sulfate (standard) ; whale chondroitin sulfate (standard) ; heparin (standard) ; control, CPC-non-precipitated fraction; control, CPC-precipitated fraction; whale chondroitin sulfate: bovine chondroitin sulfate. TABLE
I
ACTIVITIES OF HYALURO~IDASE, B-GLUCUROSIDASE
AGU
SERUM ANDURINEOFCONTROLAND
AND
AMIKO
ACID
(L_kPI IS THE
ARYLAMIDASE
PATIENTS
The activities for urinary enzymes are calculated per standard creatinine concentration moles/l. Each figure is the mean of two determinations.
of IO m-
LAP* Sevum Controls:
AGU
I 2 3
0.070
0.146 0.462
0.126
0.117
* Normal range: serum S-z?. mu/ml,
0.000 0.008
0.006 0.000 0.012
10.8 10.8
L'rzm?
1.9 3.3 5.6
urine 0.6-4.7 U/115. Clin. Chim. Acta,
36
(1972)
431-437
I’ALO,
436
SAVOLAINEN
DISCUSSION
AADG residues occur in nature linking carbohydrate
groups to protein chains.
The protein chain is continued on by peptide bonds on either side of the asparagine moiety in the usual way, while the carbohydrate chain is attached to the N-acetylglucosamine residue4. Decreased a.ctivity of AADGase leads into an abnormal urinary excretion of AADG and into aspartylglucosaminuria. AADG has been the main target of the screening techniques aimed at the detectian of AGU3. On the basis of the present investigation it seems as if the new acidic carbohydrate would be equally suitable for screening purposes. It is excreted in rather constant amounts while the excretion of AADG varies from one patient to the other. However, its role in the pathogenesis of AGU is even more obscure than that of AADG. It is definitely different from the glucose, galactose and hydroxylysine-containing glycopeptide observed in the urine of some neuroblastoma patients16; interestingly enough, this peptide was thought to be present in the glomerular basement membrane and it was expected to be found in patients suffering from kidney or collagen disease. Electron microscopic data have suggested that the cause of the urinary excretion of AADG in aspartylglucosaminuria may be the extrusion of lysosomes into the kidney tubules (Arstila et al., submitted for publication). It is possible that the other compound is excreted by the same mechanism. In any case, both of them are metabolites of the catabolic pathway of glycoproteins and have been found thus far only in the patients with AGU. Despite the several gargoyl-like clinical features enough bicchemical evidence has been gathered to delineate AGU as an independent metabolic disorder, especially different from the mucopolysaccharidose.9. The significance of the extra spot observed in the glycosaminoglycan analysis is at present unknown although it may well reflect the generalised connective tissue disorder. There is thus by far no evidence to support the inclusion of AGU into the group of mucolipidoses17. The lysosomal nature of AGU is now well established5. Increased activity of fi-D-glucuronidase in liver, kidney and brain has been reported, as has also been the case with a number cf other acid hydrolases. The increased activity of this enzyme in urine is in good agreement with these results and fits well into the concept of a generalised lysosomal disorder. However, the increased LAP activity supports more the theory of a tubular leakage as mentioned above. On the other hand, these two theories do not necessarily exclude each other but may reflect the multidimensional nature of the disease. The very large lysosomes observed in AGU biopsy specimens contain some unidentified storage material which is at present under investigation (Palo and Savolainen, to be published). The fact that it appears electron-lucent in liver and kidney and more electron-dense in brain5 may indicate differences in its biochemical composition in various tissues. Its relation to AADG and to the newly discovered acidic carbohydrate compound remains to be determined. ACKNOWLEDGEMEiXTS
This investigation was supported by grants from the National for Medical Sciences and from Kehitysvammaliitto r.y., Finland. Clin. Chim.
Ah,
36 (1972)
431-437
Research
Council
SERUM
AND
URINE
437
IN ASPARTYLGLUCOSAMINURIA
We wish to express for technical assistance.
our appreciation
to Miss K. Lindberg
and Mr. P. MIBrGnen
REFERENCES F. R. J. R. J. R.
A. JENNER AND R. J. POLLITT, Biochem. J., 103 (1967) 48P. J. POLLITT, F. A. JENNER AND H. MERSKEY, Luncet, ii (1968) 253. PALO AI\‘D K. MATTSSON, J. Mental Deficiency Res., 14 (1970) 168. J. POLLITT AND F. A. JENNER, Clin. Chim. Acta, 25 (1969) 413. PALO, P. RIEKKINEN, A. ARSTILA AND S. AUTIO, Neurology, in Press. J. POLLITT AND F. A. JENNER, in J. D. ALLAN AEII D. N. RAINE (Eds.), Some lnhevited Disorders of Brain and Mznscle, E. & S. Livingstone, Edinburgh-London, 1969, p. 80. J. PALO AND K. MATTSSON, J. Chvomatog., 50 (1970) 534. J. PALO, J. K. VISAKORPI, J. PERHEENTUPA .%NDT. LOUHIMO, Stand. J. Clin. Invest., 25 (1970) 61. 9 R. S. ERSSERAPI‘D J. W. T. SEAKINS, Natzlve, 223 (1969) 1388. 10 M. BRENXER, A. NIEDERUTIESER AND G. PATAKI, in E. STAHL (Ed.), Diinnschichtchvomatographic, ein LaboratoriulnshandbzIch, 2. Ed., SPringer-Verlag. Berlin-Heidelberg-New York, 1967, P. 720. B. A. LEWIS AND F. SMITH, ibid., p. 795. K. LAUNIALA, J. PERHEEP~TUPA, J. VISAKORPI AND N. HALLMAN, Pedia!Yics, 34 (1964) 615. 13 L. S. WOLFE, J. CALLAHAN, J. S. FAWCETT, F. ANDERMIINN AND C. R. SCRIVER, Neurology, 20 (1970) 23. ‘4 L. LIPPIELLO AND H. J. MANKIN, Avuzl. Biochem., 39 (1971) 54, 1970, 15 Biochemica Katalog. Boehringer Mannheim GmbH, Mannheim, Germany, 16 H. SHIMIZU AND E. H. LABROSSE. Clin. Chim. Acta, 22 (1968) 623. 17 J. W. SPRAXGI~R AND H.-R. WIEDERMANN, Humangenetik, 9 (1970) 113. II I2
Cl&. Chim. Acta, 36 (1972) 431-437