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An improved method for determination of vitamin K
ANALYTICAL
BIOCHEMISTRY
An Improved
75, 305-307 (1976)
Method
for Determination
of Vitamin
K
Although the determination of vitamins K from the difference between the spectra of oxidized and reduced forms in the uv region has been described (1,2), most of the authors still determine the content of vitamin K in extracts from natural materials on the basis of the absorption of the oxidized form. Arguments for the time-consuming chromatographic separation of vitamin K and its determination on the basis of the absolute spectrum are apparently problems connected with the quantitative transformation of vitamin K into the reduced state. The purpose of this note is to describe the conditions which ensure a complete reduction of menaquinone’ and stability of the reduced form during the measurement. The most suitable milieu for reduction of menaquinone in ethanolic solution is generally assumed to be a mild acidic medium (2,3). A slightly acidic medium of about pH 5, produced by addition of 1% vol of acetate buffer to ethanolic solutions of vitamin Kz, markedly increases the amount of reduced form after the addition of borohydride compared with the reduction performed in the absence of any buffer. The reduction is considered to be complete when further addition of borohydride does not increase the absorption of the reduced form anymore. We found that an equilibrium between oxidized and reduced vitamin K was dependent on vitamin K and proton concentrations; no further change upon addition of borohydride could be observed. Large amounts of borohydride lead to shifts of pH and thus to unreproducible results. Repeated, small additions of KBH, (5 ~1 of 0.1 M aqueous solution) preserve the pH value, but the reduction is very tedious. It could be assumed that the use of a buffer at another pH will be able to produce the conditions required for a rapid and complete reduction. In acetate buffers in the region of pH 3.8-5.0 this requirement was not fulfilled. When a 0.2 M malonate buffer at pH 6.2 was used, the MK reduction was very rapid. After the second addition of borohydride, more than 90% of the MK was reduced but, at the same time, rapid oxidation took place. Very good results can be obtained by the reduction of MK in the slightly alkaline region. In Tris-HCI buffer, the extent of MK reduction increased as the pH was increased from 7.0. In the presence of buffer of pH 9.0 the reduction could be reliably completed. When a buffer of a higher pH (9.7) was used, no reduction ‘Abbreviation used: MK-6, a homolog of vitamin KP.
menaquinone
with six isoprenic units in the side chain,
305 Copyright All rights
0 1976 by Academic Press. Inc. of reproduction in any form reserved.
306
SHORT COMMUNICATIONS
FIG. 1. Course of the reduction of menaquinone. To 3 ml of a 15 PM solution of MK-6 in the sample cuvette containing 1% vol of 1 M acetate or Tris-HCI buffer, 5 /.d of 0.1 M aqueous solution of KBH, were successively added, and the difference in absorption was measured 5 min after borohydride additions. (a) Ethanolic medium at pH 5.0. Values calculated both from the disappearance of oxidized vitamin K, i. e., the difference at 27Onm, (%d - GJ210 “In = -15.0 rnM-l cm-l, and from the formation of reduced vitamin K. i.e., the difference at 245 nm, (E,~ - e,JzrJ “,,, = 28.0 mM-’ cm-l, were identical (a). (b) Ethanolic medium at pH 9.0. Values obtained as before (a). (cd) 50% aqueous propanol at pH 9.0. Points (e) are calculated from the increase of the peak at 244.5 nm caused by reduced MK. Points calculated from the difference at 271 nm are not identical (0).
