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Measurement of serum glycosaminoglycans by laser nephelometry
Clinicu Chimicu A&z, Elsevier/North-Holland
CCA
116 (1981) 369-380 Biomedical Press
369
1932
Measurement of serum glycosaminoglycans by laser nephelometry Christine Depurtnlent
Warren
* and Gerald
Manley
of Chemitzll Puthologv, T0rhu.v Hospitul, Torqu0.y TQ.? 7AA (I/. K.) (Received
March 27th, I98
I)
Summary A simple, sensitive micromethod for the assay of serum hyaluronic acid and chondroitin sulphates is presented. The method is based on the binding of the quaternary ammonium salt, cetylpyridinium chloride (CPC) to serum polyanions, and quantitation of the complexes by laser nephelometry. Measurement of the CPC complexes in serum before and after digestion with specific enzymes enables quantitation of hyaluronic acid and chondroitin sulphates in less than 100 ~1 serum. Using this technique, hyaluronic acid is detectable in a small number of normal human sera at concentrations up to 4 mg/l, and chondroitin sulphates are consistently present at concentrations ranging from 2 to 25 mg/l.
Introduction Glycosaminoglycans (GAGS) are widely distributed in human tissues, reaching high concentrations in connective tissue ground substance, synovial fluid, hyaline cartilage and cornea. Their excretion in urine has been extensively studied [ 1,2] and has led to the development of laboratory methods for the diagnosis and delineation of inborn errors of glycosaminoglycan metabolism [3,4]. Less dramatic distortions of GAG excretion have been reported in such diverse disorders as rheumatoid arthritis [5], Werner’s syndrome [6] and disseminated neoplasm [7]. Serum GAGS have been less extensively studied, mainly because available methods have been complex, time-consuming or have required large volumes of blood for the assay [8,9]. A recently published method requiring only small samples of blood (100 ~1) offers only semiquantitative assay and cannot identify individual GAGS [lo]. Nonetheless, reports of serum GAGS in normal individuals and various disease states are beginning to accumulate in the literature [8- 131. A simple micromethod allowing quantitation of individual serum GAGS would add impetus to such work.
* To whom correspondence
0009-898
should
I /8 1/OOOO-0000/$02.50
be addressed. 0 198 1 Elsevier/North-Holland
Biomedical
Press
370
The ability of quaternary ammonium salts such as cetyltrimethylammonium bromide (CTAB) and cetylpyridinium chloride (CPC) to form relatively insoluble complexes with GAGS was thoroughly investigated by Scott [14]. In 1956, Di Ferrante [15] described a turbidimetric method for quantitating pure solutions of GAGS using CTAB. The reaction has been employed in several laboratory methods for urinary GAGS [4] but has not yet found acceptance in the study of serum GAGS, largely because many other serum components (for example acid glycoproteins) also form complexes with quaternary ammonium salts. Orskov [16] used CTAB instead of antiserum in an immunoelectrophoretic technique, and showed neat precipitation arcs with negatively charged polysaccharides. This suggested the similarity of the interaction between quaternary ammonium salts and acidic polyanions to antibody-antigen reactions and that sensitive immunological techniques such as laser nephelometry might be used for quantitating these complexes. Several enzymes capable of digesting individual GAGS with a high degree of specificity have now been isolated [ 17,181. Di Ferrante [ 151 showed that the turbidity which developed when hyaluronic acid and chondroitin sulphate were added to CTAB was completely abolished if the GAGS were incubated with testicular hyaluronidase prior to the assay. Saito et al. [19] described a micromethod for measuring total and individual chondroitin sulphates in urine using specific chondroitinases. Many workers have used these enzymes as an aid to the identification of individual GAGS in various biological materials [20,21]. The possibility of combining the complexing ability of CPC, the specificity of enzymes and the sensitivity of laser nephelometry in developing a new method for the assay of serum GAGS is explored in this paper. Materials and methods Materials Cetylpyridinium chloride and ovine testicular hyaluronidase were obtained from British Drug Houses Ltd., Poole, U.K. Streptomyces hyaluronidase and chondroitinase ABC were obtained from Miles Laboratories, Slough, U.K. Reference samples of hyaluronic acid were obtained from British Drug Houses (HA’) and Miles Laboratories (HA2). Chondroitin-4-sulphate (CSA), chondroitin-6-sulphate (CSC) and dermatan sulphate (CSB) were obtained from Miles Laboratories. Normal human serum was obtained from 50 healthy blood donors by courtesy of the S.W. Regional Blood Transfusion Service, and from 20 healthy laboratory staff volunteers. The reference serum employed in this study was Calibrator 1, Atlantic Antibodies, supplied by American Hospital Supply (UK) Ltd., Station Road, Didcot, Oxon. The Hyland laser nephelometer PDQ system was supplied by Travenol Laboratories. Basic assay: measurement of CPC complexes in serum Blank reagent: 0.15 &101/l NaCl prepared for intravenous use by Boots Company, Nottingham, U.K., was used as the basic diluent. CPC reagent: 2.8 mmol/l (0.18, w/v) CPC in 0.15 mol/l NaCl was prepared by dissolving 100 mg CPC in 100 ml 0.15 mol/l NaCl.
