Summary
A quantitative cytochemical method for the measurement of β-galactosidase activity in cultured human skin fibroblasts has been developed using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside as the indigogenic substrate. The method relies upon the oxidation of the primary reaction product by ferro/ferricyanide during which an insoluble indigo dye is generated as the final reaction product. The reaction was linear with time up to 60 min using the final cytochemical standard procedure. The enzyme showed maximum activity at pH 4.0 to 4.1. The concentration optima of indigogenic substrate and potassium ferro/ferricyanide were 3.67 mM and 3.13 mM respectively. The presence of sodium chloride activated β-galactosidase up to 100 mM, but was inhibitory above that concentration. The enzyme was inhibited by N-ethylmaleimide, N-acetyl-D-galactosamine and heparin. The enzyme molecules were shown to diffuse out of the cells using media without a suitable inert colloid stabilizer. However, diffusion was completely prevented by using polyvinyl alcohol (PVA) grade G18/140. Air-drying of cells was essential to make the cell membrane permeabel to the substrate and, thereby, to avoid a pronounced lag phase. However, in a biochemical analysis, air-drying itself caused a decrease in enzyme activity to 43% of the control. Even after air-drying lysosomal latency could still be demonstrated by using PVA grade G04/140.
Control persons, one carrier of and two patients with β-galactosidase deficiency were easily identified as belonging to three separate groups by using the cytochemical assay.
It is proposed that the quantitative cytochemical approach may also be applied to cultured human amniotic fluid cells or chorion biopsies giving a rapid prenatal diagnosis of β-galactosidase deficiency due to the small number of cells needed in the analysis.
Similar content being viewed by others
References
Altman FP (1975) Quantitation in histochemistry: a review of some commercial available microdensitometers. Histochem J 7:375–395
Altman FP, Chayen J (1965) Retension of nitrogenous material in unfixed tissue sections during incubation for histochemical demonstration of enzymes. Nature 207:1205–1206
Bitensky L, Chayen J (1979) Cytochemical methods for studying lysosomes. In: Quantitative cytochemistry and its applications. Academic Press, London New York, pp 49–61
Butcher RG (1982) Grades of PVA and enzyme retension in tissue sections. Histochem J 14:165–168
Callahan JW, Gerrie J (1976) GM1-ganglioside and lactosylceramide β-galactosidase from rabbit brain: inhibitor and substrate compitition studies. Can J Biochem 54:803–812
Cotson S, Holt SJ (1958) Studies in enzyme cytochemistry IV. Kinetics of aerial oxidation of indoxyl and some of its halogen derivatives. Proc R Soc B 148:506–519
Galjaard H (1980) Quantitative cytochemical analysis of (single) cultured cells. In: Ciba Foundation Symposium 73 (new series) Trends in Enzyme Histochemistry and Cytochemistry. Excerpta Medica, Amsterdam Oxford New York, pp 161–180
Hansen TL, Andersen H (1983) Succinate dehydrogenase activity in cultured human skin fibroblasts and amniotic fluid cells. Histochemistry 78:211–225
Hansen TL, Christensen E, Brandt NJ (1982) Studies on pyruvate carboxylase, pyruvate decarboxylase and lipoamide dehydrogenase in subacute necrotizing encephalomyelopathy. Acta Paediatr Scand 71:263–267
Henderson B, Loveridge N, Robertson WR (1978) A quantitative study of the effects of different grades of polyvinyl alcohol on the activities of certain enzymes in unfixed tissue sections. Histochem J 10:453–463
Heyworth CM, Neumann EF, Wynn CH (1981) The stability and aggregation properties of human liver acid β-D-galactosidase. Biochem J 193:773–779
Ho MW, O'Brien JS (1970) Stimulation of acid β-galactosidase activity by chloride ions. Clin Chim Acta 30:531–534
Holt SJ, Withers RFJ (1958) Studies in enzyme cytochemistry V. An appraisal of indigogenic reactions for esterase localization. Proc R Soc B 148:520–532
Kaback MM, Sloan HR, Sonneborn M, Herndon RM, Percy AK (1973) Gm-gangliosidosis type I: In utero detection and fetal manifestations. J Pediatr 82:1037–1041
Kirkeby S, Blecher SR (1978) Studies on the oxidizing system in Holt's medium for histochemical demonstration of esterase activity. Acta Histochem 62:44–56
Lake BD (1974) An improved method for the detection of β-galactosidase activity, and its application to GM1-gangliosidosis and mucopolysaccharidosis. Histochem J 6:211–218
Lehninger AL (1975) Biochemistry, second ed. Worth Publishers, New York, p 294
Lilford R, Maxwell D, Coleman D, Czepulkowski B, Heaton D (1983) Diagnosis, four hours after chorion biopsy, of female fetus in pregnancy at high risk of Duchenne muscular dystrophy. Lancet 2:1491
Lojda Z, Gossrau R, Schiebler TH (1979) Enzyme histochemistry. A laboratory manual. Springer, Berlin Heidelberg New York, pp 145–154
O'Brien JS (1983) The gangliosidoses. In: Stanbury JB, Wyngaarden JB, Fredericson DS, Goldstein JL, Brown MS (eds) The metabolic basis of inhereted disease. McGraw-Hill, New York, pp 945–969
Okada S, Kato T, Yutaka T, Yamano T, Yabuuchi H (1983) Application of epithelial cells in culture to the biochemical studies of lysosomal hydrolase deficiencies. Clin Chim Acta 128:169–172
Patel V, Goebel HH, Watanabe I, Zeman W (1974) Studies on GM1-gangliosidosis, type II. Acta Neuropathol 30:155–173
Robertson WR (1980) A quantitative study of N-acetyl-β-glucosaminidase activity in unfixed tissue sections of the guinea-pig thyroid gland. Histochem J 12:87–96
Robertson WR, Frost J, Høyer PE, Weinkove C (1982) 20α-Hydroxysteroid dehydrogenase activity in the rat corpus luteum; a quantitative cytochemical study. J Steroid Biochem 17:237–243
Rushton AR, Dawson G (1977) The effect of glycosaminoglycans on the in vitro activity of human skin fibroblast glycosphingolipid β-galactosidase and neuraminidases. Clin Chim Acta 80:133–139
Stoward PJ (1980) Criteria for the validation of quantitative histochemical enzyme techniques. In: Ciba Foundation Symposium 73 (new series) Trends in Enzyme Histochemistry and Cytochemistry. Excerpta Medica, Amsterdam Oxford New York, pp 11–27
Tietze C (1979) Induced abortion: A fact book, third ed. The population council, New York
White A, Handler P, Smith EL, Hill RL, Lehman IR (1978) Principles of biochemistry, 6th ed. McGraw-Hill, New York, p 1150
Yarborough DJ, Meyer OT, Dannenberg AM, Pearson B (1967) Histochemistry of macrophage hydrolases. III. Studies on β-galactosidase, β-glucuronidase and aminopeptidase with indolyl and naphthyl substrates. J Reticuloendothelial Soc 4:390–408
Zaman G, Chayen J (1981) An aqueous mounting medium. J Clin Pathol 34:567–568
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lund-Hansen, T., Høyer, P.E. & Andersen, H. A quantitative cytochemical assay of β-galactosidase in single cultured human skin fibroblasts. Histochemistry 81, 321–330 (1984). https://doi.org/10.1007/BF00514326
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00514326