M ] ~ C H A N I S M O F R E S A Z U R I N R E D U C T I O N I N M I L K O F L O W

B A C T E R I A L C O N T E N T

R. 54. MOYER AND J. J. R. CAMPBELL Division of Animal Science, Dairying and Animal Husbandry

The University of British Columbia, Vancouver, Canada

SU~I~[AR¥

An accurate quantitative method of determining resazurin reduction has been developed. The method depends on the extraction of the dye with n-butanol and the spectrophotometric estimation of the oxidized and reduced forms. The reduc- ing capacity of milk was assessed by adding an excess 2,6-dichlorophenolindo- phenol having an Elo within the Eh range of fresh milk and determining the amount converted to its reduced fol~n. In individual samples of milk the rate of resazurin reduction depended on the concentration of ascorbic acid and the reduc- tion rate decreased with time as the ascorbate concentration decreased. However, milk from different sources differed widely in reduction rates at the same constant ascorbate concentrations. This observation, along with the decreased rate of reduction on dilution of milk, indicated that some factor in milk in addition to ascorbate acted to reduce resazurin. I t is visualized that zones of high hydrogen ion activity occur at the surface of bacteria, leucocytes, and fat globules. Resazurin diffusing into these zones in the presence of ascorbate becomes reduced and remains reduced on diffusion back into the milk plasma. Thus, the rate of reduction would depend on the number and pI-I of the zones and the ambient ascorbate concentration. I t was concluded that the zones acted by virtue of their pH and not their Eh.

The present investigation was concerned with the mechanism of resazurin reduction by the naturally occurring reducing systems of milk in the absence of significant bacterial activity. Resazurin reduction has been reported to be virtually independent of the aerobic electrode potential of milk, although the dye had some influence on measured potential (6,9). The rate of resazurin reduction is very sensitive to pathological and abnormal physiological condi- tions of milk (11, 14), the reducing agent in these abnormal milks apparently being asso- ciated with, and possibly loosely bound to, leucocytes (6).

The visual methods employed to determine resazurin reduction may be satisfactory for estimating the bacteriological quality of com- mercial samples of milk but were found to be inadequate for detailed study of reduction rates. Methods such as the iodometric method outlined by Twigg (16) determine the amount of reso- rufin in a given batch of resazurin, and require large quantities of dye. Since, in milk, a small quantity of dye is used as an indicator, and since quantitative separation from interfering substances is difficult, the assay would be im- practical, if not impossible. The spectrophoto- metric method for sinmltaneous estimation of

Received for publication April 4, 1963.

resazurin and resorufin presented by De Baun and de Stevens (2) required some modification before it could be applied to milk.

A method for assessing the reducing capacity of milk in the presence of resazurin was de- veloped. Since aerobic electrode potential does not correlate with rate of resazurin reduction in milk, potentiometric Eh measurements were considered of no use for this purpose unless carried out anaerobically. However, since resaz- urin is reduced immediately by the anaerobic potential of milk, it was expected that this mechanism of reduction differed from that oc- curring aerobically. Since the Eh of fresh milk, in the absence of extensive bacterial ac- tivity, ranges from 0.2 to 0.3 v (4, 7), it was possible to assess the degree of poising of the system by adding an excess of an oxidant having Elo in this range and measuring the extent o£ reduction. From preliminary experi- ments it was found that 2,6-diehlorophenolindol- phenol, which has Elo of 0.247 v at pH 6.6 (3), was partially decolorized on addition to milk and that the blue sodium salt of the dye could be extracted from milk with n-butanol.

EXPERII~ENTAL PROCEDURES

Bacterial numbers. Milk was obtained from selected healthy cows of the University herd and standard methods plate counts routinely

897

898 1¢. H. M O Y E R AND J. J . R. C A M P B E L L

carried out to determine bacterial numbers. The bacterial numbers of the milk as used in these experiments ranged from 800-3,000 per milli- liter.