371
The blank and CPC reagents were filtered Corp., CA, U.S.A.) prior to use.
through
a 0.4 pm filter (Nucleopore
Procedure Standards were prepared by diluting the reference serum 1: lOO- 1 : 1600 with 0.15 mol/l NaCl. Test sera (20 ~1) were diluted 1 : 200 with 0.15 mol/l NaCl. Aliquots (1 .O ml) of standards and tests were added to 1.0 ml CPC reagent and 1.0 ml 0.15 mol/l NaCl, mixed, and incubated at 25°C for 2 h. The laser nephelometer was set up according to the Hyland manual. The CPC reagent was used as “antibody blank”. Working at sensitivity 2, zero light scattering was set on 0.15 mol/l NaCl and maximum light scattering was set on the 1: 100 dilution of reference serum incubated with CPC reagent (top standard). A standard curve was constructed by plotting the relative light scattering (RLS) of the standards after blank subtraction, against CPC binding, the RLS of the top standard being taken as 100% CPC binding. After measuring the RLS of the tests, the percentage CPC binding of the test sera were read from the standard curve and multiplied by 2 to take account of the 1 : 200 dilution of the test sera compared with the 1: 100 dilution of the top standard. Thus percentage CPC binding of sera is the RLS of CPC-incubated sera expressed as a percentage of the RLS of CPC-incubated reference serum. A control, consisting of a 1: 200 dilution of pooled human serum, was processed with each batch. Enzyme digestion Serum samples (25 ~1) were incubated for 18 h at 37°C with (a) 5 ~1 0.15 mol/l NaCl; (b) 5 ~1 0.15 mol/l NaCl containing Streptomyces hyaluronidase, 5 TRU; (c) 5 ~1 0.15 mol/l NaCl containing 5 pg ovine testicular hyaluronidase; (d) 5 ~1 0.15 mol/l NaCl containing 0.1 units chondroitinase ABC. After incubation, sera were diluted 1: 200 with 0.15 mol/l NaCl and CPC binding measured as described above. The CPC binding of the enzyme-incubated serum was subtracted from the CPC binding of the 0.15 mol/l NaCl-incubated serum to give the CPC binding attributable to the enzyme-labile material in the serum. Standard curves were prepared by measuring the CPC binding of sera to which hyaluronic acid, chondroitin-6-sulphate, chondroitin-4-sulphate and dermatan sulphate had been added at concentrations ranging from 6.25-200 mg GAG/I serum. The CPC binding of the original serum was subtracted from the CPC binding of the GAG-enriched serum, and CPC binding attributable to the added GAG was plotted against the concentration of GAG added in mg/l serum. Glycosaminoglycan-enriched sera were also subjected to enzyme digestion as described above. Comparison of the laser nephelometric method with the carbazole reaction Lipids were removed from 20 normal sera by solvent extraction and the dried residues were digested with papain. The uranic acid concentrations were then determined by the standard method of Bitter and Muir [22]. Parallel to this procedure the GAG concentration of the 20 normal sera were determined by the laser nephelometric method using chondroitinase ABC.
372
Results Investigation
of nephelometric
assay
Basic assay
The concentration of the final reaction components and the sensitivity of the nephelometer were adjusted to achieve RLS greater than 20 for normal serum tests. With these conditions the CPC reagent blank had a RLS of less than 1 and the normal serum blanks less than 5. The precision of the assay was shown to be similar to that for immunonephelometric protein methods. The between-assay coefficient of variation for the control serum was 7.6% with a mean of 86% CPC binding. The mean value is near the upper limit of the normal range of CPC binding for normal human sera (see below). Effect of varying c~n~entr~ti~ns of CPC
Standard curves using varying concentrations of CPC showed that the sensitivity of the reaction was similar for 0.005% to 0.0258, slightly less sensitive with more concentrated CPC solutions and considerably decreased with solutions containing only 0.0025% CPC (Fig. 1). An antigen excess type of phenomenon appeared to occur with test samples containing more than 50% CPC binding when the concentration of CPC was less than 0.1%. Higher CPC concentrations were not investigated due to difficulties with the solubility of CPC at greater ~ncentrations. Time course of CPC reactions The time course of the reaction
is shown in Fig. 2. It can be seen that CPC
0025
200.
0.01 a0075 0.005
155 J1 OL
KK3.
0.1
!m. QOO25
60 % Of
Serum
El0 pod
Fig. 1. The effect of varying the CPC concentrations (%) on the light scattering produced by a series of dilutions of a human serum pool.
373
minutes
Fig. 2. The change
in light scattering
with time of the reaction
of 0. I ‘%CPC with human
serum.
binding occurs rapidly for the first 20 min, is complete by 60 min and the complexes are stable for at least 3 h. The incubation time before the measurement of light scattering, provided it was greater than 60 min, was therefore not critical. Parallelism Since a large variety of serum polyanions complex with CPC, it was essential to establish that test sera behaved in a similar fashion to the reference material, particularly when the concentration of some of the polyanions may be altered in disease. It was shown that the reference material, normal and pathological sera (A, B, and C) had parallel dilution curves (Fig. 3). Addition of GAGS to sera The percentage CPC binding was shown to be proportional to the amount of GAG added (Figs. 4, 5). Addition of GAG to the pooled serum did not increase the light scattering of the test blank. The percentage CPC binding was also measured with chondroitin sulphate and hyaluronic acid solutions added to saline and human albumin solutions (40 and 80 g/l). The nature of the diluent had no significant effect on the amount of CPC binding produced by the GAG. Comparison of the amount of CPC binding produced with identical concentrations of individual GAGS showed that chondroitin-6 and 6-sulphates and hyaluronic acid produced similar amounts of CPC binding whereas dermatan sulphate produced approximately half that of the other chondroitin sulphates (Table I). CPC Binding of normal human serum The total CPC binding of 70 normal human
sera gave a mean value of 77% with a
374
B
?l oc
Reference
150.
serum
C Norma I 100.
50.
60 % dllutlon
Fig. 3. Dilution curves derived with 0.1% CPC reagent.
from testing reference
range of 54- 100% of the reference Fig. 6. Effect of enzyme digestion The percentage CPC binding
40
Fig. 4. The relationship
60
between
material,
100 of serum
normal
and pathological
serum value. The individual
of test blanks
120
the 56 CPC binding
sera (A, B and C)
values are shown in
before and after addition
160 mg cs
of enzyme
200 I 1 Serum
and the concentration
of hyaluronic
acid (HA2).