of MK by borohydride occurred. If MK was dissolved in 50% aqueous propanol instead of ethanol (4), the reduction with borohydride proceeded much more rapidly (Fig. 1). It is also possible to add borohydride in one large dose or even in the solid state. Figure 1 also shows that in 50% propanol at pH 9.0 the reduction does not proceed in only one step (curves c and d). The shape of the differential spectrum after addition of borohydride is unlike that at pH 5.0, where the disappearance of the oxidized form is associated with the appearance of a corresponding amount of the reduced form. At pH 9.0, both in ethanol and aqueous propanol, the reduced form appears later. I3y 10 min after the borohydride addition, the values are close together. The amount of MK in a sample was calculated from the maximal difference between the peak of absorption of the reduced form at 244.5 nm and the minimum caused by the disappearance of the oxidized form at 271 nm, (ered - l &44.5 “,,, - (ered - E,,.& “,,, = 43.4 rnM-l cm-‘. The slight shifts of the absorption maxima of MK in 50% aqueous propanol in comparison with the ethanolic solution (3) were accompanied by fine differences in the difference extinction coefficients. The presence of the semiquinone radical during reduction at pH 9.0 was proved on the basis of the EPR spectrum of the reaction mixture and by absorption in the visible region. This cannot, however, explain the dif-
SHORTCOMMUNICATIONS
307
ferent character of the differential spectrum during the first minutes after the addition of borohydride. The changes in the spectrum of the reduced form dependent on pH showed that the dissociated form of reduced vitamin K also arises during the course of reduction. According to our calculations, about 15% of the disappeared MK,, was present in this form in the first minute after borohydride addition. The conclusion reached regarding the easy reducibility of vitamin K2 with six isoprenic units in a slightly alkaline medium was also verified for other quinones of the vitamin K type. The reduction of vitamin K, (menadione) in an ethanolic medium was also quantitative at pH 8. The reduced K vitamins formed were quite stable during the whole measurement. The procedure described here is a suitable compromise permitting a complete and rapid reduction of K vitamins and was successfully tested by vitamin K, determination in crude lipid extracts of S~aphyfococcus epidermidis and by desmethyl vitamin KsfrornStreptococcusfaecalis . The error of estimation was always less than 3%. ACKNOWLEDGEMENT Menaquinone homologs were obtained from F. Hoffmann-La courtesy of Prof. 0. Wiss.
Roche, Basel, through the
REFERENCES 1. 2. 3. 4.
Crane, F. L. (1959) Plant Physiol. 34, 128. Lester, R. L., White, D. C., and Smith, S. L. (1964) Biochemistry 3, 949. Kriiger, A., and Dadak, V. (1969) Eur. J. Biochem. 11, 328. Schnorf, U. (1%6) Der Einfluss von Substituenten auf Redox-Potenzial und Wuchsstoffeigenschaften von Chinonen, Thesis, Eidgen. Techn. Hochschule, Zurich.
LUDMILA KRIV~~NKOVA VLADIM~RDADAK Department of Biochemistry University of J. E. Purkynt? Kotlcifskb 2, 611 37 Brno, Czechoslovakia Received October 23, 1976; accepted May 4, 1976
BIOCHEMISTRY
An Improved
75, 305-307 (1976)
Method
for Determination
of Vitamin
K
Although the determination of vitamins K from the difference between the spectra of oxidized and reduced forms in the uv region has been described (1,2), most of the authors still determine the content of vitamin K in extracts from natural materials on the basis of the absorption of the oxidized form. Arguments for the time-consuming chromatographic separation of vitamin K and its determination on the basis of the absolute spectrum are apparently problems connected with the quantitative transformation of vitamin K into the reduced state. The purpose of this note is to describe the conditions which ensure a complete reduction of menaquinone’ and stability of the reduced form during the measurement. The most suitable milieu for reduction of menaquinone in ethanolic solution is generally assumed to be a mild acidic medium (2,3). A slightly acidic medium of about pH 5, produced by addition of 1% vol of acetate buffer to ethanolic solutions of vitamin Kz, markedly increases the amount of reduced form after the addition of borohydride compared with the reduction performed in the absence of any buffer. The reduction is considered to be complete when further addition of borohydride does not increase the absorption of the reduced form anymore. We found that an equilibrium between oxidized and reduced vitamin K was dependent on vitamin K and proton concentrations; no further change upon addition of borohydride could be observed. Large amounts of borohydride lead to shifts of pH and thus to unreproducible results. Repeated, small additions of KBH, (5 ~1 of 0.1 M aqueous solution) preserve the pH value, but the reduction is very tedious. It could be assumed that the use of a buffer at another pH will be able to produce the conditions required for a rapid and complete reduction. In acetate buffers in the region of pH 3.8-5.0 this requirement was not fulfilled. When a 0.2 M malonate buffer at pH 6.2 was used, the MK reduction was very rapid. After the second addition of borohydride, more than 90% of the MK was reduced but, at the same time, rapid oxidation took place. Very good results can be obtained by the reduction of MK in the slightly alkaline region. In Tris-HCI buffer, the extent of MK reduction increased as the pH was increased from 7.0. In the presence of buffer of pH 9.0 the reduction could be reliably completed. When a buffer of a higher pH (9.7) was used, no reduction ‘Abbreviation used: MK-6, a homolog of vitamin KP.
menaquinone
with six isoprenic units in the side chain,
305 Copyright All rights
0 1976 by Academic Press. Inc. of reproduction in any form reserved.
