Studies on zinc and angiotensin-converting enzyme

Clinica Chimica Acta, 217 (1993) 213-216 © 1993 Elsevier Science Publishers B.V. All rights reserved. 0009.8981/93/$06.00

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CCA 05552

Short Communication

Studies on zinc and angiotensin-converting enzyme Joseph E. Buttery and Sandra Stuart Department of Clinical Chemistry, The Queen Elizabeth Hospital, Woodville, South Australia 5011 (Australia) (Received 13 January 1993; accepted 15 March 1993)

Key words: Kinetic enzyme assay; Zinc inhibition; Zinc activation; Zinc and EDTA blood angiotensinconverting enzyme

Introduction

Angiotensin-converting enzyme (ACE, EC 3.4.15. I) is a zinc-requiring metalloenzyme which is responsible for the conversion of angiotensin 1 to angiotensin If. It is a membrane-bound glycoprotein localised mainly in the endothelial cells of pulmonary capillaries. Its main clinical application is the diagnosis and management of satcoidosis. Plasma ACE activity is elevated with active sarcoidosis but about a third of patients with this disease have plasma ACE activity within the reference range. High ACE activities are also found in Gaucher's disease, hyperthyroidism, silicosis, asbestosis, alcoholic liver disease and primary biliary cirrhosis. EDTA is a powerful inhibitor of ACE and zinc has been reported to restore the ACE activity [1,2]. There are conflicting reports of zinc causing either activation [3] or inhibition [1,4] of plasma ACE activity or having no effect [5]. We have therefore readdressed this problem by examining the effect of added zinc on ACE activity in heparinised plasma samples and also on EDTA blood samples. Materials and Methods

Plasma ACE activity was measured by the kinetic spectrophotometric method of Ronca-Testoni [6] as optimised by us [7]. The method involves hydrolysis of the subCorrespondence to: Dr. J.E. Buttery, Department of Clinical Chemistry, The Queen Elizabeth Hospital, Woodville, S.A. 501 I, Australia.

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strate N-[3.(2-furyl)acryloyl]-L-phenylalanylglycyiglycine (FAPGG) to N-[3-(2furyi)acryloyl]-L-phenylalanine (FAP) and glycylglycine. The substrate concentration was maximised at 1.0 mmol/l in 80 mmol/I borate buffer at pH 8.2 (37°C). The measurement wavelength was 345 nm to ensure linearity and the assay used a 1:9 plasma to reagent ratio. Effect of zinc on ACE activity in plasma Heparinised blood was used in this study. For zinc activation, each plasma sample (0.9 ml) was treated with 0.1 ml of 10 mmol/l ZnCI:, solution. After about 5 rain, the plasma was centrifuged and assayed, together with the untreated sample, for ACE activity according to the method of Roulston and Allan [3]. The enzyme activity was monitored at 345 nm with the Beckman DU-70 spectrophotometer (Beckman Instruments Inc., Pale Alto, CA 94304). Effect of zinc on ACE activity in EDTA blood Blood samples for this study were collected in two tubes, a heparinised tube and an EDTA tube for each specimen, in the prescribed volumes. The plasma was separated and stored at -40"C until assayed. Varying amounts of stock ZnCI2 solution (100 retool/l) were added to aliquots of an EDTA plasma sample to obtain 1.5, 3.0, 5.0, 7.5 and 10.0 mine/! zinc. The samples were centrifuged and assayed, together with their heparinised plasma samples, for ACE activity by our optimised method [7]. The optimal zinc concentration thus determined was further assessed for its efficacy to restore ACE activity of EDTA blood samples. Results and Discussion

Effect of zinc on ACE activity in plasma Roulston and Allan [3] reported a 2-fold increase in the plasma ACE activity with 1.0 mmol/I zinc. We repeated this study but were unable to obtain significant difference in the results for zinc activated and non-activated samples. The mean ± I S.D. ACE activity for zinc treated and untreated samples were 53 ± 24 IU/I and 54 • 24 IU/I (n = 13), respectively. Huggins et al. [4], on the other hand, r~ported a 40% inhibition of ACE activity with !.0 mmol/I zinc and Maguire and Price [I] observed a 53% inhibition of ACE activity with 1.0 retool/! zinc in the reagent mixture. We cannot reconcile our findings with those of Rouiston and Allan despite having the same reaction conditions for the ACE assay. Using our optimised method [7], we also showed no increase in ACE activity with 1.0 mmol/I zinc in plasma. No enhancement of ACE activity with zinc was observed by Wong and Kinniburgh [5] either. The inhibition of ACE activity by zinc observed by Maguire and Price [I] was contrary to our findings. We therefore investigated this problem using their reaction conditions. The zinc concentration was 1.0 mmol/i but this was in the reaction mixture and is therefore much higher than the concentration used by Roulston and Allan. We monitored our assay at 345 nm with a DU-70 spectrophotometer. Twelve serum samples were tested with and without zinc. Six of the samples showed inhibition of ACE by zinc, ranging from 41 to 66% (mean 50%), thus agreeing