375
40
Fig. 5. The relationship
60
between
120
160 mgAA
the % CPC binding
200 /l serum
and the concentration
of chondroitin
sulphate
(CSA).
were identical. Varying the incubation time between 3 h and 3 days had no significant effect on the results of enzyme digestion, and an overnight incubation was considered most convenient. Doubling the concentrations of enzymes used in the digestion also had no significant effect on the results, although 0.02 units/p1 chondroitinase ABC was necessary for digesting the highest concentration of added GAG. Chondroitinase ABC completely destroyed the increased CPC binding due to added chondroitin sulphates, and most of that due to added hyaluronic acid (85% HA’ and 97% HA2). Ovine testicular hyaluronidase destroyed approximately 70% of the CPC binding due to CSA, but had no measurable effect on CSB or CSC. It destroyed approximately 90% of the CPC binding due to added HA2, but only 60% of that due to added HA’.
TABLE
I
INCREASE GAG ( 100
IN CPC BINDING
PRODUCED
BY THE ADDITION Increase
mg/l
serum)
Hyaluronic acid’ Hyaluronic acid’ Chondroitin-4-sulphate Dermatan sulphate Chondroitin-6-sulphate
30 21.5 26.5 12.5 2-l
OF GAG
in ‘%CPC binding
TO HUMAN
SERUM
316
20
40
60 ‘7. CPC
Fig. 6. Distribution
-
bIndIng
of CPC binding in 70 normal sera
L CSF
-
t2
30
P
0
5 20 x v 10 z
l-_dk SA CSB CSC Hd
HA’
Fig. 7. Effect of enzyme digestion on increased CPC binding due to addition of GAGS to ,serum (100 mg/l). The columns show % CPC binding due to CSA, CSC and HA 0 before, and W after, digestion with (a) chondroitinase ABC, (b) ovine testicular hyaluronidase and (c) Srrepromyces hyaluronidase. Methodological details are given in the text.
Streptomyces hyaluronidase had no effect on the increased CPC binding due to added chondroitin sulphates. It destroyed 77% of the CPC binding due to added HA*, but only 40% of that due to added HA’ (Fig. 7). When 24 normal sera were incubated with Streptomyces hyaluronidase, between 0 and 2% of the CPC binding was destroyed. Prior incubation of normal sera with chondroitinase ABC and ovine testicular hyaluronidase gave similar results, removing OS-9% CPC binding. This was approximately equivalent to a hyaluronic acid concentration in normal serum of between 0 and 4 mg/l, and chondroitin sulphate concentration of 2-25 mg/l. The individual values are shown in Fig. 8. The GAG concentrations of normal sera as determined by the laser nephelometric method showed good agreement with the hexuronic acid levels analysed by the method of Bitter and Muir [22]. Conversion of hexuronic acid levels to GAG was achieved by using a multiplication factor of 3 [lo]. The mean GAG concentration of 24 normal sera by laser nephelometry was 14 mg/l with a range of 2-25 mg/l compared with a mean GAG concentration by the carbazole reaction of 12 mg/l with a range of 6-21 mg/l. Discussion The use of laser nephelometry binding to CPC offers considerable
Fig. 8. Concentrations
of hyaluronic
to measure serum polyanions as a result of their advantages of sensitivity and precision compared
acid and chondroitin
sulphate (mg/l)
in 24 normal sera.
37x
with turbidimetric measurements. The behaviour of the CPC reagent in the nephelometric assay with respect to precision, sensitivity, time course and concentration required, showed many similarities with that of antibodies in nephelometric assays for proteins. The ratio of CPC to serum that was found to be optimal in this study was surprisingly high, but the appearance of “antigen excess” type phenomena with doubling dilutions of serum at lower concentrations of CPC made it necessary to employ the highest convenient concentration of CPC in this assay. Possibly the polyanion-CPC complexes form more stable suspensions, with less tendency to aggregate, at the higher concentrations of CPC. Concentrations of CPC in excess of 2.8 mmol/l are unsuitable for use in routine laboratories, since the CPC precipitates from solution if the temperature falls below 20°C. The time course of the reaction offers convenient flexibility in the routine laboratory, with complete stability between 1 and 3 h. The finding that only between 1 and 11% of the CPC binding of normal serum could be attributed to chondroitin sulphates and hyaluronic acid, suggests that either appreciable quantities of other GAGS are present in human serum, or that non-GAG molecules are reacting with CPC under the conditions employed in this assay. Murata et al. [9] in their detailed studies of the GAGS in 161 of pooled human plasma, concluded that CSA was the major GAG present followed by much smaller concentrations of CSC, hyaluronic acid, heparan sulphate and CSB. Thus it appears that much.of the CPC reactive material in human serum is of a non-GAG nature. Preliminary studies with neuraminidase suggest that some of this non-GAG, CPC-reactive material is neuraminidase-labile and probably represents the acid glycoproteins of human serum. The addition of GAGS to serum produced a linear increase in CPC binding up to a concentration of 200 mg/l serum. The assay was extremely sensitive and could accurately measure as little as 3 mg GAG/l serum. Minor increases in serum GAGS have been reported in rheumatoid arthritis [ 1I], osteoarthritis [ 131, inflammation [23], gout [24], and renal disease [ 121. More substantial increases have been reported in mucopolysaccharidoses [lo]. The simple laser nephelometric method, which requires only 100 ~1 serum, is well suited to explore serum GAGS in various pathological conditions. The relatively low CPC binding of CSB under the conditions of this assay is difficult to explain. Possibly it is related to the molecular configuration of CSB, where the inward facing carboxyl group of the iduronic acid moiety may be unable to participate in the CPC reaction. The failure of Streptomyces hyaluronidase to remove all the CPC binding attributable to the presence of added HA in serum is interesting. The specificity of Streptomyces hyaluronidase for HA is well documented [18] and its inability to abolish all the CPC binding of commercial samples of hyaluronic acid suggests the presence of impurities in these samples. Electrophoretic studies of the HA samples used in this study showed the presence of small quantities of other GAG including chondroitin sulphates which explains the inability of Streptomyces hyaluronidase to digest it completely, and also explains the greater activity of testicular hyaluronidase and chondroitinase ABC in abolishing its CPC binding.
