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Resazurin reduction in milk Moyer, Rudy H. 1962

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- i -RESAZURIN REDUCTION IN MILK by RUDY H. MOYER B.S.A., University of B r i t i s h Columbia, 1958 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science i n A g r i c u l t u r a l Microbiology i n the D i v i s i o n of Animal Science We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1962 In presenting this thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of fl/J/ryj/jL. $c fgA'c £ The University of British Columbia, Vancouver 8, Canada. Date - i i -ABSTRACT Methods were developed to quantitatively measure resaz-u r i n reduction and the reducing capacity of milk. The method for resazurin determination involved butanol extraction of the dye from milk and measurement of the o p t i c a l density of extracts at 582 mji and 6l£ m>i a f t e r saturation with sodium bicarbonate; that f o r reducing capacity was based on reaction of 2 , 6 - d i c h l o r o -phenolindophenol with milk at i t s normal pH. For the l a t t e r determination excess indophenol was added and the quantity of dye remaining estimated spectrophotometrically at 660 mji i n butanol extracts saturated with sodium bicarbonate. This assay could be applied i n the presence of resazurin or r e s o r u f i n , since these compounds had n e g l i g i b l e absorbance at 660 mji. Both of these methods were reproducible i n milk and t h e i r respective accuracies estimated at greater than 90 percent. The quantitative assay f o r resazurin was employed i n order to study the behavior of resazurin in"milk and the systems i n fresh milk responsible f o r reduction of the dye. Results ob-tained on aging whole and skim milk were used to demonstrate that the resazurin reduction dealt with i n the present i n v e s t i -gation was due to the reducing systems of milk rather than to b a c t e r i a l a c t i v i t y . These r e s u l t s showed that the reducing system was l e s s stable and more sensitive to temperature of aging i n skim than i n whole milk. Measurement of rates of r e s -azurin reduction by various f r a c t i o n s of normal milk showed that the major portion of the reducing a b i l i t y of whole milk was associated with the cream. The aqueous phase from centrifuged - i i i -warm gravity cream had a greater a b i l i t y to reduce resazurin than did the whole milk from which the fr a c t i o n s were derived. The rate of resazurin reduction by milk decreased with incuba-t i o n at 37°C, however, i n the presence of s u f f i c i e n t numbers of bac t e r i a , an acceleration of rate with incubation was noted. The point at which washed suspensions of added bacteria became s i g n i f i c a n t i n reduction was demonstrated as a change i n slope of logarithmic p l o t s of dye reduction rates. Resazurin was shown to have a d e s t a b i l i z i n g influence on the reducing capacity of milk. This Influence was c a t a l y t i c and dependent on t o t a l concentration of dye; rate of i n a c t i v a -t i o n being constant f o r a given dye concentration. Evidence was presented to show that the component of the reducing system that was inact i v a t e d was ascorbic acid. The influence of f r a c -t i o n s , obtained from passage of resazurin through a s i l i c i c acid column, suggested that t h i s c a t a l y t i c e f f e c t was probably due to the dye as such, rather than to a r t i f a c t s i n the commercial dye preparation used. Examination of the reducing capacity of milk f r a c t i o n s before and afte r treatment with ascorbic acid oxidase indicated that resazurin reduction was brought about by that part of the reducing capacity that could not be accounted f o r as free ascorbic a c i d . In mastitic samples, th i s element of the reducing capacity was concentrated completely i n the f a t and centrifuged sediment. It was concluded from these investigations that the reduc-ing system of milk existed as a measurable en t i t y at any given time rather than as a continuous evolution of electrons from - i v -some slow enzymatic reaction. This system consisted of the measurable ascorbic acid of the milk, which occurred i n the plasma, and some reducing agent bound to s t r u c t u r a l compon-ents of the cream and sediment. The measurable ascorbic acid accounted f o r approximately 80 percent of the reducing capacity but was concluded to have l i t t l e influence on resazurin reduc-t i o n . I t was concluded that the bound reducing agent depended on s t r u c t u r a l elements i n the milk f o r i t s a b i l i t y to reduce resazurin, and that i t l o s t t h i s a b i l i t y on d i s s o c i a t i o n from whatever p a r t i c l e i t occurred on. It was postulated that t h i s reducing agent was ascorbate and that i t occurred bound to leucocytes and other c e l l u l a r debris i n the milk i n situations analogous, to i t s reported occurrence i n blood. Attempts to i d e n t i f y t h i s reducing agent as ascorbate were unsuccessful i n t h i s i n v e s t i g a t i o n , but the techniques employed were probably inadequate. -V -TABLE OF CONTENTS INTRODUCTION AND HISTORICAL ASPECTS 1 MATERIALS AND METHODS 8 I MILK 8 II RESAZURIN REDUCTION 8 I I I REDUCED ASCORBIC ACID 9 A T i t r a t i o n 9 B Paper Chromatography 9 C Oxidation to Dehydroascorbic Acid 9 D Ascorbic Acid Oxidase 9 IV FREE SULFHYDRYL GROUPS 10 PRELIMINARY EXPERIMENTS 11 I RESAZURIN REDUCTION 11 A Oxidation-Reduction Aspects 11 B Limitations of Vis u a l Estimates 12 C Spectrophotometric Estimation 15 II MEASUREMENT OF REDUCING CAPACITY OF MILK 29 EXPERIMENTAL 36 I GENERAL OBSERVATIONS 36 A Decrease i n Rate of Resazurin Reduction on Aging 36 B Reduction Rate i n Milk Fractions 37 C Poising by Dye 39 D Influence of Dye Concentration on Reduction Rates , Ip. E Influence of pH and Bacteria on Rate of Reduction [j.2 - v i -II REDUCING SYSTEM OP MILK \i$ A Behavior of Resazurin at Low pH 1+5 B Influence of Resazurin I4.6 C S t a b i l i t y of Ascorbate I4.8 D Influence of Reduced Dye on Reducing System of Milk $0 E S i l i c i c Acid Treatment of Resazurin 52 P Reducing Capacity of Milk Fractions $1+ G Ascorbic Acid Oxidase 56 GENERAL DISCUSSION 59 I INTERACTION OF RESAZURIN WITH REDUCING SYSTEM 59 II STABILITY OF REDUCING SYSTEM 61 III NATURE OF REDUCING SYSTEM 63 SUMMARY AND CONCLUSIONS 67 BIBLIOGRAPHY 69 - v i i -INDEX TO FIGURES FIGURE 1 Spectra of resazurin and r e s o r u f i n i n butanol saturated with sodium bicarbonate To follow page 15 FIGURE 2 C a l i b r a t i o n curves f o r resazurin assay To follow page 20 FIGURE 3 Spectrum of 2,6-dichlorophenolindophenol i n butanol saturated with sodium bicarbonate To follow page 29 FIGURE ij. Standard curve for measuring reducing capac-i t y To follow page 30 FIGURE 5 (a and b) Influence of.storage at various temperatures on a b i l i t y of milk to reduce resazurin To follow page 36 FIGURE 6 Reduction rate i n milk f r a c t i o n s To follow page 37 FIGURE 7 Poising by dye To follow page L|_0 FIGURE 8 Influence of dye concentration on reduction rates To follow page I4.X FIGURE 9 (a and b) Influence of pH and bacteria on rate of reduction To follow page lili FIGURE 10 Behavior of resazurin at low pH To follow page \\$ FIGURE 11 Influence of resazurin on reducing capacity To follow page Ij.7 FIGURE 12 Influence of resazurin on ascorbate i n EDTA To follow page FIGURE 15 Spectra of resazurin fract i o n s i n butanol saturated with sodium bicarbonate To follow page - i x -ACKNOWLEDGMENT I would l i k e to thank Professor J.J.R. Campbell for h i s patient assistance during the course of th i s work and the Canada Department of A g r i c u l -ture f o r f i n a n c i a l support i n the form of EMR grants 92 and 111. -1-INTRODUCTION AND HISTORICAL ASPECTS The present i n v e s t i g a t i o n was concerned mainly with res-azurin reduction i n the absence of s i g n i f i c a n t b a c t e r i a l a c t i v -i t y and was undertaken i n an e f f o r t to determine mechanism of reduction by the naturally occurring reducing systems of milk. Since i n t e r e s t i n resazurin reduction i n milk has been focused on use of the dye as an indicator of sanitary condition, the bulk of the research on t h i s topic has been centred on assess-ing milk produced under abnormal p h y s i o l o g i c a l or pathological udder conditions and on subsequent b a c t e r i a l contamination. A great deal of the work has involved comparison of resazurin reduction with other tests such as b a c t e r i a l and c e l l counts, catalase, methylene blue reduction, and potentiometric measure-ments. In the following summary, no e f f o r t w i l l be made to d e t a i l this work since i t has recently been reviewed (1). The reducing systems operative i n normal and mastitic milks have also been the subject of a recent review (2). Those observa-tions having a d i r e c t bearing on the present i n v e s t i g a t i o n w i l l be considered b r i e f l y . Usually, these were passing observations made i n the course of some study r e l a t e d to assessing hygenic quality of milk and were not investigated beyond their f i r s t recording. As a r e s u l t , the following summary of these observa-tions i s b r i e f and discontinuous with respect to time and con-text . The use of resazurin as an indicator f o r determining the sanitary condition of milk was f i r s t studied i n North America by Ramsdell et a l . (3) i n 1935, though the in d i c a t o r had received some attention i n t h i s regard i n Europe p r i o r to that time. These investigators observed that resazurin offered several advantages over the more electronegative methylene blue used i n the well established reductase t e s t . Resazurin became r e -duced much more r e a d i l y than did methylene blue thus giving a more rapid assessment of b a c t e r i a l a c t i v i t y . One of the more important observations made, however, was that resazurin was very sensitive to p h y s i o l o g i c a l l y abnormal and pathological conditions of milk; rapid reduction c o r r e l a t i n g well with high catalase content and high c e l l count. Samples of this type showed rapid i n i t i a l dye reduction a f t e r which l i t t l e change took place u n t i l b a c t e r i a l a c t i v i t y was evident. In attempt-ing to correlate potentiometrically measured Eh values with degree of resazurin reduction, these workers found that there was a wide range of Eh values corresponding to a single shade of resazurin color i n normal skim milk. If abnormal milk was added, the zones of reduction were shi f t e d to more e l e c t r o p o s i -tiv e values, r e s u l t i n g i n more rapid reduction of the dye. In these i n i t i a l investigations, the observation was made that the yellow carotenoid pigments of the emulsion phase of the milk decreased the i n t e n s i t y of the blue color of the dye, making v i s u a l observation of color change d i f f i c u l t i n some whole milk samples. Strynadka and Thornton, i n 1938 (li) , using leucocytes obtained from blood and repeatedly washed i n ph y s i o l o g i c a l s a l i n e , could obtain no s i g n i f i c a n t a l t e r a t i o n i n reduction time of methylene blue when suspensions of these were added to raw -3-milk. In addition, they could not obtain good correlations between leucocyte counts of milk samples and a b i l i t y to reduce methylene blue and concluded that leucocytes were not of p a r t i c -u l a r importance i n the reductase t e s t . The influence of resazurin on the Eh of milk was reported by Johns and Howson i n 19UX) (5). They found that the time-p o t e n t i a l curves f o r milk containing resazurin d i f f e r e d from those of p l a i n milk or milk containing methylene blue, the curves for resazurin-containing milk showing a sharper i n i t i a l decrease i n Eh followed by a f l a t t e n i n g of the curves aft e r the dye had reached the pink stage. Decreasing dye concentrations resulted i n curves of the same shape but showing the decrease i n Eh at an e a r l i e r time. They also noted that resazurin reduc-t i o n occurred i n a shorter time and at a higher Eh with lower dye concentrations. In addition, hourly inversion of tubes during incubation shortened reduction time, the incorporation of oxygen by t h i s practice having no effect on the time-potential curves i n the early stages and only a transient e f f e c t l a t e r . In further studies concerning the behavior of resazurin i n milk i n 19lil, Johns (6) found that resazurin could show marked changes i n color with no decrease i n p o t e n t i a l of the milk. Using continuous aeration to maintain a high Eh i n one series of samples, he showed that rate of resazurin reduction was the same for comparable unaerated samples i n which the Eh decreased by as much as 0.2 v o l t . This observation, along with data on the influence of d i l u t i o n of normal milk with leucocytes and centrifuge sediment from abnormal milk enabled him to suggest that resazurin reduction depended on substances present i n abnormal milk which had no influence on oxidation reduction p o t e n t i a l . In connection with t h i s observation, he pointed out that washing leucocytes i n ph y s i o l o g i c a l saline d e f i n i t e l y decreased t h e i r reducing a c t i v i t y , while washing i n normal milk had less e f f e c t . In the same series of experiments, Johns observed an increase i n p o t e n t i a l of approximately 0.06 v o l t on 5 hours incubation of normal milk with added resazurin. Since control samples f a i l e d to show t h i s trend, the author concluded that the increase was due to the presence of resazurin. McBride and Golding ( 7 ) , i n 1 9 5 1 , found that i f the res-azurin test was conducted on quarter samples of milk within four hours of milking i t measured leucocyte a c t i v i t y only. They also observed that mastitic milk could reduce the dye to the c o l o r l e s s form without the lag period found by e a r l i e r workers ( 3 , 5 ) . The reducing properties of normal and abnormal milk were reported by Nilsson i n the years 1950 to 1957 and were reviewed by the same author i n 1959 ( 2 ) . She found that mastitic milk contained a reducing system associated with the f a t , which was destroyed by heating to 85°C for 5 minutes. In testing sub-strates f o r the reducing enzymes of milk, she found that only those f o r xanthine oxidase produced a f a l l In p o t e n t i a l com-parable with that c h a r a c t e r i s t i c of mastitic milk. The d i f f e r -ence between normal and mastitic milk with