Isolation and characterization of glycosaminoglycans from brain of children with protein-calorie malnutrition.

The uronic acid containing glycosaminoglycans (GAGs) were isolated from the brains of 1-year-old and 4-year-old kwashiorkor children and characterised by constituent analyses. A marked reduction is the total GAG concentration of brain was noticed in both cases of kwashiorkor. In the 1-year-old kwashiorkor brain, hyaluronic acid is the most predominant GAG (73.5 per cent) whereas heparan sulphate, chondroitin sulphates and low sulphated chondroitin sulphate constituted less than 10 per cent. In the 4-year-old kwashiorkor brain, the proportion of hyaluronic acid was 27.5 per cent, low sulphated chondroitin sulphate 31.2 per cent, chondroitin sulphates 28.3 per cent and heparan sulphate 10 per cent. This marked reduction in the concentration as well as qualitative changes in GAG in protein-calorie malnutrition as compared to the normal is discussed in relation to brain function. THE OCCURRENCE of various glycosaminoglycans (GAGS) in brain has been established by the work of several laboratories. Although the functional significance of GAGs in brain has yet to be clearly understood, several reports indicate their important role in the brain function. MARGOLIS (1967), by showing that only a small portion of GAG in brain is of connective tissue origin, suggested that GAGS may have a distinct role in brain as compared with other organs. Earlier study in this laboratory (SINGH and BACHHAWAT, 1968) indicated the variation in the levels of these compounds in brain with age, suggesting their role in the process of myelination and brain maturation, as postulated by BRANTE in 1958. The recent work from this laboratory (GEORGE, SINGH and BACHHAWAT, 1969) has shown that the GAGS-sulphotransferase preparations from rat and human brain of various age groups differ in the nature of sulphation process, the ratio of N-sulphation to 0-sulphation decreasing with the maturation of brain. In the mucopolysaccharidosis, Sanfilippo syndrome, which is associated with severe mental retardation, the accumulation of heparan sulphate in brain has been established (GEORGE and BACHHAWAT, 1970). The important role of GAGS such as hyaluronic acid and chondroitin sulphate in brain function is evident from the experiment of CUSTOD and YOUNG (1968) who observed in adult cat, after lateral ventricular injection of testicular hyaluronidase, neurological impairment in addition to a reduction in the levels of hyaluronic acid and chondroitin sulphate. A recent study from this laboratory (VASAN and BACHHAWAT, 1971) shows a decrease in the concentration of hyaluronic acid and low sulphated GAGs in rat brain in experimental allergic encephalomyelitis. Considering the above reports and the findings that there is an impairment of the active sulphate synthesis in rat liver on a low protein diet (LEVI, GELLER, ROOT and WOLF, 1968) and high urinary excretion of Institute of Child Health, Calcutta. Abbreviation used: GAG, glycosaminoglycan. 1913 1914 E. V. CHANDRASEKARAN, K. L. MUKHERJEE and B. K. BACHHAWAT low-sulphated chondroitin sulphate in kwashiorkor (CHERIAN, CHANDRASEKARAN and BACHHAWAT, 1970), it would be of interest to study the nature of GAGS in brain in protein-calorie malnutrition. The present investigation reports on the isolation and characterization of glycosaminoglycans in brain of two children of different ages in protein-calorie malnutrition. M A T E R I A L S A N D M E T H O D S Papain (crude) and pronase B were received respectively from Central Food Technological Research Institute, Mysore and California Biochemical Corporation, U.S.A. Cetyl pyridinium bromide was obtained from Mann Research Laboratory, U.S.A. DEAE-Sephadex was supplied by Pharmacia, Uppsala, Sweden and chondroitin-6-sulphate containing 10 per cent chondroitin-4-sulphate by Miles Research Laboratory, U.S.A. Sigma Chemical Company, U.S.A. supplied glucosamine-HC1, galactosamine-HCI, hyaluronic acid from human umbilical cord (Grade I) and testicular