294 - A closed cell for voltammetric measurements of oxygen consumption

Bioelectrochenristry and Bioenergetics 7 (rgSo) J_ EZecfroanal Elsevier

Chem_ 116 (rgSo)

Sequoia

S.A..

Lausanne

-

Printed

in Italy

294 - A Closed Cell for Voltammetric Consumption by 0 Istituto 00 Istituto

5p$3

=pp+

L. SILVESTROSIO, C_ SARTORP

BXeasurements of Oxygen

and G. FRIJESEO

di Clinica Jkdica Generale V. UniversitjL di Roma. Italy di Lstologia ed Embriologia, Facoltjl di Scieuze. Universitg

di Roma,

Italy Manuscript

received

Sovember

~1st

rgig

In order to carry out our research on the effects of seminal plasma on human spermatozoa it was necessary to determine the rate of osygen Conseconsumption by the aminosidases present in seminal plasma. quently we designed a type of cell which, although simple and easy to use, gave such satisfying results as to induce the publication of this short paper_ The cell in question (Fig_ I) is cylindrical and is thermostated. iwo platinum wires (B,C) one working as counter-electrode and the other as reference electrode, are sealed in the wall of the cell_ The use of platinum as reference electrode was possible because our measurements are concerned only with the peak current and not with the potential at which oxygen reduction occurs. The working electrode (A), introduced into the cell filled with sample, is supported on a ridge in the cell (H) and sealed with an O-ring_ The volume of the sample in the cell (about 1.0 cm3) should be enough to cover the ridge H so that the introduction of the working electrode expels any excess of the sample. This in turn ensures a completely filled and hermetically closed cell compartment G_ The cell is kept closed by a weight (brass cylinder E) fitted to the upper part of the glass tube carrying the working electrode. A small magnetic teffon-coated stirrer (not shown in the figure) is included in the cell in order to homogenize the sample_ The cell tightness and the reproducibility of the measurements were checked over a period of z4 hours by voltammetric measurements carried out at costant temperature on buffered solutions previously equilibrated with a gas mixture composed of 5 o/o 0, + 93 o/o N=_ The voltammetric readings, collected by a mu& tipolarograph (AMEL model, 471). were taken following the single sweep method (swee-prate of IOO mV s-l). Under these conditions the capacitive component was negligible_ The proportionality between peak current and oxygen content in the sample in question was checked using mixtures containing different ratios of 0,/N,_ Some results obtained utilizing a working electrode with a 5 m& surface and seminal plasma ilt toto at 37 & 0.05 OC are shown in Table r.

o3oz-4g58/So/oo5+oogS

Q

rgSo

Elsevier

Sequoia

S-A_

Silvestroni,

Sartori

and

Frajese

Schematic view- of the voltammetric cell. A :~vorking electrdde; B: reference electrode; I;: water jacket; G:sample; Crcounterelectrode: D: stoppingO-rin,.o- E: brassblock; WI ridge_

The sensitivity of the apparatus used was about 5 x 10-9 A per mm2 of electrode surface and per torr of oxygen, whereas the response time was negligibly small. The volt ammetric readings (each during about 5s) were carried out at time-intervals established individually for each_ case (from 5 to 20 minutes)_ The oxygen consumption due to this measuring technique (taken for each reading on the same solution) was about 50 times less than that found in continuous readings at a costant potential (0-1 -+ 0.3 $/h.r)_ For our research we employed four identical cells, each one connected to the muItipoIarograph, with which different specimens, of differently treated portions of the same sample, were followed simuitaneously. Fig_ z illustrates the patterns, recordered over a period of 50 min, of oxygen consumption in human seminal plasma z%ztoto (curve I), in samples of the same plasma to which putresciue (concentration IO-= &I)

.

-.

Closed Cell for Voltammetric Tab. I.

Proportionality betiveen O,-pressure PO: Torr 3S 76 159 22s

Xeasurements

57

and peak current intensit>r_ I

CrA 0.90

1.72 3-48 5-X”

has been added (curve 2)’ and in samples of the same plasma (at the same concentration of putrescine as for curve 2) plus semicarbazide (concentration IO-= M) (curve 3)_ The same figure shows that after 30 minutes the oxygen concentration decreased by 40 Oh in the seminal plasma AL toto, by IOO o/o in that containing putrescine. and by IO o/o

IFig_3. Osygen consumption by human seminal plasma at 37 “C.

Curve 3- : sample containin,o putrescine and semicarbazide x0-3 M_

Curve I : untreated sample. IO-= _\I_ Curve 3 : sample containing both putrescine

These results in that containing both putrescine and semicarbazide. agree with the data which indicate that putrescine is a substrate with a very high affinity for the aminoxidases which are believed to be mainly responsible for the consumption of oxygen by human seminal fluid [I, 21

and that semicarbazide is a specific inhibitor [3]_

These results prove

58 therefore

Silvestroni. Sartori and that

consumption

the

method

of oxygen

reported

iu small

here

samples

Frajese is suitabie

of biological

for measuring

the

materials.

3efereims [I] E-A_ ZEJXER. Hek_ Cl,irn_A&z 24 (19+x) 1x7 [z] E. H~LLTA, P. PL'LKUXEK. K. ELFVIG, J_ JBNXE, I3iochem.J. 145 (1975) 373 [3] J_ JHXSE. E. H~LLIA, P. HAARAXES. K_ ELFIG. CZin._Chinr. Rcta 48 (1973) 393