regard to t h i s enzyme system was found to be the presence of guanase and higher l e v e l s of xanthine oxidase a c t i v i t y i n mastitic samples. - 5 -In analyses f o r the products of t h i s enzyme system, she noted an increase of 0.10 mg per ml i n the u r i c acid content after drop i n p o t e n t i a l of mastitic milk. In normal milk there was no f a l l i n p o t e n t i a l and no increase i n u r i c a c i d . She also found that the l e v e l of other acids was the same i n both types of milk and concluded that the substrates could not have consisted of aldehydes but were probably precursors of xanthine or hypoxanthine. In t h i s series of i n v e s t i g a t i o n s , the observation was also made that leucocytes from mastitic milk did not, i n themselves, have any reducing a b i l i t y when added to pasteurized milk. The author concluded that the r e -ducing a c t i v i t y found by e a r l i e r workers to be associated with high leucocyte counts was an i n d i r e c t r e l a t i o n s h i p and that high leucocyte count r e f l e c t e d an udder condition that permitted escape of blood substances into the milk. The resazurin reducing a b i l i t y of leucocytes was r e -examined i n I960 by Campbell and Phelps ( 8 ) . They found that removal of bacteria and leucocytes from milk by centrifuging had l i t t l e influence on rate of resazurin reduction i f the fat was reincorporated. Leucocytes obtained from bovine blood and washed i n p h y s i o l o g i c a l saline had to be added to milk i n concentrations of 1 X 10^ per ml to obtain rapid resazurin reduction. When leucocytes freshly i s o l a t e d from bovine blood were held i n milk at 37°C for 1+ hours before addition of r e s -azurin, they l o s t most of t h e i r a b i l i t y to reduce the dye. I f obtained from milk and washed i n p h y s i o l o g i c a l saline or i n normal milk, addition of I | X 10^ leucocytes per ml of milk -6-produced only a s l i g h t acceleration of resazurin reduction rate. These authors also observed that when leucocytes i s o l a t e d from milk were suspended i n fresh bovine plasma f o r 2 hours they regained a b i l i t y to reduce resazurin, their a c t i v i t y being comparable with that of those f r e s h l y i s o l a t e d from bovine blood. These r e v i t a l i z e d leucocytes did not lose reduc-ing a c t i v i t y on repeated washing. Disruption of freshly i s o -l a t e d leucocytes by sonic o s c i l l a t i o n , followed by treatments with ribonuclease or desoxyribonuclease destroyed a large part of t h e i r a b i l i t y to reduce resazurin. Prom t h i s , the authors concluded that leucocytes, even when disrupted and degraded, did not supply substrates to the xanthine oxidase system i n s u f f i c i e n t quantity to account f o r rapid resazurin reduction. The influence of the xanthine oxidase system on resazurin reduction by normal milk was reported by Campbell and Keur i n 1961 (9). They could f i n d no c o r r e l a t i o n between the concentra-t i o n of xanthine oxidase i n milk and rate of resazurin reduction. Pasteurization eliminated a l l resazurin reducing a c t i v i t y but had no e f f e c t on xanthine oxidase, while added xanthine oxidase i n h i b i t o r s had no e f f e c t on rate of resazurin reduction. This b r i e f h i s t o r i c a l summary contains several s a l i e n t points concerning the mechanism of resazurin reduction i n milk i n the absence of s i g n i f i c a n t b a c t e r i a l a c t i v i t y . Resazurin reduction was v i r t u a l l y independent of the aerobic electrode p o t e n t i a l of milk, though the dye had some influence on meas-ured p o t e n t i a l (5,6). Rate of resazurin reduction was very sensitive to abnormal phy s i o l o g i c a l and pathological conditions -7-of milk (3,7). The reducing agent appearing i n these abnormal milks appeared to be associated with, and possibly bound loosely t o , the leucocytes present (6), though i t was not the leucocytes themselves (2,4,8). The xanthine oxidase was present i n high concentration but lacked a substrate In normal milk (9). In mastitic milk, there appeared to be substrates f o r t h i s en-zyme (10). It i s evident from the foregoing that a resazurin reducing system exists i n normal milk. Under abnormal udder conditions, either t h i s system becomes more active, or a new reducing system appears. The present i n v e s t i g a t i o n was concerned with deter-mining the nature of these reducing systems and whether or not they were separate systems. -8-MATERIALS AND METHODS I MILK Machine-drawn samples of milk from i n d i v i d u a l cows of the University herd, predominantly Ayrshire, were used f o r most of the work described. No e f f o r t was made to d i f f e r e n t i a t e be-tween milk from d i f f e r e n t quarters of the udder or d i f f e r e n t stages of the milking procedure. Any sample represented a complete milking, including strippings. I f there was evidence of c l i n i c a l m a s t i t i s , the affected quarter was hand-milked and the milk either discarded or used separately. I t was found that there was l i t t l e day to day v a r i a t i o n In the reducing pro-p e r t i e s of milks from i n d i v i d u a l cows, any s i g n i f i c a n t changes i n the fresh milk occurring gradually over an i n t e r v a l of sev-e r a l weeks. There was, however, considerable v a r i a t i o n between milks from d i f f e r e n t cows and mixed samples drawn from randomly selected ten-gallon cans often d i f f e r e d s i g n i f i c a n t l y from one another. Random mixed samples were often used as controls for a series of i n d i v i d u a l samples as well as to show that a given observation was not peculiar to a p a r t i c u l a r cow but applied more generally. II RESAZURIN REDUCTION Resazurin reduction was estimated v i s u a l l y according to the procedure i n Standard Methods f o r the Examination of Dairy Products (11). Incubation was at 37°C and samples were kept under observation f o r the f i r s t 30 minutes. They were r e -examined at 30 minute i n t e r v a l s with inversion of tubes at each examination. The time required f o r the color of the sample to -9 -reach that of the Munsell 5 P 7 A standard was recorded as being the reduction time. A l l samples were generally l e f t at 37°C for 2 to 3 hours regardless of reduction time. I l l REDUCED ASCORBIC ACID A. T i t r a t i o n The reagents and standards f o r the procedure l i s t e d i n O f f i c i a l Methods of Analysis of the Association of O f f i c i a l A g r i c u l t u r a l Chemists (12) were used. The t i t r a t i o n procedure was according to Sharp (13) , except that metaphosphoric a c i d -acetic acid solution was used i n place of N/lO s u l f u r i c a c i d to p r e c i p i t a t e proteins p r i o r to t i t r a t i o n . B. Paper Chromatography The method advocated by Block, Durram, and Zweig was used. The solvent system was phenol, acetic acid, water. The 2 ,6-dichlorophenolindophenol spray was used to locate ascorbic acid and was followed by the amraoniacal s i l v e r n i t r a t e spray, which developed a new series of spots on chromatograms of milk extracts. C. Oxidation to Dehydroascorblc Acid S o l i d ascorbic acid was oxidized to dehydroascorbic a c i d with 0.01 N iodine according to the method outlined by Kohman and Sanborn (l£). The dehydroascorblc acid was d i l u t e d and used within 10 minutes of i t s preparation. D. Ascorbic Acid Oxidase Concentrated cucumber juice was prepared according to Sharp et a l . (16). This crude enzyme preparation was not p u r i -f i e d further and was found to be s u f f i c i e n t l y active f o r -10-removal of ascorbic acid from milk. IV FREE SULFHYDRYL GROUPS Free sulfhydryl groups were estimated using the n i t r o -prusside procedure of Patton and Josephson (17). The standards and milk samples were kept i n ice u n t i l reagents had been added to a l l tubes. They were then placed at room temperature for 10 minutes before comparison. I t was found that maximum color developed i n 10 minutes but faded r a p i d l y a f t e r approximately 15 minutes at room temperature. -11-PRELIMINARY EXPERIMENTS I RESAZURIN REDUCTION A. Oxidation-Reduction Aspects Resazurin reduction takes place in"two stages: xxxr —• xcowxor 4 i O H Resazurin Resorufin Dihydroresorufin (blue) (pink) (colorless) Only the second stage, the reduction of res o r u f i n to dihydro-r e s o r u f i n , i s reversible at an i n e r t electrode or under phys-i o l o g i c a l conditions.. Twigg (18) showed that on t i t r a t i o n of resazurin with leuco-rosinduline at pH values between 6 and 7 i n the absence of oxygen, approximately 15 percent of the res-o r u f i n formed was simultaneously reduced to dihydroresorufin. This reduction occurred at Eh values higher than that of a res o r u f i n solution at the same concentration. Although t h i s simultaneous reduction has not been demonstrated i n a phys-i o l o g i c a l system, i t presumably occurs to some extent depending on the nature of the system. At pH values greater than 3.0 two electrons per molecule are transferred i n the reaction, resorufin dihydroresorufin. This i s t y p i c a l of nearly a l l reversible dyes. The calculated value of pK 0 f o r the system i s 6.93 and of E Q i s +0.380 v o l t s . Twigg (18) found f a i r l y good agreement between calculated and observed values of E^ except at pH 6.£83, 6.867 and 7.1).31. He attributed the deviation to low s o l u b i l i t y c h a r a c t e r i s t i c s , when the dye concentration was 1 .5 X 10~^M. Since the f i r s t of -12-these pH values i s i n the range of normal milk, and since the concentration of resazurin recommended f o r milk t e s t i n g i s 2.1+ X 10~^M, i t i s possible that there i s some depression of the E'Q value of t h i s stage of the reduction. This i s probably of l i t t l e p r a c t i c a l importance because the v a r i a t i o n observed by Twigg was s l i g h t and because the c r i t i c a l concentration of re s o r u f i n would not be reached u n t i l about h a l f the added res-azurin had been reduced. The EQ of the dye system at this pH i s -0.016 v o l t . Since the reduction of resazurin to r e s o r u f i n i s i r r e v e r s -i b l e i n phy s i o l o g i c a l systems, i t i s impossible to determine an E^ value f o r the reaction. I t has, however, been estimated at +0 .050 v o l t at pH 7.0 (19). This value has no significance i n an i r r e v e r s i b l e system and reduction i s a normal f i r s t order decay reaction, the rate depending on Eh, i f the system i s homogeneous and constant with respect to Eh. B. Limitations of Visual Estimates The v i s u a l methods employed to determine resazurin reduc-ti o n may be sat i s f a c t o r y for estimating the b a c t e r i o l o g i c a l quality of commercial samples of milk but they are not adequate for a detailed study of reduction rates. The color that i s observed v i s u a l l y i s a function of the r a t i o of the blue r e s -azurin and pink r e s o r u f i n . In some milks, p a r t i c u l a r l y those having a high f a t content, the color i s obscured and a l l that i s observed i s a gray color having shades of blue and pink. Vi s u a l estimation of extent of reduction i n these milks i s very d i f f i c u l t and, i n some cases, impossible. This i s not a -13-major problem i n estimating dye reduction i n mixed milks but becomes quite important when dealing with milk from i n d i v i d u a l cows, where the int e n s i t y of dye color varies with the sample and often d i f f e r s greatly from that of the color standard. In addition to the uncertainty of v i s u a l color estimation, there i s some uncertainty regarding the amount of colored dye present i n solution. The 15 percent simultaneous conversion of resorufin to dihydroresorufin found by Twigg (18), during resazurin reduction i n a r e l a t i v e l y uncomplicated system, indicates that t h i s conversion proceeds rea d i l y and i s not solely a function of the Eh of the system. It i s possible that t h i s e f f e c t also occurs i n milk, and equally possible that i t s magnitude varies from one sample to the next. This would change the r a t i o of resazurin to resorufin and give a fals e estimate of amount of reduction based on a v i s u a l observation. However, due to the ease of reoxidation of dihydroresorufin by atmospheric oxygen, inversion of tubes immediately p r i o r to color estimation would minimize t h i s e f f e c t . Another factor which could decrease the e f f e c t i v e concentration of res o r u f i n i n the system i s i t s low s o l u b i l i t y i n aqueous media i n the pH range of milk. Twigg (18) attributed the deviation observed f o r the EQ value of the resorufin/dihydroresorufin system at pH 6 . 