Resazurin reduction. Commercial resazurin tablets, certified by the Biological Stain Com- mission to contain approximately ]1 mg dye, were used (8, 15). According to Standard ~Y[ethods (15), each tablet makes 200 ml of the solution to be employed in a one plus ten dilution with milk for visual estimation of resazurin reduction. The resulting final con- centration in milk is approximately 2.4 × 10 -' M. The concentration varies very slightly from one batch of stock solution to the next, as does the initial concentration of resorufin (8, 16). I f the stock solution from one randonfiy chosen tablet is used as an arbitrary standard for calibration of the method, subsequent batches, made from different tablets, can be standard- ized against the original calibration data. This permits direct comparison of data obtained at different times and the use of different dye sources. Although the coefficients calculated from Figure 2 are based on the assumption that the dye tablet used contained 11 mg resaz- urin, which is an admitted approximation, they serve to standardize the method and place the measurement of reducing rates on a quanti- tative basis.

Preliminary tests of a number of solvents revealed that n-butanol extracted the dye fairly efficiently and separated from the milk on centrifuging. The absorption spectra of resaz- urin and resorufin in butanol are shown in Figure 1. The curves were obtained by adding standard resazurin solution to butanol and shaking in the presence of excess sodium bi- carbonate• The butanol layer was cleared by filtration and read against butanol which had been given similar treatment except that dis- tilled water was substituted for dye solution. To obtain the curve for resorufin, the resazurin

,, ' RESORUFIN 060~ I

0 450

URIN

~ .$OG

0.15(

400 500 600 WAVELENGTH (m,w}

FIG. 1. Spectra of resazurin and resorufin in butanol saturated with sodium bicarbonate.

0.900

0.600

g

0.300 0

RESORUFIN , ~ 582 m R

/ /

/ /

AT

I I l 0 . 0 4 8 0 . 0 9 6 0 . 1 4 4

CONCENTRATION (,u,M }

:FIG• o. Calibration curves for the method• Dye at each concentration was in 9.0 ml butanol saturated with sodium bicarbonate.

solution was reduced with sodium isoascorbate prior to addition of butanol and treated as above. The Em.x for resazurin and resorufin in butanol, saturated with sodium bicarbonate, were 615 and 582 m/z, respectively. The blanks, butanol shaken with distilled water and sodium bicarbonate, with and without added sodium isoascorbate, showed no absorption at either of these wavelengths when read against distilled water. The addition of sodium bicarbonate did not influence the absorption spectra o£ resaz- urin and resorufin extracted from milk at pH 6.6 or from phosphate buffer at pK 6.4, but did markedly increase the optical density at the points of maximum absorbance.

Since resazurin changes color from violet to orange in the p i t range 6.5 to 3.8 (12), its a bsorbance at more alkaline reactions was also expected to vary with pH. The variation in absorbanee over the range pH 6.2 to 7.0 is shown in Column A of Table 1. The readings in Column B were the result of saturating the extracts from Colmnn A with sodimu bicarbon- ate, and show that the variations in the values of Column A were due to absorbance rather than to the actual amounts of dye present. The data as a whole illustrate that extraction of dye from phosphate buffer is constant through- out the physiological pl:I range of milk and that absorbanee increases with rising pH.

Calibration curves for resazurin and reso- rufin in butanol saturated with sodium bicarbon- ate were obtained on a fresh]y prepared dye solution (Figure 2). Increments of dye and

RESAZURIN I~EDUCTION IN M I L K

TABLE 1 Optical density of resazurin and resorufin extracted from phosphate buffer of varying p i t

899

B A Extract saturated with

Extract ~ sodium bicarbonate b

pH of buffer ]%esazurin Resorufin Resazurin Resorufin

(582m~) (615 m~) (582m~) (615¢n~) (582m~) (615~n~) (582m~) (615m~) 6.2 .193 .273 .232 .009 .295 .529 .690 .025 6.3 .2'15 .330 .250 .010 .295 .520 .690 .024 6.4 .221 .350' .291 .011 .295 .520 .690 .024 6.5 .235 .377 .34~ .013 .295 .520 .690 . 0 ~ 6.6 .245 .404 .300 .015 .295 .520 .690 .024 6.8 .290 .~70 .529 .015 ,295 .520 .690 .025 7.0 .298 .482 .550 .02~ .295 .520 .590 .024 tt~O .197 .288 .3'67 .015 .295 .520 .690 .025

0.5 ml resazurin solution or 0.5 ml resorufin added to 5.0 ml ] .0% phosphate buffer at above pH values in test tubes: held at 37C for 39 rain; added to 18 nil n-butanol in 9-g Babcock cream test bottles; shaken 10 min at room temperature; centrifuged, a~ld optical density of butanol layer measured.

