Changes in Human H exosddase Isoenzyme Distribution in Body ...

1 downloads 0 Views 205KB Size Report
*Department of Clinical Chemistry, St. Thomas' Hospital, London SEl7EH, U.K., and ... Ellis et al. (1975~) demonstrated that the hexosaminidase isoenzyme ...
566th MEETING, CAMBRIDGE

241

Bonnell, J. A., Ross, J. H. & King, E. (1960)Br. J. Ind. Med. 17, 69-80 Dance, N.,Price, R. G., Cattell, W. R., Lansdell, J. & Richards, B. (1970)Clin. Chim. Acta 27, 87-92

Ellis, B. G., Price, R. G. & Topham, J. C. (1973)Chem.-Biol. Interact. 7, 101-113 Nishizumi, M.(1972)Arch. Eno. Health 24,215-225 Nomiyama, K., Sato, C . & Yamamoto, A. (1973)Toxicol. Appl. Pharmacol. 24,625-636 Pierce, R. J., Price, R. G. & Fowler, J. (1975)Biochem. SOC.Trans. 3, 1031-1033 Robinson, D., Price, R, G. & Dance, N. (1967)Biochem. J. 102, 533-538 Severi, A.(1896)Arch. Sci. Med. 20,293 Tucker, S. M.,Boyd, P.J. R., Thompson,A. E. & Price, R. G. (1975)Clin. Chim. Acta 62,333339

Wellwood, J. M., Ellis, B. G., Hall, J. H., Robinson, D. & Thompson, A. E. (1973)Br. Med. J. 2,261-265

Willis, J. B. (1962)Anal. Chem. 34,614-617

Changes in Human H e x o s d d a s e Isoenzyme Distribution in

Body Fluids during the Course of Tissue Damage SUSAN M. TUCKER* and ROBERT G. PRICE? *Department of Clinical Chemistry, St. Thomas' Hospital, London S E l 7 E H , U.K., and t Department of Biochemistry, Queen Elizabeth College, University of London, Campden Hill, London W8 7AH, U.K. Two principal forms of hexosaminidase are present in human tissues and were first isolated from spleen by Robinson & Stirling (1968). The acidic form A and the basic form B can be separated by a variety of techniques including ion-exchange chromatography and gel electrophoresis. It has been demonstrated that the A component of serum can be distinguished from the tissue A component of liver by its susceptibility to neuraminidase treatment (Swallow et al., 1974). The tissue A and serum A" forms can be separated by ion-exchange chromatography (Ikonne & Ellis, 1973), since the ASform is eluted by a lower molarity of NaCl than the tissue A form. Further minor intermediate forms I1 and Izare also found in serum (Price &Dance, 1972) and are present in varying amounts in other tissues. Ellis et al. (1975~)demonstrated that the hexosaminidase isoenzyme distribution in urine varied in patients with a range of renal diseases. Loss of kidney hexosaminidase into the urine changed the form-A: form-B ratio and the percentage contribution of form B to the total hexosaminidase activity increased with the severity of the renal lesion. ) an The change in isoenzyme pattern found in this study (Ellis et al., 1 9 7 5 ~prompted investigation of the isoenzyme composition of other body fluids in relation to tissue damage. In the present study we have compared the hexosaminidase isoenzyme patterns of normal urine, serum and synovial fluid on DEAE-cellulose chromatography with the elution profiles obtained with synovial fluid from rheumatoid arthritics, urine from renal patients and serum from patients with hepatic disease. The relative elution of the tissue A form was determined by using kidney homogenates. Fresh urine samples were collected without preservatives, and used immediately or stored at 4°C. Blood was collected without coagulant, allowed to clot and serum separated immediately. Synovial fluid was obtained from patients having routine joint aspirations. Kidneys were obtainedpost mortem or as a result of unplaced donor transplants and homogenates (lo%, w/v) were prepared in water by using a smooth glass PotterElvejhem homogenizer (Ellis et al., 1975~).DEAE-cellulose columns were prepared and used as described previously by Ellis et al. (1975~).Column fractions were assayed for hexosaminidase activity by the automated fluorimetric method of Tucker et al. (1975) and the sodium content, which was used to measure the NaCl gradient, was determined by flame photometry. The total enzyme activity corresponding to each peak was determined by integrating the area under each peak (Ellis et al., 1975~).Samples were also analysed by using a rapid automated ion-exchange system based on that described by Ellis et al. (19756).

Vol. 5

242

BIOCHEMICAL SOCIETY TRANSACTIONS

Table 1. Activity of hexosaminidase isoenzymes in body jluids and kidney Activities of each isoenzyme is expressed as a percentage of the total recovered hexosaminidase activity. Results are the mean and the number of samples is given in parentheses. Form I represents the sum of the I1 and Iz forms. Isoenzyme activity (%) Form

...