6 to low s o l u b i l i t y . His dye concentration was 1 .5 X lCT^M while that i n milk i s 2.1+ X 10~^M. If one considers that the volume occupied by fat globules and c o l l o i d a l p a r t i c l e s i n milk i s excluded to the dye, the actual dye concentration approaches 3.0 X 10~^M, depending largely on the f a t content of the milk. -14-Once the system i s saturated with r e s o r u f i n , the color change of the sample depends on the decrease of resazurin concentra-t i o n alone, since only that resorufin which i s i n solution contributes to the pink color. During the early stages of reduction, the color change depends on Increase of resorufin as well. This complicates the study of reduction rates, par-t i c u l a r l y since the magnitude of the e f f e c t i s probably variable and dependent on the f a t content of a given sample. For detailed study of rates of reduction, an accurate, quantitative method permitting assay of up to twenty samples at one time i s needed. Methods such as the iodoraetric method outlined by Twigg (18) were designed to determine the amount of re s o r u f i n present i n a given batch of resazurin, and require f a i r l y large quantities of dye. In milk, a small quantity of dye i s used as an indicator and quantitative separation from i n t e r f e r i n g substances, as well as the assay i t s e l f , would be-come impractical, i f not impossible. De Baun and de Stevens (20) have developed a spectrophotometrie method fo r the simultaneous estimation of resazurin and resorufin i n studies with erythro-cytes and succinic dehydrogenase of r a t l i v e r . The method, as presented, requires some modification before i t can be applied to milk. An extraction step has to be added because milk, unlike erythrocyte or enzyme preparations, cannot be c l a r i f i e d by centrifuging or f i l t e r i n g . I f the dye can be extracted from the milk q u a n t i t a t i v e l y , and i f the absorption spectra of the two colored forms are s u f f i c i e n t l y d i f f e r e n t and d i s t i n c t i n -15-the solvent, i t should be possible to devise a method f o r accurately estimating degree of resazurin reduction i n milk. C, Spectrophotometry Estimation Commercial resazurin t a b l e t s , c e r t i f i e d by the B i o l o g i c a l Stain Commission to contain approximately 11 mg dye and to be s u f f i c i e n t l y pure and uniform f o r milk testing (11,21), were used. According to Standard Methods (11), each tablet makes 200 ml of the solution to be employed i n a 1 plus 10 d i l u t i o n with milk f o r v i s u a l estimation of resazurin reduction. This makes the f i n a l concentration i n milk approximately 2.1+ X 10~^M. The concentration varies very s l i g h t l y from one batch of stock solution to the next as does the i n i t i a l concentration of re s -o r u f i n (18,21). If the stock solution from one randomly chosen tablet were used as an a r b i t r a r y standard for c a l i b r a t i o n of the method, subsequent batches, made from d i f f e r e n t t a b l e t s could be standardized against the o r i g i n a l c a l i b r a t i o n data. This would permit d i r e c t comparison of data obtained at d i f f e r -ent times and the use of d i f f e r e n t dye sources. Although the c o e f f i c i e n t s calculated from figure 2 are based on the assump-t i o n that the dye tablet used contained 11 mg resazurin, which i s an admitted approximation, they serve to standardize the method and place the measurement of reducing rates on a quan-t i t a t i v e b a s i s . Preliminary t e s t s of a number of solvents revealed that n-butanol extracted the dye f a i r l y e f f i c i e n t l y and separated from the milk on centrifuging. The absorption spectra of r e s -azurin and r e s o r u f i n i n butanol are shown i n figure 1. The curves were obtained by adding standard resazurin solution to t Resorufin i Resazurin 500 Wavelength (mu.) Figure 1 Spectra of Resazurin and Resorufin i n butanol saturated with sodium bicarbonate -16-butanol and shaking i n the presence of excess sodium bicarbon-ate. The butanol layer was cleared by f i l t r a t i o n and read against butanol which had been given s i m i l a r treatment, except that d i s t i l l e d water was substituted for dye s o l u t i o n . In order to obtain the curve f o r r e s o r u f i n , the resazurin solution was reduced with sodium Isoascorbate p r i o r to addition of bu-tanol and treated as above. The E max for resazurin and res-oruf i n i n butanol, saturated with sodium bicarbonate, were 6l£ and £82 up respectively. The blanks, butanol shaken with d i s -t i l l e d water and sodium bicarbonate, with and without added sodium isoascorbate, showed no absorption at either of these wavelengths when read against d i s t i l l e d water. The addition of sodium bicarbonate did not influence the absorption spectra of resazurin and resorufin extracted from milk at pH 6.6 or from phosphate buffer at pH 6.ij., but did markedly increase the o p t i c a l density at the points of maximum absorbance. Since resazurin changes color from v i o l e t to orange i n the pH range 6.5 to 3.8- (22) , i t s absorbance at more alkaline r e -actions was also expected to vary with pH. The v a r i a t i o n i n absorbance over the range pH 6.2 to 7.0 i s shown i n column A of table 1. The readings i n column B were the r e s u l t of satur-ating the extracts from column A with sodium bicarbonate, and show that the variations i n the values of column A were due to absorbance rather than to the actual amount of dye present. The data as a whole i l l u s t r a t e that extraction of dye from phosphate buffer i s constant throughout the p h y s i o l o g i c a l pH -17-range of milk and that absorbance increases with r i s i n g pH. I t would appear l o g i c a l to cal i b r a t e the method f o r a number of pH values and then measure reduction and dye recovery at the pH of the sample being examined. While the o p t i c a l density measurements of samples from phosphate buffer were s u f f i c i e n t l y reproducable to provide the c a l i b r a t i o n data, r e p l i c a t e ex-tracts from the same sample of milk tended to d i f f e r consider-ably i n absorbance, making readings u n r e l i a b l e . Since these r e p l i c a t e extracts from milk produced a common o p t i c a l density on saturating with sodium bicarbonate, the v a r i a t i o n was prob-ably the r e s u l t of s l i g h t differences i n pH. Milk containing p a r t i a l l y reduced resazurin probably also contains some col o r -l e s s dihydroresorufin. It i s remotely possible that some of the colorless compound remains, even a f t e r shaking i n a i r . Shaking i n sodium bicarbonate should ensure that the equilibrium re s o r u f i n -—^dihydroresorufin i s s h i f t e d f a r to the l e f t , the E Q values being: (18) EQ = -0.021 v at pH 6.583 -0.163 v at pH 8.338 -0.231 v at pH 9.186 Saturating with sodium bicarbonate, besides eliminating e f f e c t s of pH and traces of dihydroresorufin on the assay, also removes some water and s a l t s from the butanol extract, leaving these at a more constant concentration and so adding to the p r e c i s i o n of the method. -18-TABLE 1 OPTICAL DENSITY OF RESAZURIN AND RESORUFIN EXTRACTED FROM  PHOSPHATE BUFFER OF VARYING pH A B * F i r s t Extract # F i r s t Extract Saturated with Sodium Bicarbonate pH of Resazurin Resorufin Resazurin Resorufin Buffer - 582mu 6l5mj* 582rmj. 6l5mR 582mu, 6l5mu, 582mu_ 6l5mR 6 . 2 . 193 .273 . 2 3 2 . 0 0 9 . 2 9 5 . 5 2 0 . 6 9 0 . 0 2 5 6 . 3 .215 . 2 3 0 . 2 5 0 . 0 1 0 . 2 9 5 . 5 2 0 . 6 9 0 .021}. 6.14, . 2 2 1 . 3 5 0 . 2 9 1 .011 . 2 9 5 . 5 2 0 . 6 9 0 .021). 6 . 5 . 2 3 5 .377 .31+8 .013 . 2 9 5 . 5 2 0 . 6 9 0 .021+ 6 . 6 .21+5 .kok . 3 9 0 . 0 1 5 . 2 9 5 . 5 2 0 . 6 9 0 .021+ 6 . 8 . 2 9 0 .14-70 . 5 2 0 .015 . 2 9 5 . 5 2 0 . 6 9 0 . 0 2 5 7 . 0 . 2 9 8 .14.82 • 550 . 0 2 0 . 2 9 5 . 5 2 0 . 6 9 0 .021+ H 2 0 .197 . 2 8 8 .367 . 0 1 5 . 2 9 5 . 5 2 0 . 6 9 0 . 0 2 5 * 0 . 5 ml resazurin solution or 0 . 5 ml re s o r u f i n (resazurin solution reduced with sodium isoascorbate) added to 5*0 ml 1% phosphate buffer at above pH values i n test-tubes: held at 37°C f o r 30 minutes: added to 18 ml n-butanol i n 9 gm Babcock cream test b o t t l e s : shaken 10 minutes at room temperature: centrifuged, and o p t i c a l density of butanol layer measured at 582 and 615 mu.. f Butanol layer from column A added to excess sodium bicarbon-ate: shaken 5 minutes at room temperature: f i l t e r e d through Whatman #1 paper and o p t i c a l density measured at 582 and 615 row,. -19-The e f f e c t of varying the shaking time of a butanol-dye -milk mixture i s shown i n table 2 . The data also serve to i l l u s -trate the p r e c i s i o n of the method. Although a shaking time of 5 minutes was found to be adequate for the extraction of the dye, a 10 minute shaking i n t e r v a l was chosen f o r subsequent determinations. TABLE 2 INFLUENCE OF SHAKING TIME ON AMOUNT OF DYE  EXTRACTED FROM MILK Time on Shaker Optical Density (Minutes) $82 mu. 6l5mu, $ .407 .320 10 .1+07 .321 15 . 4 0 6 .320 20 .407 .321 25 .Ij.06 .320 30 .1|07 .320 0 . 5 ml dye solution plus 5 . 0 ml milk held at 37°C for 60 min-utes i n test tubes and then added to 9 . 0 ml n-butanol i n 9 gm Babcock cream test b o t t l e s : shaken on mechanical shaker at room temperature f o r times indicated: centrifuged 10 minutes at room temperature: butanol layer decanted to clean f l a s k s containing excess sodium bicarbonate: shaken 5 minutes at room temperature: f i l t e r e d through Whatman fl paper and o p t i -cal density read at $Q2 and 615 mu.. C a l i b r a t i o n curves f o r resazurin and r e s o r u f i n i n butanol saturated with sodium bicarbonate were obtained on a f r e s h l y prepared dye solution (figure 2 ). Increments of dye and water were added to 9 gm Babcock cream test bottles to a t o t a l volume -20-of 0 . 6 ml. Sodium isoascorbate was added to one series of fl a s k s to reduce the resazurin to re s o r u f i n . The remaining procedure was as outlined f o r table 2, except that the single phase r e s u l t i n g from the f i r s t shaking was not centrifuged; sodium bicarbonate being added d i r e c t l y to the o r i g i n a l f l a s k s a f t e r shaking 10 minutes. The stock solution from which these data were obtained was the ar b i t r a r y standard described, and subsequent solutions of dye were corrected to i t . Since i t was more convenient f o r subsequent work to calibrate the method d i r e c t l y i n terms of quantity of dye added to milk, e x t i n c t i o n coeffecients f o r the dye i n butanol were not obtained. The constants derived from the slopes of these c a l i b r a t i o n curves were used to determine the r e l a t i v e quantities of res-azurin and re s o r u f i n i n an unknown mixture (23). The c a l c u l a -tions were ca r r i e d out, and the f i n a l r e s u l t s expressed, i n terms of jmM resazurin or r e s o r u f i n i n the sample. I t should be noted that the c a l i b r a t i o n was c a r r i e d out on 0 . 6 ml samples rather than on the ml samples which would usually be em-ployed In measuring resazurin reduction i n milk. The smaller volume was completely miscible with butanol and was used f o r standardization to eliminate any p a r t i t i o n e f f e c t s due to the presence of two phases. Calculation of the r e l a t i v e concentrations of resazurin and resorufin i n unknown mixtures was as follows: 0.900 0.0UB 0 . 0 7 2 0 . 0 9 6 0 . 1 2 0 0 .II4.I4. Concentration (}*M) Figure 2 C a l i b r a t i o n curves for the method. D y e at each concentration was i n 9 . 0 ml butanol saturated with sodium bicarbonate. - 2 1 -D]_ = o p t i c a l density at 615 mu. due to resazurin. D 2 r o p t i c a l density at 615 m due to re s o r u f i n . D 3 = o p t i c a l density at 582 mu. due to resazurin. DI4. = o p t i c a l density at 582 mu, due to re s o r u f i n . R = concentration resazurin (u.M per sample). R' = concentration r e s o r u f i n (u.M per sample). Dl = : K]_R D 3 = K 3R D 2 = : K 2 R ' Dk = K^R' Kl = D X/R = 1+.82 Each value was calculated K 2 = D 2/R' = . 0 2 as an average of 6 readings = Dg/R i . e . at 6 d i f f e r e n t con-= 2 . 5 7 centrations % = V R ' = 6.1i|. E 6 1 5 = Di + D 2 - K]_R + K 2R X - observed OD at 615 mu. E 5 8 2 = D3 + % = KgR + JK[|R' = observed OD at 582 mju. .*. R = i % E 6 l 5 - K 2 E 5 8 2 K l % - K 2K 3 = 0 . 2 0 8 E 6 1 5 - 0 . 0 0 7 E^ 8 2 = pM resazurin i n sample r / = K ! E 5 8 2 - K 3 E 6 1 5 * 1 % - K 2K 3 = 0 . l 6 i i . Ec^82 - O.O87 = M M r e s o r u f i n i n sample TABLE 3 -22-EPPECT OP INCUBATION TIME ON DYERECOVERY Time to Incubation Reduce Milk to 7 A 30 Minutes or std. by Buffer v i s u a l Resaz-- Resor- Total System assay u r i n u f i n 0 * M ) (MM) 0*M) Cow #1 60 rain. .061 .040 .101 2 70 min. .066 .033 .099 3 90 min. .076 .025 .101 4 130 min. .076 .030 .106 5 175 min. .078 .022 .100 .}+% Phosphate Buffer at pH 6.4 (.001 .107 .107 with added Sodium isoascorbate Phosphate .107 <.001 .107 Buffer at pH 6.4 time at 37°C 90 Minutes 150 Minutes Resaz- Resor- Total Resaz- Resor- T o t a l u r i n u f i n u r i n u f i n (JIM) OAM) (}lM) (>4M) ( J I M ) .034 .069 .103 .023 .079 .102 .040 .062 .102 .031 .075 .106 .057 .047 .104 .043 .057 .100 .050 .048 .098 .042 .062 .104 .062 .042 .104 .052 .053 .105 (.001 .106 .106 (.001 .106 .106 .108 (.001 .108 .108 (.001 .108 Conditions were as outlined i n table 2: 0.5 ml resazurin solution containing 0.12 uM dye was added to 5.0 ml milk. The recovery range was Q2% to 90%. ' -23-The recovery of dye from milk and phosphate buffer a f t e r incubation i s shown i n table 3. Vi s u a l observations of dye color are included as well. Milk from both Ayrshires and Holsteins i s represented. Dye recoveries from both milk and buffer were low, but that recovered a f t e r 30 minutes incubation was comparable with that a f t e r 150 minutes. This indicated that l o s s of dye resulted from a p a r t i t i o n e f f e c t of some sort rather than destruction. Since recoveries from milk were com-parable with those from buffer, adsorption of dye to protein or interference from milk f a t did not s i g n i f i c a n t l y influence the butanol extraction. Butanol and water are mutually soluble to some extent {2lx.) , and part of the apparent dye loss could be due to a net gain i n volume of the butanol layer. I f t h i s gain i n volume were to account for a l l of the apparent dye l o s s , i t would have to amount to 10$ i n the case of extracts from buffer, and up to 20$ f o r extracts from milk. An estimate of d i l u t i o n of butanol extracts from various volumes of buffer, milk, and gravity cream was obtained (table 4). The difference between 10 ml and the volume of butanol r e t r e i v -able from a 10 ml portion of extract was taken to represent the d i l u t i o n . The net gain i n volume of the butanol after extrac-t i o n of dye from 1 0 . 0 ml buffer and saturating the extract with sodium bicarbonate was 9 . 2 $ . When a solution of dye i n butanol, such as that used f o r standardization, was d i l u t e d to the same extent, the apparent dye loss was 8 . 0 $ . This accounted f o r the low dye recovery calculated f o r extracts from buffer. The d i l u t i o n of butanol by whole milk, 11.8$, only accounted f o r a -2k-10% loss of dye, while apparent losses up to 1&% were encoun-tered f o r whole milk i n table 3. This difference could not be due solely to d i l u t i o n by milk f a t because losses up to Ij.C$ were found with milk f a t i n table 5 , while gravity cream could only account f o r a 12% l o s s on the basis of d i l u t i o n i n table k-The milk f a t used i n table 5 was, however, much more concen-trated than the gravity cream i n table 1+, making the actual d i f -ference less than the difference between these values ind i c a t e s . I t i s evident from the foregoing that the o r i g i n a l c a l i -bration could not be used d i r e c t l y to calculate dye recoveries unless d i l u t i o n of the extract was considered as well and some additional allowance made f o r milk f a t . Since milks from d i f f e r e n t sources d i f f e r i n fat content, i t would be d i f f i c u l t to obtain reproducible d i l u t i o n values f o r d i r e c t c a l c u l a t i o n . The apparent dye recovery from whole milk, skim, and milk f a t i s shown i n table Equal quantities (0.06 u.M) resazurin and resorufin were added to each sample and the dye extracted immediately. The extraction was c a r r i e d out before any s i g n i f i -cant resazurin reduction occurred to change the r a t i o of the two forms of the dye. The decrease i n recovery shown f o r milk fat was more rapid than i t would have been f o r gravity cream, but i t i l l u s t r a t e d that, even at recoveries as low as 60%, the r a t i o , resazurin/resorufin, remained constant regardless of source of extract. TABLE 4 - 2 5 -DILUTION OP BUTANOL EXTRACT FROM DIFFERENT SOURCES Source of Extract * Buffer (ml) 1.0 2.0 4 . 