b Butanol layer from Column A added to excess sodium bicarbonate; shaken 5 rain at room temperature; filtered through Whatman #1 paper and optical density measured.

water were added to 9-g Babcock cream test bottles to a total volume of 0.6 ml. Sodium isoascorbate was added to one series of flasks to reduce the resazur in to resorufin. The mix- tures were shaken on a mechanical shaker fo r 10 rain at room tempera tu re ; excess sodium bi- carbonate was added to the flasks and shaking continued fo r 5 rain. The mixtures were filtered through W h a h n a n #1 pape r and the optical densi ty read. The stock solution f rom which these data were ob t a ined was the a rb i t ra ry s tandard described, and subsequent solutions of dye were corrected to it. Since it was nmre convenient fo r subsequent work to calibrate the method direct ly in terms of quant i ty of dye added to milk, extinction coefficients fo r the dye in butanol were not obtained.

The constants derived f rom the slopes of these calibration curves were used to determine t h e relative quanti t ies of resazurin and reso- rufin in an unknown mixture (17). The calcu- lations were carr ied out, and the final results expressed, in terms of t ~ resazur in or reso- rufin in the sample. I t should be noted tha t the calibration was carried out on 0.6-ml sam- pies ra ther than on the 5.5-ml samples usually employed in measur ing resazur in reduct ion in ndlk. The smaller volume was completely mis- eible with butanol and was used fo r s tandardiza- t ion to eliminate any par t i t ion effects due to the presence of two phases.

Calculation of the relative concentrat ions of resazurin and resorufin in unknown mixtures was as follows :

D~ = optical densi ty at 615 m~ due to resaz- urin.

D p -~-

D ~ - ~

D~ =

optical densi ty at 615 mt~ due to reso- rufin.

optical densi ty at 582 mff due to resaz- urin.

optical densi ty at 582 mt~ due to reso- r u i n .

concentrat ion resazur in (ff~1 pe r sam- p le) .

concentrat ion resorufin (t~[ per sam- ple) .

D~ : K~R D~ = Kj,rt

D~_ : K2R ~ D~ = K~R ~

K~ = D~/R : 4.82 Each value was calculated as an

K~, = D , / R ~ .02 average of six readings, i.e., at six different con- centrat ions

K3 : D~/R = 2.57

K , : D , / R ~ : 6.14

E61~ = D1 + D~. = K 1 R + K~.R ~ = ob- served OD at 615 m/~

E~s~. = D~ + D~ = K~R + K~R ~ = ob- served OD at 582 m/~

K~Eo~ - - K ._E~ . ' . 1 ~ =

: 0.208-E~5 -- 0.007 E~,2 : tt~t resaz- ur in in sample

R J- :

: 0.164 E.. . - - 0.087 E,~ ---- ~ reso- rufin in sample

900 i%. }I. MOYEI~ AND J. J. 1~. CAMPBELL

TABLE 2

Effect of incubation time on dye recovery Incubation time at 37 C

Time to reduce to 7/4

Milk or standard buffer by visual system assay

30 min 90 min 150 rain

Resaz- Reso- Resaz- Reso- l~esaz- Reso- urin rufin Total urin rufin Total urin turin Total

Cow 1 60 min Cow 2 70 min Cow 3 90 min Cow 4 130 min Cow 5 175 min 0.4% Phosphate

buffer at pII 6.4 with added sodium isoascorbate

0.4% Phosphate buffer at pH 6.4

(g~) .061 .040 .101 .034 .069 .103 .023 .079 .102 .066 .033 .099 .040 .062 .102 .031 .075 .106 .076 .025 .101 .057 .047 .104 .043 .057 .100 .076 .030 .106 .050 .048 .098 .042 .062 .104 .078 .022 .100 .062 .042 .104 .052 .053 .105

.001 .107 .108 .091 .106 .107 .001 .106 107

.107 .001 .108 .108 .001 .109 .108 .001 .109

0.5 ml resazurin solution containing 0.12 gM dye was added to 5.0 ml milk, and the mixture incubated at 3~ C in test tubes for the times indicated. They were then added to 9.0 ml n-butanol in 9-g tlabcock cream test bottles; shaken on a mechanical shaker at room temperature for 10 rain; centrifuged 10 rain; butanol layer decanted to clean flasks containing excess sodium bicarbonate; shaken 5 min at room temperature; filtered through Whatmaa #1 pape~ and optical density read. The recovery range was 82 to 90%.