hyaluronidase Type IV. Other reagents of analytical grade. The brains from 1-year-old and 4-year-old kwashiorkor children were obtained at autopsy. The 1-year-old patient (female) had a body weight of 3.3 kg and was oedematous, which is an indication of severe kwashiorkor. The other patient (male) had a body weight of 6.2 kg and developed oedema at later stage. The delipidation of the brains and the isolation of glycosaminoglycans were carried out as described earlier (SINGH and BACHHAWAT, 1968). Analyses. The modification of the carbazole reaction (DISCHE, 1947) by BIITER and MUIR (1962) was used to estimate uronic acid. This reaction was run with and without borate to identify dermatan sulphate. The total hexosamine, galactosamine, sulphate, non-acetylated hexosamine (N-sulphate), chondroitin-6-sulphate and hyaluronidase resistant GAG were estimated as described earlier (CHANDRASEKARAN and BACHHAWAT, 1969). All the analysis were carried out in triplicate. R E S U L T S The levels of the three-GAG-fractions isolated by employing the cetylpyridinium procedure are given in Table 1. Uronic acid 0.91 mg/g of lipid free dry tissue is present in the I-year-old kwashiorkor brain whereas it is only 053 mg/g in the 4-year-old brain. The level of GAG/lipid free dry tissue of both fractions I and I1 are correspondingIy higher (by 70-73 per cent) in the 1-year-old kwashiorkor brain when compared to the other brain and hence the values of ratio 1/11 for both brains are similar. Fraction I11 is extremely low in both cases. TABLE I.-LEVELS OF VARIOUS GLYCOSAMINOGLYCAN FRACTIONS IN ~-YEAR-OLD AND YEAR-OLD KWASHIORKOR BRAINS pg uronic acid/g lipid free dry tissue Kwashiorkor Fraction Fraction Fraction Total Ratio brain I I1 I11 1/11 1 -year-old 542.5 3484 14.1 905 1.56 4-year-old 310.7 206.8 16.1 5336 1.50 Fractions I, I1 and I11 are GAG fractions obtained from cetyl pyridinium bromide-GAG complex by extracting with 0.4 M; 1.2 Mand 2.1 M-NaCI, respectively. In Table 2, the levels of the DEAE-Sephadex fractions are shown. The concentration of Fraction I-A in both is around 12 times that of Fraction I-B. Next to Fraction I-A, Glycosaminoglycans in protein-calorie malnutrition 1915 TABLE 2. LEVELS OF GLYCOSAMINOGLYCANS IN VARIOUS DEAE-SEPHADEX FRACTIONS FROM 1-YEAR-OLD AND 4-YEAR-OLD KWASHIORKOR BRAINS pg uronic acid/g lipid free dry tissue Ratio Kwashiorkor brain I-A I-B 11-A 11-B I-A/I-B 11-A/II-B 1-year-old 502.3 40.2 335 13.4 12.5 25.0 4-year-old 287.7 23.0 206.8 12.5 Fraction I was further subfractionated on DEAE-Sephadex column and fractions I-A and I-B were obtained by eluting with 0.5 Mand 0.9 M-Nacl. Similarly, Fraction I1 was subfractionated on DEAE-Sephadex column and Fractions 11-A and 11-B were obtained with 9.9 M-NaCI and 1-25 M-NaCl in 0.01 N-HCI. Fraction 11-A is predominant in both brains. Fraction 11-B occurs in low concentration in 1-year-old kwashiorkor brain whereas it is absent in 4-year-old brain. The analytical data of the various GAG fractions isolated from the kwashiorkor brains are reported in Table 3. Table 4 presents the analytical data of the hyaluronidase resistant GAG fractions. Fraction I-A. This fraction from both brains consists of equimolar hexosamine with respect to uronic acid. The amount of galactosamine is low in the case of I-year-old kwashiorkor brain whereas it is higher than that of glucosamine in the 4-year-old brain. Sulphate is present in this fraction from both brains and its concentration is TABLE 3.-THE ANALYTICAL DATA OF THE VARIOUS GLYCOSAMINOGLYCAN FRACTIONS FROM KWASHIORKOR BRAINS As percentage total GAG in the fraction Galactosaminet Age group and Chondroitin-6Hyaluronidase fraction Hexasamines* Glucosamine Sulphate sulphate resistant Kwashiorkor brain (1 -year-old) : I-A 1 -03 0.1 0.26 I-B 0.66 Glucosamine 1.34 57 11-A 0.76 0.36 1 -43 23 11-B 1.31 0.30 1 -04 Nil 111 0.70 3.9 1.03 44.0 24 Kwashiorkor brain (4-year-old): I-A 1.05 1.39 0.44 61 I-B 0.99 0.91 1-32 11-A 0.60 2.00 0.89 24 111 0.50 7.00 0.89 48.0 11 * Expressed as molar ratio of uronic acid. t Estimation of glucosamine and galactosamine was carried out by the method described in the text. 1916 E. V. CHANDRASEKARAN, K. L. MUKHERJEE and B. K. BACHHAWAT TABLE 4.