0 10 .0 Whole Milk (ml) 1.0 2.0 4 . 0 1 0 . 0 Skim Milk (ml) 1.0 2.0 4 . 0 10.0 Gravity Cream (ml) 1.0 2.0 4 . 0 10 .0 * . 0 i l $ phosphate, pH 6 . 4 V o l . BuOH (ml) per Extract A f t e r Sat-uration with NaHCOg 9.73 9 .24 9.08 9.08 9.72 9 .42 9.13 8 .82 9.66 9.26 9.03 9.10 9.96 9 .50 8.78 8.63 Percent D i l u t i o n (100 minus V o l . BuOH per 100 ml extract) 2.7 7.6 9.2 9.2 2.8 5.8 8.7 11.8 3 . 4 7.4 9.7 9.0 0 .4 5.0 12.2 13.7 Materials shown i n f i r s t column added to 18 ml butanol i n Babcock f l a s k s and shaken 10 minutes: centrifuged and butanol layer decanted: excess NaHCOg added to butanol extract and shaken 5 minutes: f i l t e r e d : 10 ml extract added to 50 ml H2O and d i s t i l l e d at 92.6°C: volume butanol calculated from weight of 25 ml d i s t i l l a t e . TABLE 5 - 2 6 ~ APPARENT DYE RECOVERY FROM DIFFERENT SOURCES Whole Milk OD Resazurin Resorufin Total Resazurin/ (ml) 582rau. 6l5mu. (JAM) ()lM) Resorufin 0 .1+28 .260 . 0 5 2 .01+8 . 1 0 0 1 . 1 1 .1+35 . 2 5 5 . 0 5 1 .01+9 . 1 0 0 1 . 0 2 .1+30 .21+8 .01+9 .01+9 . 0 9 8 1 . 0 3 .1+30 .21+2 .01+8 . 0 5 0 . 0 9 8 1 . 0 k .1+30 .21+0 .01+8 . 0 5 0 . 0 9 8 1 . 0 5 .1+30 .238 .01+8 . 0 5 0 . 0 9 8 1 . 0 .m Milk (ml) 0 .1+20 .260 . 0 5 2 .01+8 . 1 0 0 1.1 1 .1+1+0 . 2 6 0 . 0 5 2 . 0 5 2 .101+ 1 . 0 2 .1+35 . 2 5 5 . 0 5 1 . 0 5 2 .103 1 . 0 3 .1+1+0 . 2 5 0 . 0 5 0 .051 . 1 0 1 1 . 0 k .1+30 .21+0 .01+8 . 0 5 0 . 0 9 8 1 . 0 5 .1+1+0 .21+0 .01+8 . 0 5 2 . 1 0 0 0 . 9 .lk Fat (ml) 0 .1+28 .260 . 0 5 2 .01+8 . 1 0 0 1.1 1 .1+00 .235 .01+7 .01+5 . 0 9 2 1 . 0 2 .380 . 2 2 0 .01+1+ .01+3 . 0 8 7 1 . 0 3 .360 . 2 1 0 .01+2 .01+1 . 0 8 3 1 . 0 k .31+0 . 2 0 0 .01+0 .038 . 0 7 8 1 . 0 5 .312 . 175 .035 .036 . 0 7 1 1 . 0 ^-obtained by centrifuging whole milk and therefore containing more f a t than gravity cream. Milk, skim, or f a t d i l u t e d to a t o t a l volume of 5 . 0 ml with water p r i o r to adding .12 ;uM dye (1:1 resazurin:resorufin mixture): assay procedure was the same as that f o r table 3 except that sam-ples were not warmed to 37°C before extraction. - 2 7 -Generally, i n whole milk, more than %0% of added dye was recovered. Of the 20$ l o s s , half could be accounted f o r by gain i n volume of the butanol extract, leaving a net 10$ loss that could not be accounted f o r . On the basis of the data i n table S i i t was considered v a l i d to assume that the r a t i o , resazurin/resorufin was the same i n t h i s 10% as i n the ext r a c t , and that recovered dye could be extrapolated to 100$ using t h i s r a t i o to arrive at a quantitative estimate of extent of reduction. This assay does not correlate exactly with the v i s u a l method. The absorbance of resazurin and resorufin varies over the normal pH range of milk and i t i s possible that the shades of color observed i n the v i s u a l method vary i n a s i m i l a r manner. Calculation of apparent resazurin and resorufin con-centrations for the data i n table 1 presents the following picture: PH Resazurin O M ) Resorufin (JAM) Resazurin/ Resorufin 6 . 3 . 0 6 9 .01+1 1 . 6 8 6.2]. . 0 7 3 .01+7 1 . 5 5 6 . 5 . 0 7 8 . 0 5 7 1 .37 6 . 6 .081+ .061+ 1.31 6 . 8 . 0 9 8 . 0 8 5 1 .15 with bicarbonate . 108 .112 0 . 9 7 Since resazurin and resorufin were at the same concentration, the r a t i o should be 1 . 0 . While i t i s true that the constants used for the above c a l c u l a t i o n are v a l i d only f o r extracts - 2 8 -saturated with sodium bicarbonate, the i l l u s t r a t i o n shows that the apparent concentration of res o r u f i n decreased more ra p i d l y with pH than did that of resazurin. I f the same reasoning were applied to the v i s u a l assay, a milk sample at pH 6 . 8 could appear more reduced than one at pH 6 . 5 * though both contained the same concentrations of dye with the same r a t i o s of resazurin/resorufIn. For purposes of comparison with the v i s u a l assay, a resazurin/resorufin r a t i o of 1.0, which was frequently obtained from samples which appeared to be reduced to the Munsell 7/1+ color standard, was accepted as being similar to the 7/4 standard. - 2 9 -II .MEASUREMENT OF REDUCING CAPACITY OF MILK It was found necessary to have an estimate of reducing capacity of milk i n the presence of added resazurin. Potentio-metric measurements of Eh, since they give no i n d i c a t i o n of poise, or capacity of the system to r e s i s t change, are of no use f o r this purpose. This i s analogous to pH gi v i n g no i n d i c a t i o n of buffer capacity. The Eh of fresh milk, i n the absence of extensive b a c t e r i a l a c t i v i t y , ranges from +0.2 to + 0.3 volt (25,26). It i s possible to assess degree of poising of the system by adding an excess of an oxidant having EQ i n t h i s range and measuring extent of reduction of added oxidant. The oxidant selected i n our experiments was the reversible Eh i n d i c a t o r , 2,6-dichlorophenolindophenol, which has Eo =+0 .2i|7 v at pH 6.6 ( 2 7 ) . I t was found from preliminary experiments that indophenol was decolorized on addition to milk and that the blue sodium s a l t of the dye could be extracted from milk with n-butanol. The problems involved i n butanol extraction of dye from milk which were encountered i n measuring resazurin reduction, were also expected to occur with indophenol. The method of extrac-t i o n and treatment of extracts were therefore exactly the same as those described f o r resazurin. The absorption spectrum of a butanol extract of indophenol from phosphate buffer (0.01$, pH 6.6) i s shown i n figure 3. Maximum absorbance occurred at 660 mp,. Spectra of extracts obtained under the same conditions from milk or cream were superimposable on that shown, except f o r concentration differences. Neither blank extracts from milk or 0.900 0.600 0.300 Wavelength (mu.) Figure 3 Absorbance spectrum of 2,6-dichlorophenolindophenol i n butanol saturated with bicarbonate. -30-cream, nor extracts of resazurin or resorufin (0.12 j^ M) had s i g n i f i c a n t o p t i c a l density at t h i s wavelength. Since the poising of normal fr e s h milk i s believed due to i t s ascorbic acid content (26,28), indophenol solutions were standardized with ascorbic acid immediately p r i o r to use. An example of such a standardization i s shown by figure l+. The indophenol solution (0.^ mg/ml) was prepared dy di s s o l v i n g the dye, along with an equal weight of sodium bicarbonate, i n water. The bicarbonate was added to ensure maximum s o l u b i l i t y of dye and was not present i n s u f f i c i e n t concentration to a f f e c t the pH of milk or buffer solution. The dye so l u t i o n was f i l t e r e d and 1.0 ml added to a series of test tubes containing 5«5 ml M/50 phosphate buffer (pH 6.6) and graded increments of ascorbic a c i d . The tube contents were mixed by inversion and, a f t e r 3 minutes, added to 10 ml butanol i n Babcock f l a s k s . The ex-t r a c t i o n and treatment of extracts were as described f o r resaz-u r i n . The f i l t e r e d extracts from solutions of low ascorbic acid concentration were dil u t e d with butanol to an o p t i c a l density i n the range 0.100 to 0.600. The o p t i c a l densities tabulated i n figure 4 were obtained by direct m u l t i p l i c a t i o n of observed 0D by the appropriate d i l u t i o n f a c t o r . The apparent dye losses encountered i n butanol extraction of r e s a z u r i n from buffer and milk (tables li and 5) probably also occurred i n butanol extraction of indophenol. The losses with the method described f o r indophenol were, however, of much less s i g n i f i c a n c e . The apparent losses, (10$), due to d i l u t i o n of butanol by water were eliminated e n t i r e l y by use of the same 0.1 0.2 0.3 O.k 0.5 0.6 Concentration Ascorbate (u.M) Figure 1| Standardization of indophenol solution with ascorbate: extracts read i n butanol saturated with sodium bicarbonate. -31-condltions f o r c a l i b r a t i o n as were used f o r te s t i n g unknown samples. The maximum losses of o p t i c a l density found i n table 5 were more severe than those normally encountered, being a de-crease of 25 percent i n the observed values. With indophenol extracts, 25 percent d i l u t i o n of the o r i g i n a l extract would cause a loss of only 5 percent i n the observed values. The difference, i n s e n s i t i v i t y to errors of extraction, between the two methods r e s u l t s from the difference i n o p t i c a l density ranges for which they were cal i b r a t e d : resazurin, OD range = 0 to 0 .7* indophenol, OD range = 0 to 3 . 0 . The reducing capacity of a sample of fresh milk i s shown by table 6 . The extracts were prepared as described f o r f i g -ure I4., except that 5«0 ml milk with 0 . 5 ml added water or r e s -azurin (resorufin) solution replaced buffer. Aliquots of the same sample of milk were used f o r a l l the readings shown. I t i s evident from these data that the method was reproducible and that neither resazurin nor r e s o r u f i n i n t e r f e r e d with reduction of added indophenol. That indophenol reduction was quantitative, and not merely a function of the Eh of the system, i s i l l u s t r a t e d by the data i n table 7 . A sample of the milk used i n table 6 was d i l u t e d with water as shown, and the observed reducing capacities com-pared with values calculated from that observed f o r the un-d i l u t e d sample. Though there was no d i r e c t evidence that ascor-bic acid was responsible f o r reduction of indophenol added to milk, the reducing capacities were expressed i n terms of -32-ascorbic acid. This was mainly to provide a scale by which milk samples could be compared. TABLE 6 REDUCING CAPACITY OF MILK (expressed as ascorbate) Milk (ml) HpO (ml) 0D at 660 mu. Equiv. Ascorbate (MM) 5-0 0.5 0.580 0.57 5 . 0 0 . 5 Resazurin (>lM/0.5 ml) 0.540 0.58 5 . 0 0.12 0.560 0.58 5 . 0 0.12 Resorufin (uM/0.5 ml) 0.540 0.58 5 . 0 0.12 0.540 0.58 5 . 0 0.12 0.560 0.58 TABLE 7 INFLUENCE OF DILUTION ON REDUCING CAPACITY Milk HpO OD at 660 mu* Equiv. Ascorbate (uM) (ml) (ml) Found Calculated 1 . 0 4 . 0 2 . 5 8 0 0 . 10 0 .11 2.0 3.0 1.900 0.26 0.23 3.0 2.0 I . 4 8 O 0 .36 0 . 3 4 4 - 0 1.0 0 . 9 6 0 O.48 0 . 4 6 5 . 0 0 0 . 5 6 0 0 . 5 7 ^Extracts were d i l u t e d with butanol to an OD i n the range 0.100 to 0.600 before reading. The value tabulated was extrapolated back to o r i g i n a l concentration. -33-On ap p l i c a t i o n of t h i s method to a wide var i e t y of milk samples, i t was found that the milk reduced a portion of the added indophenol i n every case, the quantity depending on the previous h i s t o r y of the sample. I f the milk was aged, held at 37°C f o r several hours, or treated with a mild oxidizing agent, i t l o s t some of i t s a b i l i t y to reduce the dye. This argues In favor of dye reduction rather than adsorption or destruction. The following discussion i s based on t h i s wider application of the method as well as on the s p e c i f i c r e s u l t s reported i n tables 6 and 7» The discussion i s presented i n an e f f o r t to explain the apparently anomalous behavior of indophenol under the conditions previously described and to show that the reduc-t i o n of the dye under these conditions i s a measure of the r e -ducing capacity, or poise, of milk. Since indophenol, having E Q = + O.2I4/7 v o l t at pH 6.6, i s quantitatively reduced by milk with Eh i n the same range, i t s reduction must be determined by factors other than the potentio-m e t r i c a l l y determined Eh of the milk. Tha f a c t that resazurin and r e s o r u f i n have no measurable e f f e c t on the indophenol-milk system helps substantiate this conclusion. The rap i d , quanti-t a t i v e reduction of indophenol indicates that milk must be strongly poised at an Eh lower than that determined by potentio-metric measurement. The Eh of fresh milk appears to be a func-t i o n of i t s oxygen content, since removal of oxygen by fl u s h i n g with nitrogen r e s u l t s i n an immediate decrease of over 0.If. v o l t i n the measured p o t e n t i a l (26). The p o t e n t i a l i n the presence of oxygen, over a 0 . 