306
SHORT COMMUNICATIONS
FIG. 1. Course of the reduction of menaquinone. To 3 ml of a 15 PM solution of MK-6 in the sample cuvette containing 1% vol of 1 M acetate or Tris-HCI buffer, 5 /.d of 0.1 M aqueous solution of KBH, were successively added, and the difference in absorption was measured 5 min after borohydride additions. (a) Ethanolic medium at pH 5.0. Values calculated both from the disappearance of oxidized vitamin K, i. e., the difference at 27Onm, (%d - GJ210 “In = -15.0 rnM-l cm-l, and from the formation of reduced vitamin K. i.e., the difference at 245 nm, (E,~ - e,JzrJ “,,, = 28.0 mM-’ cm-l, were identical (a). (b) Ethanolic medium at pH 9.0. Values obtained as before (a). (cd) 50% aqueous propanol at pH 9.0. Points (e) are calculated from the increase of the peak at 244.5 nm caused by reduced MK. Points calculated from the difference at 271 nm are not identical (0).
of MK by borohydride occurred. If MK was dissolved in 50% aqueous propanol instead of ethanol (4), the reduction with borohydride proceeded much more rapidly (Fig. 1). It is also possible to add borohydride in one large dose or even in the solid state. Figure 1 also shows that in 50% propanol at pH 9.0 the reduction does not proceed in only one step (curves c and d). The shape of the differential spectrum after addition of borohydride is unlike that at pH 5.0, where the disappearance of the oxidized form is associated with the appearance of a corresponding amount of the reduced form. At pH 9.0, both in ethanol and aqueous propanol, the reduced form appears later. I3y 10 min after the borohydride addition, the values are close together. The amount of MK in a sample was calculated from the maximal difference between the peak of absorption of the reduced form at 244.5 nm and the minimum caused by the disappearance of the oxidized form at 271 nm, (ered - l &44.5 “,,, - (ered - E,,.& “,,, = 43.4 rnM-l cm-‘. The slight shifts of the absorption maxima of MK in 50% aqueous propanol in comparison with the ethanolic solution (3) were accompanied by fine differences in the difference extinction coefficients. The presence of the semiquinone radical during reduction at pH 9.0 was proved on the basis of the EPR spectrum of the reaction mixture and by absorption in the visible region. This cannot, however, explain the dif-
SHORTCOMMUNICATIONS
307
ferent character of the differential spectrum during the first minutes after the addition of borohydride. The changes in the spectrum of the reduced form dependent on pH showed that the dissociated form of reduced vitamin K also arises during the course of reduction. According to our calculations, about 15% of the disappeared MK,, was present in this form in the first minute after borohydride addition. The conclusion reached regarding the easy reducibility of vitamin K2 with six isoprenic units in a slightly alkaline medium was also verified for other quinones of the vitamin K type. The reduction of vitamin K, (menadione) in an ethanolic medium was also quantitative at pH 8. The reduced K vitamins formed were quite stable during the whole measurement. The procedure described here is a suitable compromise permitting a complete and rapid reduction of K vitamins and was successfully tested by vitamin K, determination in crude lipid extracts of S~aphyfococcus epidermidis and by desmethyl vitamin KsfrornStreptococcusfaecalis . The error of estimation was always less than 3%. ACKNOWLEDGEMENT Menaquinone homologs were obtained from F. Hoffmann-La courtesy of Prof. 0. Wiss.
Roche, Basel, through the
REFERENCES 1. 2. 3. 4.
Crane, F. L. (1959) Plant Physiol. 34, 128. Lester, R. L., White, D. C., and Smith, S. L. (1964) Biochemistry 3, 949. Kriiger, A., and Dadak, V. (1969) Eur. J. Biochem. 11, 328. Schnorf, U. (1%6) Der Einfluss von Substituenten auf Redox-Potenzial und Wuchsstoffeigenschaften von Chinonen, Thesis, Eidgen. Techn. Hochschule, Zurich.
LUDMILA KRIV~~NKOVA VLADIM~RDADAK Department of Biochemistry University of J. E. Purkynt? Kotlcifskb 2, 611 37 Brno, Czechoslovakia Received October 23, 1976; accepted May 4, 1976