I 4 4 10 20

B 17 13 28 10

Kidney (5) Urine (1 0) Synovial fluid (2) Serum (5)

A”

A

-

79

62

-

70

83

13’Oi

26

61

I

I

I

88

120

159

1

I

190

“aCl] (m)

Fig. 1. Resolution of hexosaminidase components of dialysed serum on DEAE-cellulose

----, Normal individual; --, patient with liver failure. Elution profiles are drawn from the recorder trace. Full details of the experimental procedures are given in the text. Hexosaminidase activity is expressed in arbitrary units. Serum (2nd) was dialysed against 0.01 M-sodium phosphate buffer, pH7.0, at 4°C for 2h, and 0.4ml was applied to the column. The total hexosaminidase activities in all the pathological samples assayed were increased up to 1 0 times the activities in normal serum, urine and synovial fluid. The relative amounts of each isoenzyme in kidney homogenates and body fluids were determined by using DEAE-cellulose chromatography. The activitiesof hexosaminidase

1977

566th MEETING, CAMBRIDGE

243

isoenzymes in urine, kidney, serum and synovial fluid are compared in Table 1. The tissue A form is predominant in urine, but the isoenzyme pattern in synovial fluid W ~ S similar to that of serum. The hexosaminidase pattern of the pathological samples resembled the tissue pattern seen in the kidney. It can be seen in Fig. 1 that the hexosaminidase pattern found in the serum of a patient with infectious hepatitis differs markedly from the normal serum pattern. There is a pronounced increase in the activity of all the isoenzymes present, particularly the B form, which is normally present in low activities. This change in isoenzyme pattern is remarkably similar to that present in pathological urine. A similar pattern was observed in synovial fluid from arthritics. The presence of the B form therefore appears to be a sensitive indicator of cell damage. In some pathological samples of synovial fluid a second A-form component was present resulting in a double peak. The double peak may be explained by the presence of both the A and the As forms in the same sample. It is worth noting, however, that there are a number of different components present in the A-form peak from leucocytes and amniotic fluid (R. B. Ellis, personal communication). The possibility of the microheterogeneity of the hexosaminidase A form should therefore be considered when interpreting pathological data. The present findings appear to be of interest in the assessment of tissue damage and in the study of the interrelationships of tissue and body fluid forms of hexosaminidase. Further study may also provide an insight into the mechanisms involved in the secretion of enzymes. We are grateful to the Endowments Committee of St. Thomas’ Hospital and the National Kidney Fund (U.K.) for financial support. Ellis, B. G., Tucker, S. M., Thompson,A. E. &Price, R. G. (1975~)Clin. Chim. Acta 64,195-202 Ellis, R. B., Ikonne, J. U. & Masson, P. K. (197%) Anal. Biochem. 63, 5-11 Ikonne, J. U. & Ellis, R. B. (1973) Biochem. J. 135,457-462 Price, R. G. & Dance, N. (1972) Biochim. Biophys. Acta 271, 145-153 Robinson, D. & Stirling, J. L. (1968) Biochem. J. 107, 321-327 Swallow, D. M., Stokes, D. C., Gorney, G. &.Harris,H. (1974) Ann. Human Genet.37,287-302 Tucker, S . M., Boyd, P. J. R., Thompson,A. E. &Price, R. G. (1975) Clin. Chim. Acta 62,333339

Disposition of Oestrone Sulphate by the Isolated Perfused Rat Kidney MICHAEL HGLLER, KARIN DENGLER and HEINZ BREUER Institut fur Klinische Biochemie, Universitat Bonn, 0-53 Bonn, Federal Republic of Germany

The important function of oestrone sulphate in the metabolism of oestrogens has been stressed (Holler et al., 1975; Ruder et al., 1972). Whereas the role of the liver in the metabolism of oestrogens has been studied intensively in vivo and in vitro, the function of the kidney in the metabolism of steroids has not been fully elucidated. Many steroidmetabolizing enzymes have been found in kidney tissue preparations; however, in most of these preparations the functional compartments of the organ have been destroyed. Therefore in order to approach physiological conditions, the isolated pulsatory perfused rat kidney was established as a suitable model for the study of steroid-hormone metabolism (Holler et al., 1976~). [6,7-3H210estrone sulphate (specific radioactivity 14.7Ci/mol; final concentration 6.17 nmol/ml) was dissolved in the perfusion medium. Two different media were used, one containing dextran (mol.wt. 70000) to make up colloid osmotic pressure (40g/l), the other containing albumin instead of dextran. Details about the perfusion technique have been described earlier (Holler et al., 1976~). Perfusions were carried out as ‘once-through’ perfusions, the outflowing medium being fractionated in lOml portions. Flow rate was 8.5-9.0ml/min per g, and systolic pressure varied between 110 and 15OmmHg, diastolic pressure between 70 and 100mmHg. Pulse rate was 155-165 per min. Two groups of

Vol. 5