0 7 5 v o l t range, depends, however, on reduced -31|-ascorbic acid content (28) . These observations indicate that Eh must be the r e s u l t of a contribution from both dissolved oxygen and the reducing system, though not necessarily the re s u l t of an i n t e r a c t i o n between them. Both oxygen and the reducing system maintain t h e i r i d e n t i t y and appear to function independently of each other, as evidenced by the behavior of the system on removal of oxygen or addition of indophenol. This type of behavior toward oxygen i s t y p i c a l of many b i o l o g i c a l reducing systems i n situations which approximate th e i r natural environment, f o r example, i n tissue preparations or c e l l - f r e e extracts. Oxidation, under these conditions, i s extremely slow, the reducing system and oxygen behaving as i f they were physi-c a l l y separated so that t h e i r respective potentials are super-imposed to produce a net Eh f o r the system as a whole, without a f f e c t i n g the actual potentials of the components. This means that the true p o t e n t i a l of the reducing system can be measured only aft e r removing the contribution to Eh due to oxygen, that i s by removing oxygen. Reducing systems of t h i s type are usu-a l l y f u l l y active toward many oxidants other than oxygen and react with them ra p i d l y and quantitatively i n the presence or absence of oxygen. This appears to be true f o r the reduction of indophenol added to milk. On the basis of the preceding d i s -cussion, the actual Eh of the reducing system of milk would be approximately -0.1 v o l t , assuming that a decrease of 0.1+ vo l t would r e s u l t on removal of the po t e n t i a l due to oxygen. Since the presence of oxygen, and hence the measurable Eh of milk, has no influence on the reaction of indophenol with the - 3 5 -reducing system, the quantity of dye needed to r a i s e the Eh of the reducing system from -0.1 v o l t to +0.2 volt i s a measure of the poise, or reducing capacity of the milk. In the method proposed f o r estimating reducing capacity, the quantity of indophenol decolorized by a sample i s measured to assign a r e l a t i v e value to the poise of the reducing system. -36-EXPERIMENTAL I GENERAL OBSERVATIONS A. Decrease i n Rate of Resazurin Reduction on Aging of Milk In early experiments, using v i s u a l estimates of resazurin reduction, i t was observed that the time taken to reduce the dye increased with age of the milk sample, provided extensive bacter-i a l growth had not occurred. In order to obtain a more quan-t i t a t i v e estimate of the aging e f f e c t , fresh milk and skim were stored at 0°, 1x9, and 12.8°C f o r 50 hours. The skim was ob-tained by centrifuging warm whole milk within 30 minutes of milking. Resazurin/resorufin r a t i o s were obtained after 5, 10, 20 and 50 hours on a l l samples. Representative r e s u l t s are shown by figure 5 ( a and b). Resazurin/resorufin r a t i o s were determined after samples had been held with 0.12 ^M dye f o r 60 minutes at 37°C. Both of the milks i n figures 5 show a rapid los s of the a b i l i t y to reduce resazurin, even when storage was at 0°C. Differences i n temperature were r e f l e c t e d to a much greater extent i n skim than i n whole milk. The break i n the curves between 5 a n < i 10 hours was probably a r e a l e f f e c t rather than an experimental a r t i f a c t . I t was l a r g e l y absent from the whole samples and much les s pronounced i n the skim from sample 1 (figure 5a) than i n that from sample 2 (figure 5b). Since a l l of the samples, for any given time, were assayed simultaneously, i t i s u n l i k e l y that re l a t e d samples should have d i f f e r e d due to random errors. The effect was also found i n other samples i n the same experiment, i t s magnitude varying as i n the examples 0 10 20 30 l+O 5 0 Hours of Storage Figure 5a (Milk 1) Influence of storage at various temperatures on a b i l i t y to reduce resazurin. Broken l i n e s represent skim and s o l i d l i n e s whole milk. Resazurin/resorufin determined af t e r incubation with dye at 37°C fo r 60 minutes. Figure 5b (Milk 2) - 3 7 -shown, and always occurring at the same point i n the curves. I t should be noted that, although milk 1, when fr e s h , r e -duced resazurin more rapidly than did milk 2, i t retained more of i t s a b i l i t y to reduce dye on storage and was les s sensitive to temperature of storage. This i s contrary to what would be expected i f rapid resazurin reduction depended on a more mobile and hence, a more l a b i l e , reducing system. In f a c t , these data indicate that the reverse may have been true, rapid reduction depending on a more stable reducing system. The major point I l l u s t r a t e d by these figures i s that the resazurin reduction observed with fresh milks, or those r e -f r i g e r a t e d for short i n t e r v a l s , was due to the normal reducing systems of milk rather than to b a c t e r i a l a c t i v i t y . Reduction due to b a c t e r i a l action would be expected to increase with stor-age time. The rapid reversal of slope shown at 20 hours by the l i n e s representing milk 2 stored at 12.8°C (5b) resulted from b a c t e r i a l a c t i v i t y . This i l l u s t r a t e s that, though milk 2 con-tained a more active b a c t e r i a l population, i t was a les s active resazurin reducer i n i t i a l l y , and also l o s t i t s reducing a b i l i t y more rapidly than milk 1. B. Reduction Rate i n Milk Fractions The differences i n reducing rate and s t a b i l i t y on storage between whole milk and skim, shown i n figure 5 , l e d to i n t e r e s t i n the dye reducing a b i l i t y of the various f r a c t i o n s of normal milk. The r e s u l t s of a t y p i c a l experiment are pictured i n f i g -ure 6. Fresh milk was permitted to cream f o r 20 hours i n a cylinder at lj. 0C. The cream was removed and a portion warmed 30 I I I I L 30 60 90 120 150 Incubation Time (Minutes) Figure 6 Reduction Rate i n Milk Fractions 1 - Gravity skim 2 - Whole milk 3 - Plasma (aqueous phase from c e n t r i -fuging warm cream) \i - Gravity cream -38-to room temperature, centrifuged, and the aqueous phase used to obtain the curve l a b e l l e d "plasma". The remaining curves i n the figure represented whole milk which had been held at l+°C i n a separate container, and the gravity cream and skim from the cylinder r e f e r r e d to above. Each point i n the figure was derived from a 5»0 ml sample, with 0 . 5 ml added resazurin solution (0.12 U.M dye), held at 37°C for the time i n t e r v a l s shown on the arithmetic scale. The spectrophotometrie assay fo r resazurin and r e s o r u f i n was applied to obtain the values p l o t t e d on the logarithmic scale. These values are tabulated as percent resazurin i n the recovered dye and were interpreted to represent percent of added dye remaining as resazurin i n the sample. The v a l i d i t y of t h i s extrapolation was discussed i n section 1 of "Preliminary Experiments". As predicted by the r e s u l t s on aging, skim reduced dye more slowly than did whole milk, the cream layer apparently accounting f o r the difference. No e f f o r t was made to re l a t e the differences i n reducing a c t i v i t y between milk and skim to the f a t content of the o r i g i n a l whole milk. The p o s i t i o n of the curve f o r plasma, i n r e l a t i o n to those for the other f r a c -t i o n s , was of considerable i n t e r e s t . Since this f r a c t i o n was obtained by centrifuging warm, gravity cream, i t contained a much greater concentration of the material which had been loosely adsorbed to the f a t globule surfaces, i . e . the r e a d i l y removable, outer layer of the f a t globule membrane (26), than did the skim or whole milk. The f a t globule membrane has been shown to contain the bulk of the enzyme a c t i v i t y as well'as - 2 9 -most of the copper and i r o n of milk (26 ) . The plasma f r a c t i o n , as obtained i n t h i s experiment, would also have contained the majority of the bacteria and leucocytes present. In view of the membrane constituents as well as the bacteria and leuco-cytes, i t i s to be expected that t h i s f r a c t i o n would have en-hanced resazurin reducing a c t i v i t y . The reducing rates p l o t t e d i n figure 6 provide some i n -formation concerning the k i n e t i c s of resazurin reduction. The p l o t s were l i n e a r over the i n i t i a l p o r t i o n , which would have been t y p i c a l of spontaneous decay or of a f i r s t order enzyme reaction. The rate, however, decreased before h a l f the added dye had been reduced. If reduction were spontaneous decay dependent on Eh, or i f the dye were involved i n electron trans-port linked to some enzyme system, the increasing concentrations of the r e v e r s i b l e , re soruf i n 7—*- dihydrore soruf i n , forms of the dye could possibly change the poise of an Eh system or mediate electron transport, so that rate of further resazurin reduction would be decreased. Other f a c t o r s , for example, depletion of a substrate, i n a c t i v a t i o n of an enzyme, or removal of a reductant through reaction with oxygen, could be presented as equally probable explanations. C. Poising by Dye The following experiment, i l l u s t r a t e d by figure 7, was designed to determine the influence of r e s o r u f i n on rate of res-azurin reduction. Resorufin was prepared by reducing resazurin solution with ascorbic acid, extracting the r e s o r u f i n with ether, and readjusting to volume after evaporation of ether. When -40-checked spectrophotometrically, t h i s solution contained 0.24 JAM r e s o r u f i n per ml and no resazurin. Resazurin and r e s o r u f i n solutions were mixed to produce dye solutions"having the r e l a -t i v e concentrations shown i n figure 7• D y Q solution (0 .5 ml) was added to tubes containing 5*0 ml milk and held at 37°0. Resazurin and resorufin were measured, and r e s u l t s expressed as described f o r figure 6. The decreasing rates of resazurin reduction with time of incubation (figure 6) had suggested that the increasing con-centrations of the r e v e r s i b l e , r e s o r u f i n d i h y d r o r e s o r u f i n , forms of the dye could have i n h i b i t e d resazurin reduction through poising of the Eh system or by mediating electron transport. That th i s was u n l i k e l y , i s shown by the curves i n figure 7. These are roughly p a r a l l e l , i n d i c a t i n g that poising was not of p a r t i c u l a r significance i n decreasing the rate of reduction. The dotted curve (above curve 1) was calculated from curve 4 by eliminating the contribution of added r e s o r u f i n to the r e -covered dye. If i t i s considered that the actual quantity of r e s o r u f i n present i n i t i a l l y i n t h i s set of samples was 20 per-cent greater than that f i n a l l y generated i n the samples repre-sented by curve 1, and that the rate of change of these two curves was comparable over the f i r s t hour of incubation, the influence of added r e s o r u f i n was n e g l i g i b l e . Concentration d i f -ferences must, however, be remembered i n comparing these two curves. The t o t a l resazurin reduced during the f i r s t hour by the system represented by the dotted curve was 9 X 10 ~^^M, 3 0 6 0 9 0 120 150 Incubation Time (Minutes) Figure ? Poising by Dye Added to 5 ml Milk 1 - 0.12 u.M Resazurin 2 - 0.096 MM Resazurin and O.02I4. « M Resorufin 3 - 0.072 uM Resazurin and O.O/4.8 )&L Resorufin I4. - O.OI4.8 JUM Resazurin and 0.072 « M Resorufin 5 - Percent resazurin remaining i n samples of curve 1+ i f re s o r u f i n added I n i t i a l l y was disregarded. -1*1-while that by the system represented by curve 1 was 22.8 X 10"^ " JJ.M, or 2 .5 times as much. The greater decrease i n reduc-t i o n rate shown by the dotted curve on incubation beyond one hour could be due to the low concentration of resazurin or, pos-s i b l y , to competition from the resorufin/dihydroresorufin system, whose concentration at one hour incubation was 1 . 55 times that of the resazurin present. D. Influence of Dye Concentration on Reduction Rates It was considered possible that the decrease i n reduction rate shown by the dotted curve i n figure 7 on incubation beyond one hour could have been due to the low concentration of r e s -azurin. The data i n figure 8 - i l l u s t r a t e that the f r a c t i o n of added resazurin reduced changed more rapidly with incubation time i f dye was present at lower concentrations than that nor-mally employed f o r milk t e s t i n g . The curves were obtained using the conditions and techniques described for figure 6, except that volumes of resazurin solution containing 0.021+, O.Oij.8, and 0.120 }XM. dye were d i l u t e d to 0 .5 ml and added to 5.0 ml milk samples. Two samples of milk are represented i n figure 8. The upper set of curves (milk 1) represent a slow dye reducer and the lower set (milk 2), a rapid reducer. In both samples, the higher dye concentrations showed greater deviations from t y p i c a l f i r s t order p l o t s than did the lower concentrations. Examination of these curves and c a l c u l a -t i o n of the actual quantities of resazurin reduced could imply that the deviation from l i n e a r i t y was due to poising by the r e s o r u f i n d i h y d r o r e s o r u f i n system. However, i t was 30 60 9 0 120 1^0 Incubation Time (Minutes) Figure 8 Influence of Dye Concentration on Reduction Rates Added to 5 nil milk: 1 - 0.02k ,uM Resazurin 2 - 0.0l|.8 JAM Resazurin 3 - 0.120 jiM Resazurin - 1 * 2 -demonstrated i n figure 7 that the interference from this system was n e g l i g i b l e i n the i n i t i a l stages of reduction and had only a small influence i n the l a t e r stages. I t i s u n l i k e l y that the resorufin/dihydroresorufin system required a 60 minute induction period before becoming f u n c t i o n a l , since the equilibrium i s supposedly f r e e l y r e v e r s i b l e . A more plausible explanation i s that both resazurin and r e s o r u f i n were capable of i n t e r f e r i n g with some reducing process i n the milk without being reduced themselves, i . e . acting c a t a l y t i c a l l y to deplete the system of a reducing substance. The explanation that the higher concentra-t i o n of resazurin depleted the milk of some reducing substance by quantitative i n t e r a c t i o n , cannot be applied to t h i s system. Though a l l samples showed a decelerated reduction rate with time of incubation, i n any given time i n t e r v a l , the actual quantity of dye reduced i n the more concentrated solutions was Ix to 5 times as great as that i n the most d i l u t e . E. Influence of pH and Bacteria on Rate of Reduction Before attempting to locate and study the actual systems responsible for resazurin reduction i n milk, i t was considered advisable to determine the influence of added b a c t e r i a l popu-l a t i o n s and small changes i n pH on rate and pattern of reduc-t i o n . Typical r e s u l t s of experiments i n t h i s regard are summarized i n figures 9 (a and b). Bacteria were grown on yeast litmus milk at 37°C for 12 to 18 hours and harvested immedi-ately before use. C e l l s were washed once i n M/50 phosphate buffer at pH 6 . 8 , resuspended i n a minimum volume of buffer and added to the milk at the same time as dye. The pH of the milk samples was dropped to 6.1+ with N-HC1 immediately p r i o r to addition of bacteria and dye. The remaining procedure was as described f o r figure 6 . In order to i l l u s t r a t e a maximum e f f e c t , the curves shown are f o r normal milk and milk at pH 6.1|, which i s below the ph y s i o l o g i c a l range of milk. Curves f o r rates within the phys i o l o g i c a l range and below pH 6 . 6 , occurred be-tween those shown, while those for pH above 6 . 