215 with the 53% inhibition obtained by Maguire and Price. The other six samples showed negative ACE results with zinc. On examination of the reaction mixture in the cuvette, turbidity was noted. The greater the turbidity the larger the negative result. Samples which showed 'inhibition' by zinc did not have visible turbidity in the reaction mixture. The turbidity due to added zinc, observed by us, is similar to the ZnSO4 turbidity test of Kunkel [8], which gives an index of the amount of v-globulin present. Hence, the more turbid the reaction, the greater the v-globulin concentration. Further investigation of the negative ACE results was carried out with six other serum samples with varying concentrations of v-globulin determined from the serum protein electrophoretic pattern. The ACE assay was performed according to the method of Maguire and Price [1], with and without zinc. Blank assays were performed using buffer and buffer with zinc instead of the substrate solution. The final absorbances of these solutions were measured and the data are presented in Table I. Zinc-associated turbidity in the ACE assay was visually graded at the end of the test and was found to increase with increasing levels of v-globulin. Tubes containing serum and buffer with zinc also showed increasing turbidity with increasing V" globulin, indicated by AAb in the Table. With normal v-globulin concentration, Zn caused slight turbidity and lowered the ACE activity. This 'inhibition' of ACE activity is probably due to the effect of turbidity on the net absorbance change (&A/min) and not a direct effect of zinc on ACE. With elevated v-globulin, the turbidity was more severe and the AA/min due to turbidity was greater than that due to the ACE assay and consequently, a negative ACE activity was measured. Hence, samples with elevated v-globulin formed turbidity with zinc during the reaction and registered negative ACE results. The sample assayed by Maguire and Price [I] most probably had normal V" globulin concentration and therefore showed 'inhibition' in the ACE measurement with zinc. We also obtained similar 'inhibition' with samples containing normal V" globulin concentrations. Samples with elevated ~,-globulin concentrations gave negative ACE results. Hence Zn does not appear to inhibit or activate ACE per se, as we have shown earlier. TABLE I Effect of T-globulinon turbidity and ACE activity Specimen

! 2 3 4 5 6

7-Globulin Turbiditya

Normal Normal Normal Raised Raised Raised

Nil Nil Nil + ++ ++

Absorbance345 nm

ACE (IU/I)

(+)Zn

(-)Zn

&Ab

Normal (+)Zn

0.206 0.248 0.165 0.212 0.457 0.832

0.163 0.213 0. !53 0.126 0.125 0.288

0.043 0.035 0.012 0.086 0.332 0.544

81 87 101 72 81 151

aTurbidity visuallygraded at the end of the ACE assay. bNegative ACE result due to turbidity in the reaction mixture.

39 26 42 -83 b

-328b -529 b

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Effect of zinc on ACE activity in EDTA blood Maguire and Price [1] showed that 7 mmoWl zinc in the presence of 6 mmol/l EDTA serum sample led to a 15% enhancement of the basal ACE activity. Roulston and Allan [3] also showed that zinc restored, in a dose-dependent manner, ACE activity in plasma to which EDTA was added. These studies represent situations which are unlikely to occur in practice, i.e. the addition of EDTA to serum or plasma samples. However, the accidental collection of blood in an EDTA container is a real problem and therefore the assessment of zinc to restore ACE activity of EDTA plasma is useful. Addition of 1.5 and 3.0 mmol/l zinc to an EDTA plasma sample restored the ACE activity to < 5%. At 5.0 mmol/l zinc, ACE was restored to about 42%, at 7.5 mmol/! to about 100% and at 10.0 mmol/I to about 90%. The concentration of zinc which appears to fully restore the ACE activity is 7.5 mmol/l. With this zinc concentration, the mean ± 1 S.D. ACE activity of the EDTA samples from blood donors was 76 • 29 IU/I (n = 29) and of the heparin samples was 78 4- 28 IU/l. There was no statistical difference between the two sets of data (t = 1.899, P = 0.07). Differences in ACE activity between the zinc-restored and heparin samples ranged from -12 IU/i to +11 IU/I (mean -2.0 IU/I). Hence the ACE activity of zinc-restored samples are generally reliable, but some care should be taken when interpreting values near the upper reference range. Conclusion Zinc does not appear to increase ACE activity in vitro. The claims of ACE activation by zinc are not substantiated in our study while those of ACE inhibition by zinc appear not to be a true inhibition but rather an effect of zinc=induced turbidity. ACE activity in plasma derived from EDTA blood samples can be restored with 7.5 mmol/I zinc.

References I 2 3 4 5 6 7 8

MaguireOA, Price CP. A continuous monitoring spectrophotometric method for the measurement of an~iotensin.4:onvertingenzyme in human serum. Ann Clin Biochem 1985;22:204-210. ReevesPG, O'Dell BL. An experimental study of" the effect of zinc on the activity of angiotensin converting enzymein serum. Clin Chem 1985;31:581-584. RoulstonJE, Allan D. Studies with an automated kinetic assayfor plasma angiotensin-converting enzyme activity and its potentiation by zinc ion. Clin Chim Acta 1987;108:!87-198. Huuins CO, Coreoran RJ, Gordon JS, Henry HW, John JP. Kinetics of the plasmaand lung anglotensin ! converting enzymes.Circ Res 19"/0;28(Suppl. I):93-101. WongYW, Kinniburgh DW. Evaluation of a kinetic assayfor angiotensinconverting enzyme.Clin Biochem 1987;20:323-327. P,onca.Testoni S. Direct spectrophotometric,ssay for angiotensin-convertingenzymein serum.Clin Chem 1983;29:!093-1096. ButteryJE, Stuart S. Assessmentand optimization of kinetic methods for angiotensin-converting enzyme in plasma. Clin Chem 1993;39:312-316. Kunkers zinc sulphateturbidity test. In: Varley H, Gowenlock AH, Bell M. eds. Practical Clinical Biochemistry. Vol. !, Sth ed. London: William Heinemann Medical Books Ltd., 1980;1057-1058.