The recovery of dye f rom milk and phospha te buffer a f t e r incubat ion is shown in Table 2. Visual observat ions of dye color are included, as well.

An est imate of the dilution of butanol ex- t rac ts f rom various volumes of buffer, milk, and grav i ty cream was obtained (Table 3). The difference between 10 ml and the volume of butanol retr ievable f rom a 10-ml por t ion of ext rac t was taken to represen t the dilution. The net ga in in volume of the butanol a f t e r extract ion of dye f rom 10 ml ~f buffer and sa tura t ing the extract wi th sodium bicarbonate was 9.2%. When a solution of dye in butanol, such as tha t used fo r s tandardizat ion, was di- luted to the same extent, the a p p a r e n t dye loss was 8.0%. This largely accounted f o r the low dye recover 7 calculated fo r extracts f rom buf- fer. The dilution of butanol by whole milk, 11.8%, accounted fo r only a 10% loss of dye, whereas a p p a r e n t losses up to 18% were en- countered fo r whole milk in Table 2. This sug- gests tha t some fac tor in addi t ion to aqueous dilution of the extract , possibly dilution by milk fat , was func t ion ing to depress dye re- coveries f rom whole milk.

I t is evident f rom the foregoing tha t the original cal ibrat ion could not be used directly to calculate dye recoveries unless dilution of the extract was considered as well and some addit ional allowance made f o r milk fa t . Since milks f rom different sources differ in fa t con-

TABLE 3

Dilution of butanol extract from different sources

Vol. butanol Per cent diln- (ml) per ex- tion (100 minus tract af ter vol. butanol

Source of saturation per 100 ml extract with NaHCOs extract)

Buffer" O~l) 1.0 9.73 ~.7 2.0 9.24 7.6 4.0 9.08 9.2

10.0 9.08 9.2

Whole milk (m~) 1.0 9.72 9.8 2.0 9.42 5.8 4.0 9.13 8.7

10.0 8.82 11.8

Skimmilk (ml) 1.0 9.66 3.4 2.0 9.26 7.4 4.0 9.03 9.7

10.0 9.10 9.0'

Gravity cream (ml) 1.0 9.96 0.4 2.0 9.50 5.0 4.0 8.78 12.2

I0.0 8.63 13.7

0.04% phosphate, p i t 6.4. Materials shown in first column added to 18 ml

butanol in Babcock flasks and shaken 10 rain; centrifuged and butanol layer decanted; excess NaIICO~ added to butanol extract and shaken 5 rain; filtered; 10 ml extract added to 5.0 ml H20 and distilled at 99.6 C; volume butanol calculated from weight of 25 ml distillate.

I%ESAZURIN R E D U C T I O N I N 1VIILK

TABLE 4

Apparent dye recovery f~om different sources

901

Resaz- ICeso- Resazurin/ OD urhl rufin Total resorufin

Whole milk (ml) (582m~) (615 m~) ( ~ ) 0 .~28 .260 .052 .048 .100 1.1 1 .435 .255 .051 .049 .100 1.0 2 .430 .2¢8 .049 .049 .098 1.0 3 .430 .242 .048 .050 .098 1.0 5 .430 .238 .048 .050 .098 1.0

Skimmfik (ml) 0 .420 .260 .052 .048 .100 1.1 1 .440 .260 .052 .052 .104 1.0 2 .435 .255 .051 .052 .103 1.0 3 .440 .250 .050 .051 .101 1.0 5 .440 .240 .0~8 .052 .100 0.9

Milk fat ~ (ml) 0 .428 .260 .052 .048 .100 1.1 1 .400 .2~5 .047 .045 .092 1.0 2 .380 .220 .044 .043 .087 1.0 3 .360 .210 .042 .041 .083 1.0 5 .312 .175 .035 .036 .071 1.0

Obtained by centrifuging whole milk and, therefore, containing more fat than gravity c r e a m .