6 , occurred above the upper curve and showed a greater decrease i n reduction rate with time of incubation. Curves f o r b a c t e r i a l a c t i v i t y were only obtained at the pH values shown but variations with pH would probably have been as predicted by the behavior of milk with no added bacteria. The constant (or accelerating) rate of reduction with time of incubation of the curves representing added b a c t e r i a , as com-pared with the decelerating rate of the controls, further sub-stantiates the e a r l i e r observations that b a c t e r i a l a c t i v i t y was normally of l i t t l e significance i n resazurin reduction by fresh milk. The curves shown represented an intermediate concentration of b a c t e r i a . Higher concentrations of these same organisms caused proportionately f a s t e r reduction, while lower concentra-tions produced curves which were barely distinguishable from the controls. Some e f f o r t was made to correlate the drop i n Eh of milk, i n the presence of added bacteria, with resazurin reduction due to bacteria. The r e s u l t s were not conclusive, and more elaborate experiments would have been necessary to demonstrate a r e l a t i o n s h i p . Oxidation-reduction systems generally, show a strong p o s i t i v e increase i n EQ with decreasing pH. According to the data of Twigg (18), dropping the pH from 6.6 to 6.1 produced an increase i n the EQ value of the resorufin/dihydroresorufin system of-f 0.03 v o l t . A change of t h i s magnitude was, however, found to be compensated f o r by a r i s e of -+0.03 v o l t i n the Eh of milk, on dropping the pH from 6.? to 6.2. The r e l a t i v e potentials of dye and milk should therefore have remained un-changed, so that the e f f e c t of pH could not be accounted f o r on t h i s basis. 30 60 90 120 150 Incubation Time (Minutes) Figure 9a (Milk 1) Influence of pH and Bacteria on Rate of Reduction 1 - Normal milk at pH 6.7 2 - pH of milk dropped to 6.1^  with N HCl 3 - Milk at pH 6.7: 2 X 106/ml un i d e n t i f i e d Streptococcus i s o l a t e d from milk added k - pH dropped to 6.I1' 2 X 106/ml un i d e n t i f i e d Streptococcus i s o l a t e d from milk added 20 30 60 90 120 Incubation Time (Minutes) Figure 9b (Milk 2) Bacteria added; 750,000/ral S. faecalIs 150 -li-5-II REDUCING SYSTEM OF MILK A. Behavior of Resazurin at Low pH The pH dependence of resazurin reduction i n milk sug-gested that useful information concerning the mechanism of reduction could be obtained by dropping the pH to lower l e v e l s . The r e s u l t s of such an experiment are shown i n figure 10 . Samples of whole milk were adjusted to the pH values shown using 6 N HCI, and held at 37°C f o r 15 minutes i n the presence of dye before butanol extraction. The extraction was ca r r i e d out at the pH of the samples and resazurin remaining estimated by the spectrophotometric method.' The values p l o t t e d i n figure 10 are percent resazurin i n the recovered dye. Since dye recover-ies i n the acid pH range were as high as those normally encoun-tered (approximately Q0% f o r a l l samples), the values p l o t t e d were interpreted as percent of added dye remaining as resazurin i n the samples. Curves very s i m i l a r to that pictured i n figure 10 were obtained using buffered ascorbic acid solutions (10 mg/litre) i n place of milk, and i t i s possible that the curve obtained from milk was nothing more than reaction of ascorbic acid with resazurin. The s i t u a t i o n depicted i s , however, the reverse of that explained by B a l l (29) f o r the system, ascorbic a c i d / methylene blue, i . e . Methylene blue can be used f o r assay of ascorbic acid at any pH except 5 . 0 , where the E 0/pH curves of the two systems i n t e r s e c t . At higher pH, the dye has E 0 below that of ascorbic a c i d , but i s reduced by the ascorbic acid system because of the low Eh generated as a r e s u l t of the 90 1 Sto fTo • u.o 3?o T7o pH Figure 10 Behavior of Resazurin at low pH pH adjusted using 6 N HCl: held at 37°C f o r 15 minutes i n the presence of dye. -1+6-I n s t a b i l i t y of dehydroascorblc acid. At pH below 5*0, the ascorbic acid system has E 0 below that of methylene blue and reduction of dye i s an obvious consequence of mixing the two systems. That a mixture of ascorbic acid and resazurin behaved i n the reverse manner indicates that the i n t e r a c t i o n was prob-ably not a simple function of Eh and pH. Whether or not resaz-u r i n showed t h i s odd behavior i n the presence of reducing agents other than ascorbic acid was not determined. The regular increase i n rate of reduction from pH 6.6 to 3.8 indicates that the reduc-ing system charted i n figure 10 was the normal reducing system i n milk. Since i t i s common knowledge that l e v e l s of reduced ascorbic a c i d , as such, do not correlate with rapid resazurin reduction, and since ascorbic acid was present i n s u f f i c i e n t concentration i n the milk to produce the curve shown i n figure 10, no advantage could be seen i n investigating t h i s p a r t i c u l a r effect any further. The observation i s included merely as an i l l u s t r a t i o n that the i n t e r a c t i o n of the reducing system of milk with resazurin probably involves more than a simple oxidation-reduction process. B. Influence of Resazurin The deceleration i n rate of resazurin reduction with time of incubation noted i n e a r l i e r experiments suggested that resaz-u r i n had some i n a c t i v a t i n g influence on the reducing capacity of milk. The magnitude of t h i s influence i s i l l u s t r a t e d i n figure 11. Reducing capacity was measured as described i n section I I of "Preliminary Experiments" and was expressed as u.M ascorbate per sample. The values plotted to produce the three upper curves - i |7-on the logarithmic scale represent ascorbate remaining at the times shown. Concentrations of resazurin (^M/sample) are shown by the two lower curves. The volume of a l l milk samples was 5 ' 0 ml with 0 .5 ml added water or resazurin solution. Res-azurin solutions contained either 0.12 pM or 0.02ij, uM dye per 0 . 5 ml. The data i n figure 11 i l l u s t r a t e that added dye had a c a t a l y t i c e f f e c t on destruction of the reducing system of the milk. C a l c u l a t i o n of half l i f e periods for the s t a b i l i t y of the reducing system under the three conditions measured pro-duces values of 576 minutes f o r the control system with no added dye, 287 minutes i n the presence of Q.Q2li i\M dye, and 155 minutes i n the presence of 0.12 pM* The reaction with added dye was not quantitative, 0.02lj. ;uM causing a loss equiv-alent to 0.17 JU.M ascorbate. This was 7*1 times the quantity of dye added. The higher dye concentration was not so e f f i c i e n t , 0.12 ;nM dye causing a loss of only 0 .3 l j . pM, or 2 .8 times the amount added. The quantities of dye reduced during t h i s i n t e r -val were 61 .5 percent of that added, or 0.015 >*M at the low concentration, and 50 percent, or 0.06 JJM at the high concentra-t i o n . I t i s evident from inspection of figure 11 that rate of destruction of the reducing system was constant f o r a given dye concentration, over a time i n t e r v a l i n which s i g n i f i c a n t con-centrations of resazurin were reduced i n the same system. On this b a s i s , oxidized resazurin could not have been the sole c a t a l y t i c agent involved. The e f f e c t observed could have been 6 0 1 2 0 1 8 0 21j.O 3 0 0 I n c u b a t i o n Time (Minutes) F i g u r e 1 1 I n f l u e n c e of R e s a z u r i n on Reducing C a p a c i t y Reducing C a p a c i t y (uM ascorbate) R e s a z u r i n Remaining (uM) 1 - No added dye k - 0 . 1 2 J I M added 2 - O.02I4. r e a a z u r i n 5 - O.02I4. added 3 - 0 . 1 2 MM r e a a z u r i n -1+8-due to the dye molecule as such, since concentrations of t o t a l dye remained constant while those of the oxidized and reduced species varied. An additional p o s s i b i l i t y i s that c a t a l y s i s resulted from an a r t i f a c t , such as a metal ion introduced with the dye and therefore varying with dye concentration. C. S t a b i l i t y of Ascorbate Milk usually contains approximately 20 mg/litre or 0.57 JAM/ 5 ml reduced ascorbic acid. The reducing capacity, as measured by a b i l i t y to decolorize neutral indophenol, was usually 0.7 pM/ 5 ml. Since any ascorbic acid present would react with the indophenol, t i t r a t a b l e ascorbate may be said to constitute an average of 80 percent of the reducing capacity of milk by t h i s measurement. I t was therefore probable that the e f f e c t of r e s -azurin on the reducing system involved oxidation of ascorbate. Several unsuccessful attempts were made to measure this e f f e c t i n buffered solutions of ascorbic acid but the ascorbate was too unstable at pH 6.6 and 37°C , even when precautions were taken to minimize contamination by metal ions. Some success was a t t a i n -ed by making up a l l solutions i n ethylenediamine tetraacetate (EDTA), though i t was known that EDTA at neutral pH was a pro-oxidant f o r ascorbate (30). Results of an experiment employing buffered solutions of ascorbate i n two concentrations of EDTA are p l o t t e d i n figure 12. Samples contained M/50 phosphate buffer at pH 6.6 and either 0.02$ or 0.2$ EDTA. Ascorbate (0.76 p\JA) was added to $.0 ml buffer followed by 0.12 uJVl resazurin solution. The f i n a l volume was 5.5 ml per sample. Ascorbate was measured using the method described f o r reducing capacity using the -i+9-general procedure outline f o r figure 11. Comparison of figures 12 and 11, neglecting differences i n arithmetic scales, shows that added resazurin had a similar a f f e c t on both ascorbate solutions and milk. In ascorbate solutions, however, no s i g n i f i c a n t resazurin reduction accom-panied loss of half the reducing system present. While i t i s true that ascorbate was much less stable i n the buffered solu-t i o n than i n milk, some resazurin reduction was expected i n the 90 minute i n t e r v a l measured. In the milk, 0 . 0 5 M M resazurin was reduced i n the f i r s t 90 minutes at the high dye concentration though the milk contained only 0 . 5 8 JUM t i t r a t a b l e ascorbate per sample. The r e l a t i v e e f f e c t s of d i f f e r e n t concentrations of resazurin on ascorbate i n the buffered solutions was similar to that obtained i n milk and i t would appear that added resazurin probably stimulated the oxidation of ascorbate i n the milk. The fact that resazurin was not reduced i n the presence of 0.76 u^M ascorbate i n buffered solutions (figure 12) leads to the con-clusion that the 0.58 #M t i t r a t a b l e ascorbate i n milk would not have caused resazurin reduction. Unless the greater s t a b i l i t y of ascorbate i n milk caused i t to reduce added dye, reduction must have been due to the 20 percent reducing capacity that could not be accounted f o r as t i t r a t a b l e ascorbate. The decreased s t a b i l i t y of ascorbate i n higher concentra-tions of EDTA shown i n figure 12 has been studied i n other systems (30). I t i s possible that i f s t i l l lower concentrations of EDTA had been used, an ascorbate solution as stable at pH 6 . 6 as the ascorbate i n milk could have been obtained. I t was found 30 60 90 Incubation Time (Minutes) Figure 12 Influence of Resazurin on Ascorbate i n EDTA Ascorbate Remaining (u.M) 1 -0.02% EDTA 2 - 0.02% EDTA, 0.12 juM resazurin 3 - 0.2$ EDTA 1+ - 0.2$ EDTA, 0.12 uM resazurin Resazurin Remaining (u.M) 5 - 0.02$ EDTA, ascorbate 6 - 0 . 2 $ EDTA, ascorbate - 5 0 -that ascorbate added to powdered skim milk was as stable as that occurring i n most normal milk. Addition of resazurin to these s t a b i l i z e d solutions resulted i n p l o t s s i m i l a r to those i l l u s t r a t e d i n figure 12 i . e . accelerated ascorbate disappear-ance i n the presence of added dye but no s i g n i f i c a n t reduction of resazurin by samples containing 0 . 7 )*M ascorbate per 5*0 ml. D. Influence of Reduced Dye on Reducing System of Milk If the dye molecule was involved i n catalysing the i n -a c t i v a t i o n of ascorbate i n milk, the oxidized and reduced forms must have been equally e f f i c i e n t , since rate of i n a c t i v a t i o n was constant f o r a given dye concentration, though the r e l a t i v e concentrations of resazurin and r e s o r u f i n varied. This r e s u l t would also have been obtained i f c a t a l y s i s had been due to a r t i f a c t s i n the dye preparation. In preparing r e s o r u f i n solutions for use i n constructing figure 7 , ascorbic acid was employed to reduce resazurin solu-t i o n s , and the r e s u l t i n g resorufin extracted with ether. This eliminated both residual ascorbic acid and i t s degradation products from the r e s o r u f i n solution. Ether extraction could, however, not be used i n the present instance. Because c a t a l y s i s could have been due to an a r t i f a c t , i t was e s s e n t i a l to test the entire dye solution rather than just the ether soluble por-tio n s . The data i n table 8 i l l u s t r a t e that ascorbate degrada-t i o n products, at a concentration greater than that occurring i n the r e s o r u f i n solution, had no influence on the reducing system of milk nor on the measurements made on this system. For preparation of r e s o r u f i n , ascorbate was added to resazurin - 5 1 -solution at 5 times the dye concentration. The system was evacuated and held at 37°C f o r 20 minutes, aft e r which, r e s -idual ascorbate was destroyed by aeration. Spectrophotometrie examination showed this solution to contain 0 . 1 2 jj.M r e s o r u f i n and no resazurin. Since no f r a c t i o n a t i o n was applied, the solution should s t i l l have contained any impurities present i n the o r i g i n a l dye. TABLE 8 INFLUENCE OF REDUCED DYE ON THE REDUCING SYSTEM Reducing Capacity Resazurin Sample (uM ascorbate) (u.M) 5 . 0 ml milk, 0 . 5 ml H2O 0 Time 120 Mins. 0 Time 120 Mins. Control . 7 9 . 7 6 + . 1 2 ^M resazurin . 78 . 5 3 . 12 . 0 6 1 + .12 JULM r e s o r u f i n . 7 9 .77 -+.7 JUM dehydroascorbate . 7 9 . 7 5 +.7 >»M dehydroascorbate + .12 u.M resazurin .78 . 5 2 .12 . 0 5 9 Reducing capacity and resazurin were measured as described previously. As shown by the data i n table 8 , reduced dye' had no a b i l i t y to decrease the reducing capacity of milk. This indicated that the ascorbic acid treatment must have reduced something i n addi-t i o n to resazurin since resazurin alone could not have been r e s -ponsible" f o r the c a t a l y t i c influence on the reducing system (figure 1 1 ) . The dye preparation must, therefore, have contained some a r t i f a c t that inactivated the reducing capacity of milk - 5 2 -but was i t s e l f i n activated by ascorbic acid treatment. E. S i l i c i c Acid Treatment of Resazurin A s i l i c i c acid column was employed i n an e f f o r t to puri f y resazurin. One commercial resazurin tablet was ground i n a mortar with repeated 10 ml volumes of ethyl acetate and s u f f i -cient 0.01 N HCl to maintain a pH below 5 . 