379
The failure of ovine testicular hyaluronidase to destroy the increased CPC binding that resulted from the addition of CSC to serum was unexpected, since the specificity of testicular hyaluronidase has been extensively studied and it is known to be active against CSC. It is possible that CSC is only partially degraded by testicular hyaiuronidase to fragments that are still capable of binding CPC. Ovine testicular hyaluronidase was also unable to degrade all the added CSA. When the enzyme concentration was increased to 10 g/l, the added CSA was completely digested, but there was still no effect on CSC. These results suggest that either the enzyme concentration used in these experiments was insufficient or that the enzyme was inhibited by the added GAG. The latter explanation seems more probable since animal hyaluronidases, in contrast to microbial hyaluronate lyases, are competitively inhibited by GAG [21]. Testicular hyaluronidase (1 g/l) and chondroitinase ABC destroyed similar percentages of CPC binding in normal human sera. This suggests that the concentration of GAGS in normal sera is insufficient to inhibit the enzyme and also confirms the results of previous studies which showed that the major GAG in human sera is CSA [9]. The total concentrations of GAGS in normal human sera revealed in this study is also in agreement with previous studies, which revealed concentrations ranging from 2-7 mg hexuronate/l, representing approximately 6- 2 1 mg GAG/l. The laser nephelometric assay of serum GAGS reported here is simple, sensitive and specific. We are at present exploring means of increasing its sensitivity to allow accurate measurement of the very small quantities of HA present in human serum, and as more highly specific enzymes become commercially available, the range and specificity of the assay can be extended. Indeed the principles of the assay can be applied to the measurement of other serum components - for example neuraminoglycoproteins. The polyanion complexing ability of quaternary ammonium salts, combined with the sensitivity of laser nephelometry and the specificity of enzymes, opens the possibility of studying serum GAGS of individual patients in a wide range of clinical conditions, studies which have previously been hindered by the tack of methods suitable for the hospital clinical chemistry laboratory. References I Manley G, Severn M, Hawksworth J. Excretion patterns of glycosaminoglycans and glycoproteins in normal human mine. J Clin Path01 1968; 21: 339-345. 2 Scott JE. Newton DJ. The recovery and characterization of acid glycosaminoglycans in normal human urine. Connect Tiss Res 1975; 3: 157- 164. 3 Manley G, Williams U. Urinary excretion of glycosaminoglyc~s in the various forms of gargoylism. J Clin Path01 1969; 22: 67-75. 4 Pennock CA. A review and selection of simple laboratory methods used for the study of glycosaminoglycan excretion and the diagnosis of the mucopolysaccharidoses. J Clin Path01 1976; 29: 111-123. 5 Loewi G. Urinary excretion of acid polysaccharide in rheumatoid arthritis and other diseases. Ann Rheum Dis 1959; 18: 239-243. 6 Goto M, Murata K. Urinary excretion of macromolecular acidic glycosaminoglycans in Werner’s syndrome. Clin Chim Acta 1978; 85: IOI- 106. 7 Manley G, Bower L, Anson A. Urinary excretion of glycosa~noglycans in disseminated neoplasm. J Ctin Path01 1978; 3 I : 447-453.
380
8 Friman C, Juvani M. Acid glyco~~nogIycans in plasma. Seand J Rheum 1977; 6: 87-91. 9 Murata K, Horiuchi Y. Molecular weight dependent distribution of acidic glycosaminoglycans in human plasma. Clin Cbim Acta 1977; 75: 59-69. IO Melet J, Hooghwinkel GJM, Giesberts MAH, Van Gelderen HH. A semiquautitative micromethod for the determination of free glycosaminoglycans in serum. Clin Chim Acta 1980: 108: I79- 188. I I Friman C, Juvani M, Skrifvars B. Acid glycosaminoglycans in plasma. Stand J Rheum 1977: 6: 177- 182. 12 Friman C, Storgards E, Juvani M. Kock 3. The glycosam~nogly~ans ~muc~polysa~h~des~ in plasma in patients with renal insufficiency. CIin Nepbrol 1977; 8: 435-439. I3 Yusipova NA, Kruik AS. Articular cartilage, blood serum gly~osaminoglycans and gtycoproteins in osteoarthritis deformans. Clin Chim Acta 1979; 94: 9-21. 14 Scott JE. Alipathic ammonium salts in the assay of acidic polysaccharides from tissues. Meth Biochcm Anal 1960; 8: 145-197. IS Di Ferrante N, Turbidimetric measurement of acid mucopolysaccharides and hyaluronidase activity. J Biol Chem 1956; 220: 303-306. 16 Orskov F. Agarose electrophoresis combined with second dimension cetavlon precipitation. Acta Path Microbial Stand 1976; Sect B. 84: 319-320. i 7 Yamagata T, Saito H, Habuchi 0. Suzuki S. Purification and properties of bacterial ~hondroitinas~s and ~hondrosulfat~es. J Biol Chem 1468; 243: 1523- f 535. 18 Ohya T, Kaneko Y, Novel hyaluronidase from streptomyces. B&him Biophys Acta 1970: 198: 607-609. 19 Saito H, Yamagata T, Suzuki S. Enzymatic methods for the determination of small quantities of isomeric chondroitin sulphates. J Biol Chem 1968; 243: I536- 1542. 20 Takeuchi J, Sobuc M, Sato E, Shamoto M, Miura K, Nahagaki S. Variation in glycosaminoglycan components of breast turnours. Cancer Res 1976; 36: 2133-2139. 21 Jourdian G, Wolfman M, Sarber R, Distler J. A specific, sensitive method for the determination of by~uronate. Anal B&hem 1979; 96: 474-480. 22 Bitter T, Muir HM. A modified uranic acid carbazole reaction. Anal B&hem 1962; 4: 330-334. 23 Kerby G. The effect of inffammation on the hexuronate-containing potysaccharides of human plasma, J Clin Invest 1958: 37: 962-964. 24 Katz WA. Accelerated connective tissue metabolism in gout. Arthritis and Rheumatism 1977; 18: Supplement, 751-756.