Milk, skim, or fat diluted to a total volume of 5.0 ml with water prior to adding .12 ~ dye (1:1 resazurin: resorutin mixture); assay procedure was the same as that for Table 2, except that samples were not warmed to 37 C before extraction.

tent, it would be difficult to obtain reproducible dilution values for direct calculation.

The apparent dye recovery from whole milk, skim, and milk fat is shown in Table 4. Equal q!aantities (0.06 ~ ) of resazurin and resorufin were added to each sample and the dye ex- tracted immediately. The extraction was car- ried out before any significant resazurin reduc- tion occurred to change the ratio of the two forms of the dye. The decrease in recovery shown for milk fat was more rapid than it would have been for gravity cream, but it illus- trated that, even at recoveries as low as 60%, the ratio, resazurin/resorufin remained con- stant regardless of source of extract.

Generally, in whole milk, more than 80% of added dye was recovered. Of the 20% loss, half could be accounted for by gain in volume of the butanol extract, leaving a net 10% loss that could not be accounted for. On the basis of the data in Table 4, it was considered valid to assume that the ratio, resazurin/resorufin was the same in this 10% as in the extract, and that recovered dye could be extrapolated to 100%, using this ratio to arrive at a quanti- tative estimate of extent of reduction.

For purposes of comparison with the visual assay, a resazurin/resorufin ratio of 1.0, fre- quently obtained from samples which appeared to be reduced to the Munsell 7/4 color stand- ard, was accepted as being similar to the 7/4 standard.

Estimation of reducing capacity. The ab- sorption spectrum o£ a butanol extract of indo- phenol from phosphate buffer (0.04%, pH 6.6) showed a maximum absorbance at 660 m~. Spectra of extracts obtained under the same conditions from milk or cream were identical to this. Since the poising of normal fresh milk is believed due to its ascorbic acid content (1, 4), indophenol solutions were standardized against ascorbic acid immediately prior to use. An example of such a standardization is shown in Figure 3. The indophenol solution (0.4 rag/ ml) was prepared by dissolving the dye, along with an equal weight of sodimn bicarbonate, in water. The bicarbonate ensured solubility of the dye and was not present in sufficient con- centration to affect the pH of milk or buffer solution. The dye solution was filtered and 1 ml added to a series of test tubes containing 5.5 ml ~ /50 phosphate buffer (pH 6.6) and increments of ascorbic acid. The tube contents were mixed by inversion and, after 3 min, added to 10 ml butanol in Babcock flasks. The ex- traction and treatment of extracts were as de- scribed for resazurin. The filtered extracts from solutions of low ascorbic acid concentration were diluted with butanol to yield an optical density (OD) in the range 0.100 to 0.600. The optical densities tabulated in Table 5 were ob- tained by direct multiplication of observed OD by the dilution factor.

902 R. if, MOYER AND J . J . R. CAMPBELL

O ~o 2 . 0 ID

Z tt]

1.0

O

I I I 0.2 0.4 0.6

CONCENTR&TION ASCORB&TE (#M)

:FIG. 3. Standardization of indophenol solution with ascorbate; extracts read in butanol saturated with sodium bicarbonate.

TABLE 5

hlfluence of dilution ou reducing capacity

Equiv. aseorbate

(~)

OD at Calcu- ~iilk tIsO 660 m~ " Found lated

(ml) Onl) 1.0 4.0 2.580 0.10 0.11 2.0 3.0 1.900 0.26 0.23 3.0 2.0 1.480 0.36 0.34 4.0 1.0 0.960 0.~8 0.46 5.0 0 0.560 0.57 ......

" Extracts were diluted with butanol to an OD ~n the range 0.100 to 0.600 before reading. The value tabulated was extrapolated back to original concentration.

I t was found that the method was repro- ducible and that neither resazurin nor resorufin interfered with the reduction of indophenol. That indophenol reduction was quantitative, and not merely a function of the Eh of the sys- tem, is illustrated by the data in Table 5. A sample of the milk was diluted with water as shown, and the observed reducing capacities compared with values calculated from the ob- served value of the undiluted sample.

