The estimation of 2,b-diaminopimelic acid in digesta

221

Summary Olaquindox - a new growth promoting feed additiv. I. The effect on piglet rearing For the first time 5 trials involving 305 piglets have been conducted during the last 4 years to examine the effect of Olaquindox, a new feed additiv. The hygienic and nutritional conditions had the optimal standard known today. Liveweight gain and feed conversion were tested. Althoug using different housing systems the performance values of the negative controlgroups were reproducible. Irrespective of different housing systems dietary concentrations of 25 up to 100 ppm Olaquindox improved partly significantly - liveweight gain in all trials. Olaquindox in a dosage of 50 ppm in the feed given to early weaned piglets of 5-1 1 kg liveweight produced an improvement of 18 O/O in daily liveweight gain and 5 O/O in feed conversion; 100/120 ppm an increase of 20 O/o respectively 7 o/o (mean values of all trials). Growing piglets of ca. 11-25 kg liveweight receiving 50160 ppm Olaquindox showed an increase of 12 O / o in liveweight gain. Feed conversion was not effected. I n 2 trials Olaquindox has been found to be as effective as Carbadox. The results of a comparison trial with Zincbacitracin showed the superiority of Olaquindox. Literatur 1. METZGER, K., 1974: Bay Va 9391 a new antibacterial agent. 1st Intersectional Congress of the Internar. Assoc. of Microbiological Societies, Tokyo, p. 75. - 2. MENKE,K. H.; KRAMPITZ, G., 1973 : Antibiotikawirkungen in nutritiver Dosierung. Ubersichten zur Tiercrniihrung, 1, 255-272. - 3. BRAUDE, R., 1974: Personliche Mitteilung. - 4. RAYNAUD, J.-P., 1974: Evaluation on 5500 young pigs of Carbadox used at 50 ppm. Economic interpretation in reference with performances of control animals or supplements used at nutritional (< 150 ppm) or therapeutic (>200 ppm) levels. Ztschr. f. Tierphysiol., Tierernahrg. u. Futtermittelkde. 32, 249-278.

Anschrifi der Autoren: Institut fur Tierzucht und Tierernahrung, 1000 Berlin 33, Briimmer-

strage 34

Department of Animal Physiology and Biochemistry, Copenhagen

The estimation of 2,6-diaminogimelic acid in digesta and faeces using acid ninhydrin reagent By V. C. MASONand S . BECH-ANDERSEN Receipt of M s . 20. 6. 71

Introduction 2,6-Diamin~opimelic acid (DAPA) is a constituent of the cell-wall mucopeptide of many species of bacteria (WORK& DEWEY1953) and it has been found in the bluegreen and green algae (WORK& DEWEY 1953; FUJIWARA & AKABORI 1954). It is not 2. Tierphysiol., TierernShrg. u. I:uctermictelkde. 36 (1976), 221-229 @ 1976 Verlag Paul Parey, Hamburg und Berlin

ISSN 0044-3565

1 ASTM-Coden: ZTTFAA

222

V . C. Mason and S . Bech-Andersen

present in the higher plants (SYNGE1953), o r in protozoa (WORK DEWEY1953; WELLER et al. 1958; VIRTANEN1967) except in trace amounts which may be due to contaminating bacteria (HUTTONet al. 1971). Muscle and liver tissue and cells of the succus entericus of sheep are devoid of this amino acid (MASON,unpublished) and sheep urine contains only traces of D A P A even though D A P A infused into the blood of these animals is excreted almost quantitatively in the urine (MASON& WHITE 1971). T h e presence of D A P A in several bacteria isolated from the intestinal digesta of farm animals (SYNGE1953) has led to its use as a marker in studies of nitrogen metabolism by bacteria in the alimentary tract of ruminant animals (WELLERet al. 1958; EL-SHAZLYa HUNGATE 1966; MASON1969; MASON8r WHITE1971; HUTTON et al. 1971). O n e of the problems associated with this technique is that the concentration of D A P A in hydrolysates of digesta and faeces is usually low relative to that of the more common amino acids, with the result that very good separations of this amino acid from the other amino acids are necessary if a high degree of accuracy is to be attained. T o achieve these separations both iton-exchange (WELLERet al. 1958) and gas chromatographic methods (SEN et al. 1969) have been employed. Ion-exchange methods have the advantage of requiring fewer preparatory steps than the gaschromatography methods, but as generaIly applied, using ninhydrin buffered to p H 5.5 (MOOREei STEIN1954), the former methods are slower 00 achieve a separation on thec olumn. Many of the disadvantages of the ion-exchange methods can be overcome, however, if acid ninhydrin reagent is used. Under these conditions D A P A molecules condense directly with ninhydrin (CHINARD1952) to fmorm a yellow product which can be measured a t 440 m p (WORK 1957). This reaction is shared by relatively few other amino acids (WORK1957), and permits the use of a more rapid short column system for D A P A determinations. Manual techniques based on this principle have been described by EL-SHAZLY HUNGATE (1966) and MASON(1969). A semi-automated Technicon amino acid analyzer procedure has also been reported by HUTTON et al. (1971), but suffers from the fact that the system must be recalibrated daily owing to the instability of the manifold tubing. T h e present paper describes a semi-automated method, involving simpler equipment, which avoids this problem.