CCA
116 (1981) 369-380 Biomedical Press
369
1932
Measurement of serum glycosaminoglycans by laser nephelometry Christine Depurtnlent
Warren
* and Gerald
Manley
of Chemitzll Puthologv, T0rhu.v Hospitul, Torqu0.y TQ.? 7AA (I/. K.) (Received
March 27th, I98
I)
Summary A simple, sensitive micromethod for the assay of serum hyaluronic acid and chondroitin sulphates is presented. The method is based on the binding of the quaternary ammonium salt, cetylpyridinium chloride (CPC) to serum polyanions, and quantitation of the complexes by laser nephelometry. Measurement of the CPC complexes in serum before and after digestion with specific enzymes enables quantitation of hyaluronic acid and chondroitin sulphates in less than 100 ~1 serum. Using this technique, hyaluronic acid is detectable in a small number of normal human sera at concentrations up to 4 mg/l, and chondroitin sulphates are consistently present at concentrations ranging from 2 to 25 mg/l.
Introduction Glycosaminoglycans (GAGS) are widely distributed in human tissues, reaching high concentrations in connective tissue ground substance, synovial fluid, hyaline cartilage and cornea. Their excretion in urine has been extensively studied [ 1,2] and has led to the development of laboratory methods for the diagnosis and delineation of inborn errors of glycosaminoglycan metabolism [3,4]. Less dramatic distortions of GAG excretion have been reported in such diverse disorders as rheumatoid arthritis [5], Werner’s syndrome [6] and disseminated neoplasm [7]. Serum GAGS have been less extensively studied, mainly because available methods have been complex, time-consuming or have required large volumes of blood for the assay [8,9]. A recently published method requiring only small samples of blood (100 ~1) offers only semiquantitative assay and cannot identify individual GAGS [lo]. Nonetheless, reports of serum GAGS in normal individuals and various disease states are beginning to accumulate in the literature [8- 131. A simple micromethod allowing quantitation of individual serum GAGS would add impetus to such work.
* To whom correspondence
0009-898
should
I /8 1/OOOO-0000/$02.50
be addressed. 0 198 1 Elsevier/North-Holland
Biomedical
Press
370
The ability of quaternary ammonium salts such as cetyltrimethylammonium bromide (CTAB) and cetylpyridinium chloride (CPC) to form relatively insoluble complexes with GAGS was thoroughly investigated by Scott [14]. In 1956, Di Ferrante [15] described a turbidimetric method for quantitating pure solutions of GAGS using CTAB. The reaction has been employed in several laboratory methods for urinary GAGS [4] but has not yet found acceptance in the study of serum GAGS, largely because many other serum components (for example acid glycoproteins) also form complexes with quaternary ammonium salts. Orskov [16] used CTAB instead of antiserum in an immunoelectrophoretic technique, and showed neat precipitation arcs with negatively charged polysaccharides. This suggested the similarity of the interaction between quaternary ammonium salts and acidic polyanions to antibody-antigen reactions and that sensitive immunological techniques such as laser nephelometry might be used for quantitating these complexes. Several enzymes capable of digesting individual GAGS with a high degree of specificity have now been isolated [ 17,181. Di Ferrante [ 151 showed that the turbidity which developed when hyaluronic acid and chondroitin sulphate were added to CTAB was completely abolished if the GAGS were incubated with testicular hyaluronidase prior to the assay. Saito et al. [19] described a micromethod for measuring total and individual chondroitin sulphates in urine using specific chondroitinases. Many workers have used these enzymes as an aid to the identification of individual GAGS in various biological materials [20,21]. The possibility of combining the complexing ability of CPC, the specificity of enzymes and the sensitivity of laser nephelometry in developing a new method for the assay of serum GAGS is explored in this paper. Materials and methods Materials Cetylpyridinium chloride and ovine testicular hyaluronidase were obtained from British Drug Houses Ltd., Poole, U.K. Streptomyces hyaluronidase and chondroitinase ABC were obtained from Miles Laboratories, Slough, U.K. Reference samples of hyaluronic acid were obtained from British Drug Houses (HA’) and Miles Laboratories (HA2). Chondroitin-4-sulphate (CSA), chondroitin-6-sulphate (CSC) and dermatan sulphate (CSB) were obtained from Miles Laboratories. Normal human serum was obtained from 50 healthy blood donors by courtesy of the S.W. Regional Blood Transfusion Service, and from 20 healthy laboratory staff volunteers. The reference serum employed in this study was Calibrator 1, Atlantic Antibodies, supplied by American Hospital Supply (UK) Ltd., Station Road, Didcot, Oxon. The Hyland laser nephelometer PDQ system was supplied by Travenol Laboratories. Basic assay: measurement of CPC complexes in serum Blank reagent: 0.15 &101/l NaCl prepared for intravenous use by Boots Company, Nottingham, U.K., was used as the basic diluent. CPC reagent: 2.8 mmol/l (0.18, w/v) CPC in 0.15 mol/l NaCl was prepared by dissolving 100 mg CPC in 100 ml 0.15 mol/l NaCl.