On application of this method to a wide variety of milk samples, it was found that a portion of the added indophenol was reduced in every case. I f the milk had been aged, held

at 37 C for several hours, or treated with a mild oxidizing agent, some of its ability to reduce the dye was lost. This argues in favor of dye reduction rather than absorption or de- struction.

nESVl~S ~tl~D mSOUSSIO~

In early experiments it was observed that, for concentrations o£ resazurin less than that recommended for milk testing, the fraction of added resazurin reduced by a given milk sample in a given time interval was essentially inde- pendent of dye concentration. As the recom- mended concentration was approached, the amount of dye present had some influence on the percentage reduced in a particular time interval. I t was, therefore, expected that resaz- urin reduction by milk followed first-order kinetics at low dye concentrations. However, efforts to plot the logarithm of the dye concen- tration remaining oxidized against incubation time produced curves similar to D and E of Figure 4. Instead of being linear, the logarith- mic rate of reduction decreased with time of incubation; the decrease in rate appearing more marked at higher dye eonceutrations. These observations suggested that either resazurin had some inactivating influence on the reducing system of milk, or the appearance of the re- versible resorufin ,~ dihydroresorufin forms of the dye with progressive resazurin reduction poised the Eh of the milk sufficiently to de- celerate further reduction.

To deternfine which of these situations ap- plied, reducing capacity in the presence of two concentrations of resazurin was followed for a 300-rain interval (Figure 4~). Reducing ca- pacity was expressed as t,~ ascorbate per sam- ple and the values plotted to produce the three upper curves on the logarithnfic scale represent tim ascorbate remaining at the times shown. Concentrations of resazuriu (till/sample) are shown by the two lower curves. The volume of all milk samples was 5.0 ml plus 0.5 nil water or resazurin solution. Resazurin solutions con- tained either 0.12 t*~ or 0.024 /x~ of dye per 0.5 ml.

The data of Figure ~ illustrate that added dye had a catalytic effect on the destruction of the reducing system of milk. Calculation of half-life periods for the stability of the reduc- ing system under the three conditions measured produced values of 576 rain for the control system with no added dye, 287 rain in the pres- ence of 0.024/x~t dye, and 155 rain in the pres- ence of 0.12 t ~ dye. The reaction with added dye was not quantitative~ 0.024 ~ causing a loss equivalent to 0.17 ~ ascorbate. This was 7.1 times the quantity of dye added. The

RESAZURIN I~EDUCTION IN MILK 903

0.50

m <[

g g~ V- ° ~:~ oJo

~w z d 8~

'~ 0.05

~ . c

60 12O 180 240 300

INCUBATION TIME (Minutes)

FIG. 4. Influence of resazurhl on reducing ca- pacity of milk.

Reducing capacity Resazurin remainhlg ( ~ ascorbate) (~[) A--No added dye D--0.12 ~ added ]3--0.024 ~ resazurin E--0.024 ~ added C--0.12 ~ resazurin

higher dye concentration was not as efficient, 0.12 ~ I dye causing a loss of only 0.34 F~ ascorbate, or 2.8 times the amount of dye added. The quantities of dye reduced during this interval were 61.5% of that added, or 0.015 ~ at the low concentration, and 50%, or 0.06 ~ at the high concentration.

In milk containing approximately 0.57 /L~ of reduced aseorbate per 5 ml, the reducing ca- pacity, as measured by ability to decolorize neutral indophenol, was 0.7 ~ / 5 ml. Since any ascorhate present was assumed to react with the indophenol, titratable ascorbate was considered to constitute 80% of the reducing capacity of the milk by this measurement. I t is, therefore, apparent from Figure 4 that the effect of resazurin on the reducing system in- volved oxidation of ascorbate.

To determine whether the decrease in rate of resazurin reduction shown by Curves D and E (Figure 4) resulted from the concurrent loss of ascorbate (Curves B and C), ascorbate levels were maintained at approximately the initial values by addition of 0.1 ~ at 30-rain intervals (Figures 5 and 6). The scales in Figures 5 and 6 are the same as those in Figure 4. Curves A, B, and C represent reducing ca- pacity in iz~ ascorbate and Curves D and E,

~)i resazurin remaining per sample. The vol- ume of all samples was 5.0 ml plus 0.5 ml water or resazurin solution. Resazurin solutions con- tained 0.105 ~ of dye per 0.5 ml. To obtain the data for Figure 6, the milk sample used to construct Figure 5 was diluted to contain 40% water and the ascorbate level was raised to that initially present.