Materials and methods a.

Equipment

T h e apparatus and materials used for this analysis include an ion-exchange resin (Amberlite IR 120/AS, particle size 28-35 p, Serva, W. Germany), a buffer pump (Accu-Flo Pump, Spinco Div., Beckman Instr. Inc., USA), a ninhydrin pump (MicroPump 2-6000, Biichler Instr. Inc., USA) a reaction coil (tephlon tubing, 23 m long, 1.1 mm internal diameter), a photometer and a recorder. Most of the early studies were performed using an LKB Multichannel Absorptiometer (5901A, LKB Products A/B, Sweden) fitted with a 3 mm cuvette and connected to a slightly modified Philips recorder (PR 3210A/100. Philips, Holland).

The estimation of 2,6-diaminopimelic acid in digesta

223

This apparatus was later replaced by a combination of a Vitatron U P M Universal Photometer (Vitatron, Holland) fitted with a Helma No. 1 7 8 - 0 s cuvette (pathway 10 mm), and a 12 channel Philips Multipoint recorder (PM 8235) fitted with the universal measuring range unit (PM 9833). T h e recorder was slightly altered by replacing the variable restistance (5M), which regulates the print interval, with a fixed resistance (107K) to give a print interval of 9.75 seconds. T h e photometer and recorder were connected in such a way that 6 of the recorder channels measure the 10 mv output from the photometer, the remaining 6 channels recording the 100 mv cutput. With this system the amino acid peaks are plotted on a linear scale, and different measuring ranges can be selected. T h e resin bed is contained in a jacketed column and is 15 cm long and 0.9 cm in diameter during operation. Its temperature is maintained at 50° C. A simple bubble trap is incorporated in the ninhydrin line between the ninhydrin reservoir and the Buchler pump. A pulse dampener, consisting of a simple glass side arm 30 cm long and 0.5 cm in internal diameter, is located in the ninhydrin line just beyond the Buchler pump. This side arm contains 3-4 ml air during operation, its blind end being sealed by a tight tephlon cock. T h e eluate and ninhydrin streams meet a t a T-junction consisting of a 3-way cock. Due to the corrosive nature of the acid ninhydrin reagent, tephlon and glass components are used throughout the system wherever possible. Small joints are made with silicon tubing, but certain qualities of this tubing have been found to react with acid ninhydrin reagent during storage, causing disturbances in the base-line of the chromatogram a t the start of a run.

b. Sample preparation A sample of faeces or freeze-dried digesta containing about 2 0 m g nitrogen is weighed into a small beaker. I n a second beaker, 0.5 ml 3Oo/o hydrogen peroxide and 4.5 ml concentrated formic acid (98-1OO0/o) are mixed a t Oo C and the performic acid solution then carefully added to the faeces or digesta sample (MOORE 1963). T h e mixture is left to react a t Oo C for 16 hours (MOORE1963) and then excess performic acid is degraded by the addition of about 0.84 g sodium pyrosulphite (NanSeOs) with a glass spoon (KRAMPITZ, personal communication). T h e mixture is quantitatively transferred to a 2-litre round-bottomed flask with 500 ml 6 N hydrochloric acid (246 ml HCI, 37O/o reagent grade, made u p to 500 ml with distilled water). T h e mixture is refluxed on a sand-bath for 23 hours, the time being taken from the moment that boiling begins. After hydrolysis the hydrolysate is immediately cooled under running tap water and filtered on a Buchner funnel through a 7 cm open filter (WHATMANNo. 41), two papers being used to ensure a clear solution. T h e filter papers are washed with a little water and the hydrolysate plus washings reduced to about 30 ml on a Buchi evaporator, the water-bath being maintained a t less than 50° C. T h e hydrolysate is transferred to a beaker and its p H adjusted to 2.22 with 7.5 N sodium hydroxide. During this process the temperature of the mixture must not exceed 3OOC. T h e hydrolysate is transferred to a 1 0 0 m l standard flask via a Scheider & Schull No. 5892 acid-washed filter paper and the solution made u p to 1 0 0 m l with pH 2.22, 0.2 N “a+] citrate buffer (MOORE,SPACKMAN 8r STEIN 1958). The hydrolysate is stored a t 4O C, but warmed to room temperature just prior to use.