371
The blank and CPC reagents were filtered Corp., CA, U.S.A.) prior to use.
through
a 0.4 pm filter (Nucleopore
Procedure Standards were prepared by diluting the reference serum 1: lOO- 1 : 1600 with 0.15 mol/l NaCl. Test sera (20 ~1) were diluted 1 : 200 with 0.15 mol/l NaCl. Aliquots (1 .O ml) of standards and tests were added to 1.0 ml CPC reagent and 1.0 ml 0.15 mol/l NaCl, mixed, and incubated at 25°C for 2 h. The laser nephelometer was set up according to the Hyland manual. The CPC reagent was used as “antibody blank”. Working at sensitivity 2, zero light scattering was set on 0.15 mol/l NaCl and maximum light scattering was set on the 1: 100 dilution of reference serum incubated with CPC reagent (top standard). A standard curve was constructed by plotting the relative light scattering (RLS) of the standards after blank subtraction, against CPC binding, the RLS of the top standard being taken as 100% CPC binding. After measuring the RLS of the tests, the percentage CPC binding of the test sera were read from the standard curve and multiplied by 2 to take account of the 1 : 200 dilution of the test sera compared with the 1: 100 dilution of the top standard. Thus percentage CPC binding of sera is the RLS of CPC-incubated sera expressed as a percentage of the RLS of CPC-incubated reference serum. A control, consisting of a 1: 200 dilution of pooled human serum, was processed with each batch. Enzyme digestion Serum samples (25 ~1) were incubated for 18 h at 37°C with (a) 5 ~1 0.15 mol/l NaCl; (b) 5 ~1 0.15 mol/l NaCl containing Streptomyces hyaluronidase, 5 TRU; (c) 5 ~1 0.15 mol/l NaCl containing 5 pg ovine testicular hyaluronidase; (d) 5 ~1 0.15 mol/l NaCl containing 0.1 units chondroitinase ABC. After incubation, sera were diluted 1: 200 with 0.15 mol/l NaCl and CPC binding measured as described above. The CPC binding of the enzyme-incubated serum was subtracted from the CPC binding of the 0.15 mol/l NaCl-incubated serum to give the CPC binding attributable to the enzyme-labile material in the serum. Standard curves were prepared by measuring the CPC binding of sera to which hyaluronic acid, chondroitin-6-sulphate, chondroitin-4-sulphate and dermatan sulphate had been added at concentrations ranging from 6.25-200 mg GAG/I serum. The CPC binding of the original serum was subtracted from the CPC binding of the GAG-enriched serum, and CPC binding attributable to the added GAG was plotted against the concentration of GAG added in mg/l serum. Glycosaminoglycan-enriched sera were also subjected to enzyme digestion as described above. Comparison of the laser nephelometric method with the carbazole reaction Lipids were removed from 20 normal sera by solvent extraction and the dried residues were digested with papain. The uranic acid concentrations were then determined by the standard method of Bitter and Muir [22]. Parallel to this procedure the GAG concentration of the 20 normal sera were determined by the laser nephelometric method using chondroitinase ABC.
372
Results Investigation
of nephelometric
assay
Basic assay
The concentration of the final reaction components and the sensitivity of the nephelometer were adjusted to achieve RLS greater than 20 for normal serum tests. With these conditions the CPC reagent blank had a RLS of less than 1 and the normal serum blanks less than 5. The precision of the assay was shown to be similar to that for immunonephelometric protein methods. The between-assay coefficient of variation for the control serum was 7.6% with a mean of 86% CPC binding. The mean value is near the upper limit of the normal range of CPC binding for normal human sera (see below). Effect of varying c~n~entr~ti~ns of CPC
Standard curves using varying concentrations of CPC showed that the sensitivity of the reaction was similar for 0.005% to 0.0258, slightly less sensitive with more concentrated CPC solutions and considerably decreased with solutions containing only 0.0025% CPC (Fig. 1). An antigen excess type of phenomenon appeared to occur with test samples containing more than 50% CPC binding when the concentration of CPC was less than 0.1%. Higher CPC concentrations were not investigated due to difficulties with the solubility of CPC at greater ~ncentrations. Time course of CPC reactions The time course of the reaction
is shown in Fig. 2. It can be seen that CPC
0025
200.
0.01 a0075 0.005
155 J1 OL
KK3.
0.1
!m. QOO25
60 % Of
Serum
El0 pod
Fig. 1. The effect of varying the CPC concentrations (%) on the light scattering produced by a series of dilutions of a human serum pool.
373
minutes
Fig. 2. The change
in light scattering
with time of the reaction
of 0. I ‘%CPC with human
serum.
binding occurs rapidly for the first 20 min, is complete by 60 min and the complexes are stable for at least 3 h. The incubation time before the measurement of light scattering, provided it was greater than 60 min, was therefore not critical. Parallelism Since a large variety of serum polyanions complex with CPC, it was essential to establish that test sera behaved in a similar fashion to the reference material, particularly when the concentration of some of the polyanions may be altered in disease. It was shown that the reference material, normal and pathological sera (A, B, and C) had parallel dilution curves (Fig. 3). Addition of GAGS to sera The percentage CPC binding was shown to be proportional to the amount of GAG added (Figs. 4, 5). Addition of GAG to the pooled serum did not increase the light scattering of the test blank. The percentage CPC binding was also measured with chondroitin sulphate and hyaluronic acid solutions added to saline and human albumin solutions (40 and 80 g/l). The nature of the diluent had no significant effect on the amount of CPC binding produced by the GAG. Comparison of the amount of CPC binding produced with identical concentrations of individual GAGS showed that chondroitin-6 and 6-sulphates and hyaluronic acid produced similar amounts of CPC binding whereas dermatan sulphate produced approximately half that of the other chondroitin sulphates (Table I). CPC Binding of normal human serum The total CPC binding of 70 normal human
sera gave a mean value of 77% with a
374
B
?l oc
Reference
150.
serum
C Norma I 100.
50.
60 % dllutlon
Fig. 3. Dilution curves derived with 0.1% CPC reagent.
from testing reference
range of 54- 100% of the reference Fig. 6. Effect of enzyme digestion The percentage CPC binding
40
Fig. 4. The relationship
60
between
material,
100 of serum
normal
and pathological
serum value. The individual
of test blanks
120
the 56 CPC binding
sera (A, B and C)
values are shown in
before and after addition
160 mg cs
of enzyme
200 I 1 Serum
and the concentration
of hyaluronic
acid (HA2).