0 . The low pH was necessary f o r extraction of resazurin by ethyl acetate. The extraction was stopped when approximately 100 ml extract had been obtained, though only a portion of the resazurin had been dissolved. The extract was concentrated to approximately 2 ml and layered on top of a small s i l i c i c acid column. The column had been packed i n petroleum ether and washed with approximately 2 bed volumes of ethyl acetate. Development was with ethyl acetate, which separated the extract into four bands. Three of these were eluted with ethyl acetate and the fourth with a 50/50 mixture of ethyl acetate/n-butanol. The f r a c t i o n s were numbered i n the sequence of t h e i r e l u -t i o n from the column. Water was added to each before evapora-t i o n of solvent and the aqueous solutions concentrated to 5«0 ml. Both f r a c t i o n s 1 and 2 were insoluble i n aqueous media or i n d i l u t e sodium bicarbonate. They appeared as o i l s heavier than water and were present at approximately 0 .2 ml each. Frac-t i o n 1 was a muddy grey color and f r a c t i o n 2 , a bright f l u o r e s -cent red, reminiscent of a very concentrated solution of res-or u f i n . Fractions 3 and \+ were completely soluble i n water and both appeared blue, f r a c t i o n 3 having much les s color than 1+. Spectra of a l l fract i o n s were obtained i n butanol saturated -53-with sodium bicarbonate under the conditions employed f o r spectrophotometric measurement of resazurin. Volumes of the solutions described added to 9.0 ml butanol were: f r a c t i o n 1, 5.0 ml; f r a c t i o n 2, 1.0 ml; f r a c t i o n 3 , 3.0 ml; f r a c t i o n II, 1.0 ml. The spectra of fract i o n s 1 , 2 and Ix are shown by f i g -ure 13. The o p t i c a l densities of f r a c t i o n 3 were too low to plot on t h i s scale. I t had one maximum coinciding with that at 580 mu, shown for f r a c t i o n 2. I t i s obvious, on comparison of these spectra with those previously obtained f o r resazurin and r e s o r u f i n (figure 1 ) , that f r a c t i o n Ix was resazurin. Calculation of the quanity of resazurin present showed that 1.0 ml contained 0.12^uM, making f r a c t i o n 1+ half as concentrated as the resazurin solution usual-l y employed. Though f r a c t i o n 2 had a fluorescent red color, i t was not r e s o r u f i n . A r e s o r u f i n solution having o p t i c a l density of 0*150 at i|75 mp. would have had a very strong absorbance at 582 mu.. A l l f r a c t i o n s were tested f o r t h e i r a b i l i t y to catalyse the decomposition of the reducing capacity of milk. Results are shown i n table 9. I t i s evident from these data that f r a c -t i o n 1+ was much more active than any of the other f r a c t i o n s . Though i t was present at only half the concentration of un-p u r i f i e d resazurin, i t s e f f e c t was almost as great. This f r a c -tion represented quite an extensive p u r i f i c a t i o n of resazurin and most a r t i f a c t s should have been removed from i t . It would appear, therefore, that the dye i t s e l f affected the reducing system. This i s at variance with the r e s u l t s obtained using . 6 0 0 Wavelength (ran,) Figure 13 Spectra of Resazurin fractions in butanol saturated with sodium bicarbonat -51+-reduced dye. However, i t i s possible that an active form of the dye, possibly a metal chelate, was ina c t i v a t e d , even under the extremely mild conditions employed for reduction i n the experiment with reduced dye. TABLE 9 INFLUENCE OF RESAZURIN FRACTIONS ON REDUCING CAPACITY Reducing Capacity (^ M ascorbate) % Remaining Sample afte r (5.0 ml milk) 0 Time 120 Mins. 120 Mins Control (0.5 ml H 2 O ) . 6 5 . 5 8 93 F r a c t i o n 1 (0.3 ml) .62 .14.9 79 F r a c t i o n 2 (0.3 ml) .62 .1+7 7 5 Fraction 3 (0.3 ml) .61 .53 8 5 F r a c t i o n 1+ (0.036 )W) .63 .39 62 Resazurin ( 0 . 7 2 ^M) .63 .36 5 7 F. Reducing Capacity of Milk Fractions In experiments on rates of resazurin reduction, i t was found that f r a c t i o n s of normal milk d i f f e r e d i n t h e i r a b i l i t y to reduce dye. The present experiment was designed to deter-mine whether or not t h i s trend persisted i n raastitic samples and how i t was re l a t e d to t o t a l reducing capacity. Results are presented i n table 10. Whole milk ( 8 0 ml) was centrifuged and the f a t , combined with sediment, made back to 30 ml with whole milk. This produced a f r a c t i o n enriched i n fat and any s o l i d residues i n the milk, but having a lower f a t content than gravity cream. Resazurin ( 0.12jnM / 0 . 5 ml) or water ( 0 . 5 ml) was added to 5 . 0 ml of each f r a c t i o n before incubation at 37°C. - 5 5 -TABLE 10 REDUCING CAPACITY OF MILK FRACTIONS Mast i t l c Milk Normal Milk Sample Reducing y Capacity Resazurin * Reducing Capacity Resazurin {jm Ai 3cprbate) (MM) (JAM. Ascorbate) . (MM) Time Time Time Time (Minutes) (Minutes) (Minutes) (Minutes) 0 60 0 60 0 60 0 60 whole + H20 .76 • 77 .61 whole +- resazurin .75 .79 .12 0 .61 .35 .12 .079 skim • + H20 .51 •kk .66 .53 skim + resazurin .51 .32 .12 .089 .65 .38 .12 .091 f a t + p e l l e t + H 20 .80 .81 .53 .1+7 f a t + p e l l e t +- resazurin .79 .80 .12 0 .53 .31+ .12 .072 ^Resazurin reduction was very ra p i d , the whole and f a t + p e l l e t samples reaching the 7/l| standard i n 10 to 15 minutes: at 30 minutes, the f a t +• p e l l e t samples were white, and whole samples were approximately 50% white. - 5 6 -Resazurin and reducing capacity were determined as described previously. In t h i s experiment, reducing capacity of the mastitic milk was abnormally high, and that of the normal milk low. Despite t h i s , the data i n table 10 i l l u s t r a t e some s i g n i f i c a n t points. The resazurin reducing a b i l i t y of the mastitic sample was almost completely i n the combined fat and p e l l e t f r a c t i o n and was l a r g e l y i n s e n s i t i v e to added resazurin, only that of the skim being inactivated by added dye. This substantiates, to some extent, the e a r l i e r observation that the free ascorbate of milk was probably the component of the reducing system i n a c t i -vated by resazurin. The reducing capacity of a l l the f r a c t i o n s of normal milk was affected by added resazurin and a l l f r a c t i o n s reduced the dye to some extent, as predicted from e a r l i e r experi-ment s. G. Ascorbic Acid Oxidase In early experiments employing v i s u a l extimates of re s -azurin reduction, i t was observed that treatment of milk with ascorbic acid oxidase increased the time needed for samples of normal milk to become reduced to the 7 / 4 color standard but had no ef f e c t on rapidly reducing milks. I t was found at that time that enzyme treatment oxidized a l l the t i t r a t a b l e ascorbate i n both normal and abnormal milks. The ef f e c t of ascorbic acid oxidase treatment on the reducing capacity and t i t r a t a b l e ascor-bic acid content of fractions of mastitic milk i s shown i n table 1 1 . The f a t and p e l l e t obtained from centrifuging 1+0 ml whole milk were each made to 5 * 0 ml with skim or powdered skim - 5 7 -milk. Ascorbic acid was measured by indophenol t i t r a t i o n i n metaphosphoric acid-acetic a c i d , and reducing capacity by the method employed i n previous experiments. The volume of a l l samples was 5«0 ml. Enzyme treatment was f o r 15 minutes at 3 7 ° C TABLE 11 Sample INFLUENCE OF ASCORBIC ACID OXIDASE  ON REDUCING CAPACITY Reducing Capacity T i t r a t a b l e Ascorbate (yM Ascorbate) C U M ) I n i t i a l Values Enzyme Treated I n i t i a l Values Enzymi Treate whole .27 . 2 2 . 0 5 skim .30 .07 . 2 6 . 0 5 f a t + skim .61+ .57 .18 . 0 5 f a t +- powder* . 5 9 . 5 3 . 0 7 . 0 5 p e l l e t + skim . 6 6 . 6 2 . 2 0 . 0 5 p e l l e t + powder* . 6 2 . 6 2 . 15 . 0 5 *Blank values f o r powdered skim milk subtracted. Inspection of the i n i t i a l values i n table 11 shows that the concentration of t i t r a t a b l e ascorbic acid was much lower than the reducing capacity i n a l l f r a c t i o n s , except skim. In this f r a c t i o n , the two measurements agreed quite c l o s e l y . In addition, the reducing capacity of skim was la r g e l y eliminated on treatment with enzyme, ind i c a t i n g that ascorbate was the major component of the reducing capacity of t h i s f r a c t i o n . In the remaining f r a c t i o n s , enzyme treatment removed almost half -58 -the reducing capacity of whole milk but had l i t t l e e f f e c t on samples derived from f a t or p e l l e t . In contrast to t h i s , the enzyme removed e s s e n t i a l l y a l l the t i t r a t a b l e ascorbic acid from a l l f r a c t i o n s . A number of unsuccessful attempts were made to i d e n t i f y ascorbic acid i n these f r a c t i o n s by paper chromatography. Since ascorbate must c e r t a i n l y have occurred i n the skim, the negative r e s u l t s meant merely that the tech-niques used were inadequate. The p o s s i b i l i t y that the portion of the reducing capacity that could not be accounted for as ascorbate was due to free sulfhydryl groups was investigated. A l l f r a c t i o n s of the mastitic milk produced a s l i g h t pink color with the nitroprusside reagent, while similar f r a c t i o n s from normal milk remained white. Since mastitic skim produced as much color with the reagent as did the f a t and p e l l e t f r a c t i o n s , i t was concluded that the differences between f r a c t i o n s noted i n figure 11 could not have resulted from presence and absence of free sulfhydryl groups. - 5 9 -GENERAL DISCUSSION I INTERACTION OF RESAZURIN WITH REDUCING SYSTEM The reducing capacity of milk was inactivated by the amounts of resazurin normally employed i n milk t e s t i n g . The half l i f e of the r e l a t i v e reducing capacities of milk and milk plus resaz-u r i n were 576 and 155 minutes respectively (figure 1 1 ) . Resaz-u r i n was also found to decrease the s t a b i l i t y of ascorbic acid i n buffered solutions containing EDTA (figure 12). This i n f l u -ence of resazurin on the reducing capacity of milk was also noted by Johns (6) who found a r i s e i n Eh of approximately 0.06 vo l t over a 5 hour i n t e r v a l i f resazurin was present i n low l e u -cocyte milk. Campbell, Phelps and Keur (28) showed that a loss of 12 mg/litre reduced ascorbic acid resulted i n a r i s e of approximately 0 . 0 7 v o l t i n the aerobic p o t e n t i a l of milk. On the basis of the s i m i l a r i t y of the reducing capacity curves i n figures 11 and 12 and of the supporting observations c i t e d , i t was concluded that the component of the measured reducing capa-c i t y that was unstable to the presence of resazurin was reduced ascorbate. Comparison of resazurin reduction rates with those for r e -ducing capacity i n figure 11 shows an obvious c o r r e l a t i o n between decrease i n rate of resazurin reduction and i n a c t i v a t i o n of r e -ducing capacity. However, t h i s was probably not a cause and ef f e c t r e l a t i o n s h i p , f o r the amount of ascorbate present i n the milk was only 0 . 5 8 j^ M and i t was found that i n buffered solu-tions of ascorbate even 0 . 7 y(K would not bring about reduction of resazurin (figures 11 and 1 2 ) . Employing v i s u a l estimates of resazurin reduction, i t was found that treatment of milk -60-with ascorbic acid oxidase, which removed a l l of the t i t r a t a b l e ascorbate, had l i t t l e influence on the resazurin reduction time of normal milk and no influence on the reduction time of milk that could reduce the dye i n less than 2 hours. I t i s therefore apparent that the concentrations of ascorbate present i n milk do not influence the rate of resazurin reduction under the nor-mal aerobic conditions employed i n milk t e s t i n g , i n spite of the f a c t that ascorbate i s oxidized during t h i s period and that this oxidation i s stimulated by resazurin. It i s doubtful that the 0.06 volt r i s e i n Eh which probably accompanied the loss of ascorbate i n figure 11 was responsible for the decreased rate of resazurin reduction. Johns (6) found the same rate of reduction i n aerated samples as i n unaerated controls, concluding that the aerobic p o t e n t i a l had no i n f l u -ence on resazurin reduction i n milk. This had also been sug-gested by the observations of Ramsdell et a l . (3), that a single shade of resazurin color could exist over a wide range of Eh values i n milk. The foregoing discussion should not be construed as mean-ing that resazurin reduction i s independent of electrode poten-t i a l . In milk, the measured aerobic potential has no influence on reduction but resazurin becomes reduced to re s o r u f i n immedi-ately on exposure to the anaerobic p o t e n t i a l which i s approxi-mately O.ij. vol t lower, as determined a f t e r flushing milk with nitrogen (26). A similar e f f e c t i s noted on removal of oxygen from solutions containing 5 to 10 mg/litre ascorbate, which i s not s u f f i c i e n t to reduce resazurin aerobically but w i l l reduce -61-i t iraraediately anaerobically. It i s therefore obvious that reduction of resazurin i s dependent on Eh. In t h i s i n v e s t i g a -t i o n , no e f f o r t was made to measure rates of reduction at f i x e d Eh values, i t being assumed that an i r r e v e r s i b l e , Eh-dependent reaction would plot as spontaneous decay with rate dependent on applied p o t e n t i a l . Since the change from resazurin to resorufin i s i r r e v e r s i b l e under p h y s i o l o g i c a l conditions, reduction can occur i n additive increments to produce a progressive rate of color change, i n an ambiant Eh at which the dye should be completely stable. If zones of low Eh occurred at the surfaces of p a r t i c l e s i n the milk, such as ba c t e r i a , leucocytes, or fat globules; any dye di f f u s i n g into these zones would become reduced and, on d i f f u -sion back into the high Eh of the milk plasma, would remain r e -duced. I t i s apparent that continuation of t h i s process would re s u l t i n a measurable reduction of t o t a l dye depending on num-ber and Eh of zones and the rate of d i f f u s i o n of dye through them. II STABILITY OF REDUCING SYSTEM The experiments on aging,, demonstrate that the resazurin reduction observed with fresh milk resulted from the normal reducing systems present rather than from b a c t e r i a l a c t i v i t y (figure 5 ) . In addition some questions with regard to the l o c a -t i o n and s t a b i l i t y of the reductants are rai s e d . Milk 1 produced a resazurin/resorufin r a t i o of 0.9 a f t e r 1 hour incubation with dye at S7°C. According to accepted views, t h i s milk would be c l a s s i f i e d