Inspection of Figures 5 and 6 shows that when the ascorbate level remained constant (Curve ]~) the logarithmic rate of resazurin reduction remained linear; whereas, when the ascorbate level was pemnitted to decrease (Curve D) the rate decelerated. This suggests that the rate of resazm'in reduction depended on

the concentration of ascorbate present and that deceleration reflected depletion of ascorbate.

Curves similar to D and E of Figures 5 and 6 were obtained using other milk samples, in- cluding aliquots from bulk tanks, i.e., linear logarithmic resazurin reduction rates at con- stant ascorbate levels, the rate depending on aseorbate for a given milk sample. However, milk from different sources varied widely in reduction rate at the same constant ascorbate

o

~ - B

o d ~ _ . - - D

~ 0.05 g

I I I | I 6 0 t 2 0 180 2 4 0 3 0 0

I N C U B A T I O N T I M E (Minutes)

FIG. 5. Resazurin reduction at constant ascor- bate level in undiluted milk. Reducing capacity ( ~ Resazurin remaining

ascorbate ) (~lv!) A--No added dye D--0.105 ~ resazurin B--0.105 ~M resazurln E--0.105 ~M resazurin C--0.105 ~M resazurin (ascorbate added

(ascorbate added at 0.0I/z~ per 30 ~t 0.1 ~ per 30 rain) rain)

~ 0 4 R. I t . M O Y E R A N D 5, J . R. C A M P B E L L

0 . 5 0

,w

= 0 , 0 5

" r " " I " " " " " " " " " ' " " " c

! . . . . .

~ . ~ %.....__

I | I ! I 6 0 (20 180 2 4 0 3 0 0

INCUBATION TIME (Minutes)

Fro. 6. Resazurin reduction at constant aseor- bate level in diluted milk. Reducing c~pacity (g.,< l~esazurin renmining

ascorbate) (~M) A--No added dye D--0.105 ~ resazurin B--0.105 ~ resazurin E--0.105 / ~ resazurin C--0.105 ~ resazurin (ascorbate added

(ascorbate added at 0.1 ~ per 30 at 0.1 ~ per 30 min) rain)

concentrations. This variation, along with the decreased reduction rate found on dilution of milk (comparison of Curves E, Figures 5 and 6), suggests that some factor in milk in addi-

9 0 tion to ascorbate acted to reduce resazurin. The fact that resazurin reduction could not be effected in ascorbate solutions buffered at pH 6.6, while the same ascorbate levels in milk produced rapid reduction, indicates that this second factor functioned in concert with ascor- ~ 7o bate rather than independently. The same con- clusion can be drawn from Curves D and E of _z Figures 5 and 6. I f Curve E of Figure 5 is ~: considered to represent the maximum reduction rate obtainable with this milk sample when ~o ascorbate was nmintained at 0.5 /z~/5.0 ml, it ~ 5o should be possible to resolve this plot into two ~:

~J components, one due to ascorbate and one due to the second factor, if the two functioned inde- pendently. Curve E of Figure 6 was obtained from samples containing some milk and, there- fore, represents a rate in excess of that due to ascorbate alone. I f the values from this curve are considered to approximate the con- tribution due to ascorbate in Curve E of Fig-

ure 5, it is possible to calculate the approxi- mate contribution due to the second factor. This is represented by the broken line in Figure 5, which intersects Curve D at 210 rain. Since this line intersects Curve D, obtained from samples having some ascorbate, the resazurin reduction rate shown by Curve E could not have resulted from independent reduction by ascorbate and the second factor, but must have resulted from interaction of the two.

I t had been found that the rate of resazurin reduction in milk was significantly accelerated by snmll decreases in pH of milk-dye systems (13), and it was believed that useful informa- tion concerning the mechanism of reduction could be obtained with studies at lower pH levels (Figure 7). Samples of whole milk were adjusted to the p i t values shown using 6 N HC1, and held at 37 C for 15 rain in the pres- ence of dye before butanol extraction. The extraction was carried out at the pH of the samples and the values were plotted as per cent resazurin in the recovered dye. Since dye re- coveries in the acid pH range were similar to those normally encountered (approximately 80% for all samples), the values were inter- preted as per cent of added dye remaining as resazurin.

When buffered ascorbic acid solutions (10 rag/liter) were used in place of milk, a curve almost identical to that of Figure 7 was ob- tained, and it is reasonable to conclude that the curve pictured in Figure 7 resulted from reac- tion of ascorbic acid with resazurin.