375
40
Fig. 5. The relationship
60
between
120
160 mgAA
the % CPC binding
200 /l serum
and the concentration
of chondroitin
sulphate
(CSA).
were identical. Varying the incubation time between 3 h and 3 days had no significant effect on the results of enzyme digestion, and an overnight incubation was considered most convenient. Doubling the concentrations of enzymes used in the digestion also had no significant effect on the results, although 0.02 units/p1 chondroitinase ABC was necessary for digesting the highest concentration of added GAG. Chondroitinase ABC completely destroyed the increased CPC binding due to added chondroitin sulphates, and most of that due to added hyaluronic acid (85% HA’ and 97% HA2). Ovine testicular hyaluronidase destroyed approximately 70% of the CPC binding due to CSA, but had no measurable effect on CSB or CSC. It destroyed approximately 90% of the CPC binding due to added HA2, but only 60% of that due to added HA’.
TABLE
I
INCREASE GAG ( 100
IN CPC BINDING
PRODUCED
BY THE ADDITION Increase
mg/l
serum)
Hyaluronic acid’ Hyaluronic acid’ Chondroitin-4-sulphate Dermatan sulphate Chondroitin-6-sulphate
30 21.5 26.5 12.5 2-l
OF GAG
in ‘%CPC binding
TO HUMAN
SERUM
316
20
40
60 ‘7. CPC
Fig. 6. Distribution
-
bIndIng
of CPC binding in 70 normal sera
L CSF
-
t2
30
P
0
5 20 x v 10 z
l-_dk SA CSB CSC Hd
HA’
Fig. 7. Effect of enzyme digestion on increased CPC binding due to addition of GAGS to ,serum (100 mg/l). The columns show % CPC binding due to CSA, CSC and HA 0 before, and W after, digestion with (a) chondroitinase ABC, (b) ovine testicular hyaluronidase and (c) Srrepromyces hyaluronidase. Methodological details are given in the text.
Streptomyces hyaluronidase had no effect on the increased CPC binding due to added chondroitin sulphates. It destroyed 77% of the CPC binding due to added HA*, but only 40% of that due to added HA’ (Fig. 7). When 24 normal sera were incubated with Streptomyces hyaluronidase, between 0 and 2% of the CPC binding was destroyed. Prior incubation of normal sera with chondroitinase ABC and ovine testicular hyaluronidase gave similar results, removing OS-9% CPC binding. This was approximately equivalent to a hyaluronic acid concentration in normal serum of between 0 and 4 mg/l, and chondroitin sulphate concentration of 2-25 mg/l. The individual values are shown in Fig. 8. The GAG concentrations of normal sera as determined by the laser nephelometric method showed good agreement with the hexuronic acid levels analysed by the method of Bitter and Muir [22]. Conversion of hexuronic acid levels to GAG was achieved by using a multiplication factor of 3 [lo]. The mean GAG concentration of 24 normal sera by laser nephelometry was 14 mg/l with a range of 2-25 mg/l compared with a mean GAG concentration by the carbazole reaction of 12 mg/l with a range of 6-21 mg/l. Discussion The use of laser nephelometry binding to CPC offers considerable
Fig. 8. Concentrations
of hyaluronic
to measure serum polyanions as a result of their advantages of sensitivity and precision compared
acid and chondroitin
sulphate (mg/l)
in 24 normal sera.
37x
with turbidimetric measurements. The behaviour of the CPC reagent in the nephelometric assay with respect to precision, sensitivity, time course and concentration required, showed many similarities with that of antibodies in nephelometric assays for proteins. The ratio of CPC to serum that was found to be optimal in this study was surprisingly high, but the appearance of “antigen excess” type phenomena with doubling dilutions of serum at lower concentrations of CPC made it necessary to employ the highest convenient concentration of CPC in this assay. Possibly the polyanion-CPC complexes form more stable suspensions, with less tendency to aggregate, at the higher concentrations of CPC. Concentrations of CPC in excess of 2.8 mmol/l are unsuitable for use in routine laboratories, since the CPC precipitates from solution if the temperature falls below 20°C. The time course of the reaction offers convenient flexibility in the routine laboratory, with complete stability between 1 and 3 h. The finding that only between 1 and 11% of the CPC binding of normal serum could be attributed to chondroitin sulphates and hyaluronic acid, suggests that either appreciable quantities of other GAGS are present in human serum, or that non-GAG molecules are reacting with CPC under the conditions employed in this assay. Murata et al. [9] in their detailed studies of the GAGS in 161 of pooled human plasma, concluded that CSA was the major GAG present followed by much smaller concentrations of CSC, hyaluronic acid, heparan sulphate and CSB. Thus it appears that much.of the CPC reactive material in human serum is of a non-GAG nature. Preliminary studies with neuraminidase suggest that some of this non-GAG, CPC-reactive material is neuraminidase-labile and probably represents the acid glycoproteins of human serum. The addition of GAGS to serum produced a linear increase in CPC binding up to a concentration of 200 mg/l serum. The assay was extremely sensitive and could accurately measure as little as 3 mg GAG/l serum. Minor increases in serum GAGS have been reported in rheumatoid arthritis [ 1I], osteoarthritis [ 131, inflammation [23], gout [24], and renal disease [ 121. More substantial increases have been reported in mucopolysaccharidoses [lo]. The simple laser nephelometric method, which requires only 100 ~1 serum, is well suited to explore serum GAGS in various pathological conditions. The relatively low CPC binding of CSB under the conditions of this assay is difficult to explain. Possibly it is related to the molecular configuration of CSB, where the inward facing carboxyl group of the iduronic acid moiety may be unable to participate in the CPC reaction. The failure of Streptomyces hyaluronidase to remove all the CPC binding attributable to the presence of added HA in serum is interesting. The specificity of Streptomyces hyaluronidase for HA is well documented [18] and its inability to abolish all the CPC binding of commercial samples of hyaluronic acid suggests the presence of impurities in these samples. Electrophoretic studies of the HA samples used in this study showed the presence of small quantities of other GAG including chondroitin sulphates which explains the inability of Streptomyces hyaluronidase to digest it completely, and also explains the greater activity of testicular hyaluronidase and chondroitinase ABC in abolishing its CPC binding.