as a rapid resazurin reducer and hence, the product -62-of an unhealthy udder. Unfortunately, leucocytes were not counted i n these samples but, on the basis of the rapid resaz-u r i n reduction, they should have been quite numerous (7). If reduction had been due to leucocytes, there should have been a more rapid decrease i n rate of reduction on storage, p a r t i c -u l a r l y at the higher temperature, the current view being that preincubation at 12.8°C for 18 hours larg e l y dissipates any reducing a c t i v i t y due to leucocytes (1). Contrary to t h i s , there was l i t t l e change i n the rapid reducer on storage and very l i t t l e influence of temperature of storage. Milk 2, on the other hand, was nearer what would be termed a normal sample but l o s t reducing a b i l i t y more rapidly on storage and was more sensitive to temperature of storage. This trend was r e f l e c t e d to a greater degree i n the skims from these two milks, which had most of the leucocytes centrifuged o f f . This type of be-havior was not unique i n the two milks presented i n figure 5 , but occurred i n a l l of the samples tested, which included some mixed milks. However, the number of samples tested was not s u f f i c i e n t to state that the behavior was general f o r milk. The reducing system i n these milks was obviously not b a c t e r i a l and, from the behavior of the skims, probably not leucocytes alone. From the difference i n reduction rates be-tween whole and skim samples, i t would appear that the reducing system was associated with the fat or sediment and was s t a b i l -ized by this association. I f , as suggested e a r l i e r , resazurin reduction occurred i n association with some structure i n the milk, i t i s l o g i c a l that skim would have reduced dye more slowly - 6 3 -than whole milk, and normal milk more slowly than high leuco-cyte milk. I t had been found i n early experiments that d i l u t i o n of milk with buffer or water greatly slowed the r a t e of resazurin reduction and that washed cream lo s t a l l i t s a b i l i t y to reduce dye. Leucocytes have been reported to lose a large percentage of t h e i r reducing ,capacity on washing i n saline or water (2,]+,6), and even on washing i n milk ( 8 ) . The tendency of milk to lose reducing a c t i v i t y on d i l u t i o n leads to the suggestion that the reducing capacity i s loosely adsorbed i n the form of diffusable chemical reducing agents whose concentration determines the Eh of the surfaces of the structures they occur on. Considered i n the l i g h t of the e a r l i e r discussion on the possible mechanism of resazurin reduction i n milk, t h i s suggestion appears more plausible than the removal of cofactors of enzyme reactions, which has been advanced as an explanation f o r the loss of a c t i v -i t y of washed leucocytes (31). I f i t i s assumed that resazurin i s reduced by chemical agents adsorbed to p a r t i c l e s i n the milk, the decreased s t a b i l i t y of the skim samples of figure 5 merely r e f l e c t s an increased rate of d i f f u s i o n of reducing agent from the surfaces of any remaining p a r t i c l e s . The fat globules and leucocytes remaining i n the skim would not be protected by the mutual aggregation which occurs i n whole milk at lower tempera-tures (32) and would, therefore, probably tend to lose adsorbed constituents more r e a d i l y . I l l MATURE OF REDUCING SYSTEM The quantity of indophenol which would be decolorized by -61+-various d i l u t i o n s of a sample of milk could be calculated from the value obtained f o r the inta c t sample (table 7 ) . The r e -action of milk with indophenol was extremely r a p i d , appearing complete on mixing, and produced the same values f o r reducing capacity at 0°C as at 37°C. Because of the quantitative nature and speed of t h i s reaction, i t was concluded that the dye gained electrons from some r e a d i l y available source i n the milk rather than through mediation of some enzyme reaction. I t was shown by comparison with ascorbic acid t i t r a t i o n that reduced ascor-bate comprised approximately 80 percent of t h i s electron source. The remaining 20 percent remained unaccounted f o r , and i t i s probable that t h i s was a chemical reducing substance with a be-havior toward indophenol and oxygen analogous, i n many respects, to that of reduced ascorbate. I t has been shown that human leucocytes and blood p l a t e l e t s contain 290 to 1+30 mg/Kg bound ascorbic acid i n a weakly d i s -sociable complex (33). Extrapolation of t h i s i n t e r r e l a t i o n -ship to bovine blood produces a hypothetical p a r t i c l e with a l l the attributes i n f e r r e d , i n the e a r l i e r discussion, to be char-a c t e r i s t i c of the resazurin reducing system of milk. In addi-t i o n to posession of the c h a r a c t e r i s t i c s postulated f o r the reducing system, these p a r t i c l e s have an incidence of occur-rence and l o c a t i o n i n milk which correlates well with rate of resazurin reduction. The observation of Campbell and Phelps (8), that leucocytes i s o l a t e d from milk regained th e i r reducing a b i l i t y on suspension i n bovine plasma, also f i t s t h i s picture since leucocytes depleted of ascorbate are known to take i t up -65-rapidly from blood plasma (3J4.) . With the exception of t h e i r behavior i n the presence of ascorbic acid oxidase, these hypothetical p a r t i c l e s , considered from the point of view of the proposed mechanism of resazurin reduction, can be used to explain a l l of the observations made i n t h i s i n v e s t i g a t i o n and most of those recorded i n the l i t e r a -ture concerning the p e c u l i a r i t i e s of resazurin reduction. The fact that i t was not possible to account for t h i s part of the reducing capacity as ascorbate by t i t r a t i o n i n metaphosphoric acid i s i n favor of the existence of the p a r t i c l e s . B u l l e r and Gushman (33) found they could not t i t r a t e the ascorbate i n t h e i r preparations i f they p r e c i p i t a t e d with metaphosphoric acid with-out f i r s t saturating the system with carbon monoxide. They attributed the loss of ascorbate to immediate oxidation by hemoglobin from laking of the few red blood c e l l s present. It has recently been shown that the blood i n vat samples of milk ranged from 1 to 15 rag/litre (35). Since the quantity of ascor-bate affected was only approximately I4, mg/litre i n normal milk, i t i s probable that there was s u f f i c i e n t hemoglobin present to cause i t s rapid oxidation on treatment with metaphosphoric acid. The resistance of the residual reducing capacity to ascor-bic acid oxidase i s more d i f f i c u l t to r a t i o n a l i z e . B u l l e r and Cushman (33) reported complete destruction of the ascorbic acid i n p l a t e l e t s and leucocytes i n the presence of enzyme, but gave no experimental d e t a i l s of t h i s phase of t h e i r work. On more careful examination of the data i n table 11, i t i s evident that the loss i n t i t r a t a b l e ascorbate was p a r a l l e l l e d by a comparable - 6 6 -loss i n reducing capacity i n a l l but the p e l l e t f r a c t i o n s . I t i s possible that the constituents of t h i s f r a c t i o n s t a b i l i z e d some acid l a b i l e intermediate generated by the enzyme. Though t h i s p o s s i b i l i t y i s admittedly remote, the ascorbic acid oxidase data was the only evidence found which was contrary to the pro-posed structures postulated as instrumental i n resazurin reduc-t i o n . -67-SUMMARY AND CONCLUSIONS Spectrophotometry methods were developed to quantitatively measure resazurin reduction and the reducing capacity of milk. Reducing capacity was assessed as the quantity of indophenol reduced by milk at i t s normal pH. The method could be used i n the presence of resazurin. These methods were employed singly and i n combination to study i n t e r a c t i o n of resazurin with the reducing system of milk. Resazurin was shown to have a d e s t a b i l i z i n g influence on reducing capacity. This influence was c a t a l y t i c and dependent on t o t a l concentration of dye; rate of i n a c t i v a t i o n being con-stant f o r a given dye concentration. Evidence was presented to show that the component of the reducing capacity that was inactivated was ascorbic acid. I t was concluded from these investigations that the reduc-ing system of fresh milk existed as a measurable en t i t y at any given time rather than as a continuous evolution of electrons from some enzymatic reaction. This system consisted of the measurable ascorbic acid of the milk, which occurred i n the plasma, and some reducing agent bound to st r u c t u r a l components of the cream and sediment. The measurable ascorbic acid account-ed for approximately 80 percent of the reducing capacity but i t was concluded to have l i t t l e influence on resazurin reduction. The bound reducing agent apparently depended on st r u c t u r a l e l e -ments i n the milk f o r i t s a b i l i t y to reduce resazurin, and that i t l o s t this a b i l i t y on d i s s o c i a t i o n from the p a r t i c u l a t e struc-ture. I t was postulated that t h i s reducing agent was ascorbate - 6 8 -and that i t occurred bound to leucocytes and other c e l l u l a r debris i n the milk i n situations analogous to i t s reported occurrence i n blood. Attempts to i d e n t i f y t h i s reducing agent as ascorbate were unsuccessful i n this i n v e s t i g a t i o n , but the techniques employed were probably inadequate. - 6 9 -BIBLIOGRAPHY 1 . Johns, C.K. Applications and l i m i t a t i o n s of quality-tests f o r milk and milk products. A review. J . Dairy S c i . jx2, 1625-1650 ( 1 9 5 9 ) . 2 . Nilsson, Gerda. Reducing properties of normal and abnormal milk and t h e i r importance i n the b a c t e r i o l o g i c a l grading of milk. Bact. Rev. 23, kl-kl ( 1 9 5 9 ) . 3 . Ramsdell, G.A., Wm.T. Johnson J r . and F.R. Evans. Investigation of resazurin as an in d i c a t o r of the sanitary condition of milk. J . Dairy S c i . 1 8 , 705-717 ( 1 9 3 5 ) . I 4 . . Strynadka, N.J. and Thornton, H.R. Leucocytes and the methylene blue reduction test. J . Dairy S c i . 2 1 , 5 6 1 - 5 6 8 (1938). 5 . Johns, C.K. and R.K. Howson. Potentiometric studies with resazurin and methylene blue i n milk. J . Dairy S c i . 23, 295-302 (19i|.0). 6 . Johns, C.K. Some aspects of the resazurin t e s t . 1 5 t h Ann. Rept. New York State Assoc. Dairy and Milk Insp., 1 7 3 - 1 8 5 (191+1). 7 . McBride, C.A. and Golding, N.S. A study of resazurin reduction i n freshly drawn raastitic-like milk. J . Milk and Pood Technol. lij., 27-30 ( 1 9 5 1 ) . 8 . Campbell, J.J.R. and Phelps, R.A. Role of leucocytes i n the reduction of resazurin i n raw milk. J . Dairy S c i . 2jj3, 187-192 (I960). 9 . Campbell, J.J.R. and Keur, Lynette B. Role of xanthine oxidase i n the reduction of resazurin by raw milk. J . Dairy S c i . '1LZ$-IL2<) ( 1 9 6 1 ) . 1 0 . Nilsson, Gerda. Studies concerning the reducing pro-perties of milk. The reducing systems of milk obtained under aseptic conditions from healthy and masti-tic cows. Ann. Roy. Agr. C o l l . Sweden 23, 73-122 ( 1 9 5 7 ) . 1 1 . Standard Methods fo r the Examination of Dairy Products. American Public Health Assoc., Inc., New York, 1 1 t h ed., 1114.-118 (I960). 1 2 . O f f i c i a l Methods of Analysis of the Association of O f f i c i a l A g r i c u l t u r a l Chemists. Ass'n of O f f i c i a l A g r i c u l t u r a l Chemists, Washington, D.C., 9 t h ed., 661 (I960). -70-12. Sharp, P.P. Rapid method f o r the quantitative deter-mination of ascorbic acid i n milk. J . Dairy S c i . 21, 85 (1938). 11+. Block, R.J. , E.L. Durrum, and a . Zweig. Paper Chromato-graphy and Paper Electrophoresis, Academic Press, Inc., New York, 2nd ed. , 173-171+, 1+07-1+08 (1958). 15. Kohman, E.P. and Sanborn, N.fl. Vegetal reduction of dehydroascorblc acid. Ind. and Eng. Chem. 29, 1195 (1937). 16. Sharp, P.P., D.B. Hand, and E.S. Guthrie. Quantitative determination of dissolved oxygen: ascorbic a c i d oxidase method. Ind. and Eng. Chem. (Analytical Edition) 13 , 593 (19i+l). 17. Patton, S., and D.V. Josephson. Observations on the app l i c a t i o n of the nitroprusside test to heated milk. J . Dairy S c i . 32, 398-1+05 (191+9). 18. Twigg, R.S. Oxidation-reduction aspects of resazurin. Nature 15£, 1+01-1+03 (191+5). 19. Lardy, H.A. ed. Respiratory Enzymes. Burgess Pub. Co., Minniapolis 15, Minn., 8l (igi+9). 20. De Baun, R.M. and de Stevens, G. On the mechanism of enzyme action XLIV. Codetermination of resazurin and resorufin i n enzymatic dehydro-genation experiments. Arch. Biochem. Biophys. ! 31, 300-308 (1951). 21. Johns, C.K. Dye concentration of resazurin t a b l e t s . A.J.P.H. 3 4 , 955 (19I+8). 22. Merck Index, Merck and Co., Inc., N.J., 6th ed., 829 (1952). 23. Umbreit, W.W., B u r r i s , R.H. and Stauffer, J.P. Manometric Techniques, Burgess Pub. Co., Minniapolis 15, Minn., 3rd ed., 229 (1959). 21+. Durrans, T.H. Solvents.' Chapman and H a l l , London, 7th ed., 102 (1957). 25. Johns, C.K.' The behavior of resazurin i n milk. Can. J . Res. 20, 336-31+6 (191+2). - 7 1 -26. Jenness, R. and Pattern, S. P r i n c i p l e s of Dairy Chem-i s t r y . John Wiley and Sons, Inc., New York, 235-237, 2 6 5 - 2 8 2 ( 1 9 5 9 ) . 2 7 . Hewitt, L.F. Oxidation-Reduction Potentials i n Bacteriology and Biochemistry. E. and S. L i v i n g -stone Ltd., 6 t h ed., Edinburgh, 21, 27 ( 1 9 5 0 ) . 2 8 . Campbell, J.J.R, R.H. Phelps, and Lynette B. Keur. Dependence of oxidation-reduction p o t e n t i a l of milk on i t s vitamin C content. J . Milk and Food Technol. 22, 31+6-31+7 ( 1 9 5 9 ) . 2 9 . B a l l , E.G. Studies on oxidation-reduction: XXIII ascorbic acid. J . B. C. 118, 219 (1937). 30. Rao, M.V., Sastry, L.V.L., Srinivasan, M., and Subrahmanyan, V. I n h i b i t i o n of oxidation of ascorbic acid by EDTA. J . S c i . Food Agr. 10, i+S6-i+i+l ( 1 9 5 9 ) . 31. Davis, J.G. The resazurin t e s t , Dairy Indust. 5 , 18-21 (191+0). 32. King, N., The Milk Fat Globule Membrane, Commonwealth Agric. Bureau, Farnham Royal, Bucks, England, 7 8 - 8 0 ( 1 9 5 5 ) . 33. B u l l e r , M.A., and Cushman, Margaret. An ascorbic acid l i k e reducing substance i n the buffy layer of centrifuged oxalated blood. J . B. C. 139, 219-226 (191+1). 31+. West, E.S. and Todd, W.R. Textbook of Biochemistry. Macmillan, New York, 2nd ed. , 77I-78O ( 1 9 5 5 ) . 35. Ramraell, C.G. The estimation of blood i n bovine milk. J . Dairy Res. 28, 131-138 (1961). 

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