\ \ \ /

_ \ // \ /

J I l l I 6 . 0 5 . 0 , 4 0 5 0 2 0

pH

]~m. 7. Behavior of resazurin at low pH; pH adjusted using 6 ~ HCI; held at 37 C for 15 rain in the presence of dye.

RESAZURIN REDUCTION IN MILK 905

It is known that levels of reduced ascorbate, as such, do not correlate with the rate of resaz- urin reduction by milk. However, ascorbate was present in sufficient concentration in the milk to produce the reduction depicted in Fig- ure 7. The implication of ascorbate, acting in concert with some second factor to reduce resazurin at a rate dependent on ascorbate con- centration in a given milk sample, which was concluded from Figures 5 and 6, along with the regular increase in rate of reduction with de- creasing pH, leads to the suggestion that the system charted in Figure 7 represented the natural reducing system of milk.

Since milk is a heterogeneous system, it is not unreasonable to visualize zones of high hydrogen ion activity occurring at the surfaces of particles such as bacteria, leucocytes, or fat globules. These zones would then have an equiv- alent p i t lower than the 6.6 measurable with a glass dectrode in the milk plasma, and any res- azurin diffusing into them in the presence of ascorbate could become reduced. Since the change from resazurin to reserufin is irrevers- ible under physiological conditions, and dye re- duced in these zones would remain reduced on diffusion back into the high pH of the milk plasma. Thus, resazurin reduction could occur in additive increments to eventually produce a measurable reduction of total dye, the rate depending on number and p i t of zones, rate of dye diffusion through them, and ambient ascot- bate concentration.

In an effort to determine whether the zones referred to were zones of high hydrogen ion activity rather than zones of low Eh, use was made of a range of tetrazolium salts. ~ Since the reduction of tetrazolium salts to formazans is irreversible under physiological conditions, the behavior of these dyes should have paral- leled that of resazurin if reduction involved Eh alone. If, however, resazurin reduction was the result of interaction of ascorbate with zones of low pH, resazurin should be reduced by milk salnples which do not reduce tetrazolium salts of comparable electronegativity, since the Eh necessary for reduction of tetrazolium salts has been shown to be independent of pH (5). Of the tetrazolium salts tested, only 3 (4,5 dimethyI thiazoly] 1-2) 2,5 diphenyl tetrazolium bromide (~ViTT) was significantly reduced in 3 hr by the natural reducing system of milk. Reduction of MTT did not parallel resazurin reduction in milk samples tested, samples which reduced resazurin rapidly having less effect on the tet-

Nutritional Biochcmlea]s--Tetrazolium Salt Kit.

razolimn salt than controls which reduced resaz- urin slowly. I t was also found that aseorbate solutions in concentrations comparable with those found in milk reduced MTT as rapidly as did the milk samples tested. This indicates that the mechanism for the reduction of the tetrazolium salt differs from that for the re- duction of resazurin. The MTT was probably reduced by the electrometrically measurable Eh of the milk, which has been shown to depend on aseorbic acid concentration and to range from 0.2 to 0.3 v (1). Since resazurin is not reduced by potentials in this range at pH 6.6, the zones of high hydrogen ion activity referred to must have been involved.

CONCLUSIONS

It is suggested that the reduced ascorbic acid of milk is important in resazurin reduction when acting in concert with some other factor in the milk. I t is postulated that this second factor is comprised of zones of high hydrogen ion activity occurring at the surfaces of the particulate elements of milk, such as bacteria, leucocytes, cellular debris, and the fat globule membrane. In addition to their suitability to the proposed mechanism of reduction, these ele- ments have an incidence of occurrence and lo- cation in milk which correlates well with the rate of resazurin reduction. Since loss of ascot- bate has been shown to be quite rapid in milk refrigerated at 4 C (1), and to be more rapid in bulk milk than in can samples (10), the loss of resazurin reducing ability by milk samples on aging would be due partly to the loss of re- duced ascorbate and partly to decreased activ- ity of the particulate elements, perhaps by aggregation.

ACKI~0W~LEDGNIENT This work was carried out under Canada De-

partment of Agriculture Grant EMt~ 1]1.

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906 R. It. MOYEI~ AND J. J. R. CAMPBELL

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