379
The failure of ovine testicular hyaluronidase to destroy the increased CPC binding that resulted from the addition of CSC to serum was unexpected, since the specificity of testicular hyaluronidase has been extensively studied and it is known to be active against CSC. It is possible that CSC is only partially degraded by testicular hyaiuronidase to fragments that are still capable of binding CPC. Ovine testicular hyaluronidase was also unable to degrade all the added CSA. When the enzyme concentration was increased to 10 g/l, the added CSA was completely digested, but there was still no effect on CSC. These results suggest that either the enzyme concentration used in these experiments was insufficient or that the enzyme was inhibited by the added GAG. The latter explanation seems more probable since animal hyaluronidases, in contrast to microbial hyaluronate lyases, are competitively inhibited by GAG [21]. Testicular hyaluronidase (1 g/l) and chondroitinase ABC destroyed similar percentages of CPC binding in normal human sera. This suggests that the concentration of GAGS in normal sera is insufficient to inhibit the enzyme and also confirms the results of previous studies which showed that the major GAG in human sera is CSA [9]. The total concentrations of GAGS in normal human sera revealed in this study is also in agreement with previous studies, which revealed concentrations ranging from 2-7 mg hexuronate/l, representing approximately 6- 2 1 mg GAG/l. The laser nephelometric assay of serum GAGS reported here is simple, sensitive and specific. We are at present exploring means of increasing its sensitivity to allow accurate measurement of the very small quantities of HA present in human serum, and as more highly specific enzymes become commercially available, the range and specificity of the assay can be extended. Indeed the principles of the assay can be applied to the measurement of other serum components - for example neuraminoglycoproteins. The polyanion complexing ability of quaternary ammonium salts, combined with the sensitivity of laser nephelometry and the specificity of enzymes, opens the possibility of studying serum GAGS of individual patients in a wide range of clinical conditions, studies which have previously been hindered by the tack of methods suitable for the hospital clinical chemistry laboratory. References I Manley G, Severn M, Hawksworth J. Excretion patterns of glycosaminoglycans and glycoproteins in normal human mine. J Clin Path01 1968; 21: 339-345. 2 Scott JE. Newton DJ. The recovery and characterization of acid glycosaminoglycans in normal human urine. Connect Tiss Res 1975; 3: 157- 164. 3 Manley G, Williams U. Urinary excretion of glycosaminoglyc~s in the various forms of gargoylism. J Clin Path01 1969; 22: 67-75. 4 Pennock CA. A review and selection of simple laboratory methods used for the study of glycosaminoglycan excretion and the diagnosis of the mucopolysaccharidoses. J Clin Path01 1976; 29: 111-123. 5 Loewi G. Urinary excretion of acid polysaccharide in rheumatoid arthritis and other diseases. Ann Rheum Dis 1959; 18: 239-243. 6 Goto M, Murata K. Urinary excretion of macromolecular acidic glycosaminoglycans in Werner’s syndrome. Clin Chim Acta 1978; 85: IOI- 106. 7 Manley G, Bower L, Anson A. Urinary excretion of glycosa~noglycans in disseminated neoplasm. J Ctin Path01 1978; 3 I : 447-453.
380
8 Friman C, Juvani M. Acid glyco~~nogIycans in plasma. Seand J Rheum 1977; 6: 87-91. 9 Murata K, Horiuchi Y. Molecular weight dependent distribution of acidic glycosaminoglycans in human plasma. Clin Cbim Acta 1977; 75: 59-69. IO Melet J, Hooghwinkel GJM, Giesberts MAH, Van Gelderen HH. A semiquautitative micromethod for the determination of free glycosaminoglycans in serum. Clin Chim Acta 1980: 108: I79- 188. I I Friman C, Juvani M, Skrifvars B. Acid glycosaminoglycans in plasma. Stand J Rheum 1977: 6: 177- 182. 12 Friman C, Storgards E, Juvani M. Kock 3. The glycosam~nogly~ans ~muc~polysa~h~des~ in plasma in patients with renal insufficiency. CIin Nepbrol 1977; 8: 435-439. I3 Yusipova NA, Kruik AS. Articular cartilage, blood serum gly~osaminoglycans and gtycoproteins in osteoarthritis deformans. Clin Chim Acta 1979; 94: 9-21. 14 Scott JE. Alipathic ammonium salts in the assay of acidic polysaccharides from tissues. Meth Biochcm Anal 1960; 8: 145-197. IS Di Ferrante N, Turbidimetric measurement of acid mucopolysaccharides and hyaluronidase activity. J Biol Chem 1956; 220: 303-306. 16 Orskov F. Agarose electrophoresis combined with second dimension cetavlon precipitation. Acta Path Microbial Stand 1976; Sect B. 84: 319-320. i 7 Yamagata T, Saito H, Habuchi 0. Suzuki S. Purification and properties of bacterial ~hondroitinas~s and ~hondrosulfat~es. J Biol Chem 1468; 243: 1523- f 535. 18 Ohya T, Kaneko Y, Novel hyaluronidase from streptomyces. B&him Biophys Acta 1970: 198: 607-609. 19 Saito H, Yamagata T, Suzuki S. Enzymatic methods for the determination of small quantities of isomeric chondroitin sulphates. J Biol Chem 1968; 243: I536- 1542. 20 Takeuchi J, Sobuc M, Sato E, Shamoto M, Miura K, Nahagaki S. Variation in glycosaminoglycan components of breast turnours. Cancer Res 1976; 36: 2133-2139. 21 Jourdian G, Wolfman M, Sarber R, Distler J. A specific, sensitive method for the determination of by~uronate. Anal B&hem 1979; 96: 474-480. 22 Bitter T, Muir HM. A modified uranic acid carbazole reaction. Anal B&hem 1962; 4: 330-334. 23 Kerby G. The effect of inffammation on the hexuronate-containing potysaccharides of human plasma, J Clin Invest 1958: 37: 962-964. 24 Katz WA. Accelerated connective tissue metabolism in gout. Arthritis and Rheumatism 1977; 18: Supplement, 751-756.