-ANALYTICAL DATA OF HYALURONIDASE RESISTANT GAG FRACTIONS Expressed as molar ratio of uronic acid Fraction N-sulphate Hexosamines Galactosamine Kwashiorkor brain (I-year-old) : I-B-HR* 0.25 2.34 0.46 11-A-HR 0-20 0.88 0.13 Kwashiorkor brain (4-year-old) : I-B-HR 0 . 3 4 2.32 0 3 2 11-A-HR 0.31 I .56 0.09 * HR, Hyaluronidase resistant. higher in 4-year-old brain, but N-sulphate was found to be absent in both cases. This fraction from both brains was digested with testicular hyaluronidase and then subjected to Sephadex G-25 filtration with water as eluent. The analysis of hexosamine indicates that the void volume material contains most of the galactosamine-containing GAGS. The void volume GAG was precipitated by cetyl pyridinium bromide, extracted with 0.4 M-NaCl containing 0.1 per cent cetyl pyridihium bromide and then precipitated with 4 vol. of alcohol. This isolated GAGwas subjected to the carbazole reaction with and without borate. Since the depression in colour was not as marked as in the case of dermatan sulphate, this galactosamine containing hyaluronidase resistant material, as yet remains to be identified. Fraction I-B. In the case of 1-year-old kwashiorkor brain, 0.66 M-hexosamine with respect to uronic acid is present and in 4-year-old brain, the molar ratio of hexosamine to uronic acid is 0.99. The concentration of galactosamine is almost equal to that of glucosamine in 4-year-old brain whereas the amino sugar is absent in this fraction of the other brain. The sulphate content of both is 1.3 molar with respect to uronic acid. The hyaluronidase resistant material is 57 per cent of this fraction in 1-year-old kwashiorkor brain and 61 per cent in the 4-year-old brain. The hyaluronidase resistant fraction of 1-year-old kwashiorkor brain contains 0.25 M-N-sulphate with respect to uronic acid and 54 per cent glucosamine, indicating that all of the glucosamine GAG is heparan sulphate. The molar ratio of N-sulphate to uronic acid is 0.34 and 70 per cent glucosamine are present in the hyaluronidase resistant portion of 4-year-old brain, indicating again that the glucosamine GAG is heparan sulphate. The presence of low amount of galactosamine in this fraction from 1-year-old kwashiorkor brain may be the cause of the inabiiity to identify it, when the whole fraction was subjected to hexosamine analysis, Further, the high total hexosamine values noticed in the hyaluronidase resistant fractions could not be explained. Fraction IZ-A. The molar ratio of hexosamine to uronic acid is 0.76 and 0.60 respectively in the case of 1-year-old and 4-year-old kwashiorkor brains. The amount of galactosamine is twice and 0.36 times that of glucosamine and sulphate to uronic acid ratio is 0.89 and 1.43 in 4-year-old and I-year-old kwashiorkor brains, respectively. In I-year-old kwashiorkor brain, the hyaluronidase resistant material is 23 per cent; the resistant material contains 13-4 per cent galactosamine only and hence, the Glycosaminoglycans in protein-calorie malnutrition 1917 resistant galactosamine GAG in the whole fraction is 3 per cent. The whole fraction consists of 26 per cent galactosamine indicating the presence of 26 per cent chondroitin sulphate. The hyaluronidase resistant material from 1-year-old kwashiorkor brain contains 86.6 per cent glucosamine and the molar ratio of N-sulphate to uronic acid is 0.2 suggesting the presence of heparan sulphate which is probably low N-sulphated. In the same way, 4-year-old brain contains 64.6 per cent chondroitin sulphate and 2.4 per cent hyaluronidase resistant galactosamine GAG. The hyaluronidase resistant material contains 90 per cent glucosamine and the molar ratio of N-sulphate to uronic acid is 0.3 indicating the occurrence of heparan sulphate which is probably low Nsulphated . TABLE 5.-THE NATURE AND AMOUNT OF GLYCOSAMINOGLYCANS IDENTIFIED IN THE VARIOUS FRACTIONS ISOLATED FROM THE KWASHIORKOR BRAINS