224

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c . Buffer and sodium hydroxide solutions T h e compositions of the two buffers used in this analysis are presented in Table 1. Any p H adjustments are made by the addition of 0.1 N HCI or N a O H . Both Table 1 Composition of citrate buffer solutions

Buffer pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sodium ion conccntration, N . . . . . . . . . . . . . . . . Composition per litre: Tri-sodium citrate di-hydrate, g . . . . . . . . . . . . . Hydrochloric acid (37 O / o reagent grade), ml . . . Thiodiglycol, ml . . . . . . . . . . . . . . . . . . . . . . . . . . . Phenol, g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BKIJ-35, g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.22

0.20

3.30 0.25

19.60 16.50

24.50

5.00

0.00 1.oo 1 .oo

1.oo 0.00

12.34

buffers are boiled under reflux for 30 minutes following their preparation and stored under paraffin. 0.20 N sodium hydroxide is prepared from extra-pure analytical grade pellets and stored under soda lime (self-indicating granules).

d. Acid ninhydrin reagent 50 g ninhydrin are weighed into a flask and dissolved in 2530 g glacial acetic acid. I n a second flask a solution of 6 M phosphoric acid is prepared by mixing 1 9 4 0 g ortho-phosphoric acid (S.G. 1.71) with 1650 g fresh double distilled water. These solutions are mixed together in a glass flask for 24 hours with the aid of a magnetic stirrer. T h e reagent is stable to both light and oxygen, but it has been our practice to store the solution in a darkened reservoir under argon. Under these conditions the acid ninhydrin reagent is stable for many months.

e. Procedure Before use the column is washed with C0,-free 0.20 N N a O H and then equilibrated with 0.20 N “a+] citrate buffer a t p H 2.22. Hydrolysate samples of 0.5-20 ml a t p H 2.22 are applied to the column and 2 ml aliquots of p H 2.22 buffer used to wash the walls of the column. p H 3 . 3 0 , 0.25 N “a+] citrate buffer is introduced and the elution started. T h e flow rates of the elution buffer and the ninhydrin reagent are 50 and 7 0 m l per hour, respectively. T h e t w o streams meet at the Tjunction, mix and flow through the reaction coil where the mixture is heated f o r 11 minutes by the surrounding vigorously boiling water. T h e resulting yellow DAPAninhydrin complex cools to room temperature and flows through the photometer where its extinction is measured at a wave-length of 440 mF.

The estimation

of

2,6-diarninopimelic acid in digesta

225

Results and discussion T h e apparatus used for this analysis is, with the exception of the Buchler pump and the Vitatron photometer-Philips Multipoint recorder combination, standard equipment of the type in general use for the semi-automated determination of amino acids in hydrolysates. T h e Buchler pump was used because, unlike more conventional equipment, it is not corroded by the acid ninhydrin solution, the ninhydrin reagent only coming into contact with glass and tephlon. This instrument has a double pump action, but only onc of the two pumping sections is used in the Table 2 2,C-Diaminopimelic acid (DAPA) concentration in hydro- present procedure. It lysates of oxidized and unoxidized duodenal and faecal operates a t 5.7 strokes per minute, compared samples from sheep with 30 strokes per minute in the case of the g DAPAll6 g N Sheep Sample origin Accu-Flo Buffer Pump. osidizcd I unoxidized Despite this difference in stroke-rate the even base0.37 0.39 13 Duodenum 0.25 0.25 653 Duodenum line and the high repro0.26 0.26 554 Duodenum of results obducibility 0.36 0.37 654 Duodenum 0.49 0.50 11 Faeces tained with this system 0.64 0.61 31 Faeces sugests that the streams 551 Faeces 0.60 0.57 from the eluate and nin0.67 0.67 65 1 Faeces hydrin lines are well mixed before they enter the reaction coil. The simple pulse dampener in the ninhydrin line helps to even out the flow, but its effect can only be detected by the Vitatron photometer-Philips recorder combination operating a t 2.5 times the sensitivity normally employed for amino acid analyses. T h e Vitatron photometer-Philips recorder combination has proved to be very suitable for recording amino acid concentrations in hydrolysates, and has the advantage that these concentrations are plotted on a linear scale. It is a potentially very sensitive system, exploitation of which is limited by the quality of the buffer and ninhydrin pumps. T h e procedure employed for the hydrolysis of faeces and digesta is basically the same as that used for the routinc analysis of feed amino acids at this laboratory (MASON 1963, modified by WEIDNER a EGGUM1966). D A P A can be measured in either an unoxidized ( M A S O N 1969) or an oxidized sample (IBRAHIMet al. 1970), evidence of the stability to oxidation of D A P A in faecal and duodenal samples being presented in Table 2. A chromatogram of a hydrolysate of oxidized sheep faeces is presented in Figure 1. With this system D A P A is eluted together with leucine, but tests with standard solutions have shown that the peak area corresponding to 1.58 p mole DAPA is not altered by the addition of 10 p mole leucine (peak area = 1.752 vs 1.749). Further studies have demonstrated that D A P A is well separated from the few other naturally occurring amino acids which give a colour reaction with acid ninhydrin. Thus, the

226

V . C. Mason and S . Bech-Andersen

peak times for some of these and D A P A were found to be: lanthionine, 30 mins.; citrulline, 32 mins.; proline, 33 mins.; cystine, 40 mins.; djenkolic acid, 49 mins.; cystathionine, 52 mins.; methionine, 56 mins.; DAPA, 67 mins.; and tyrosine, 97 mins. Oxidation and hydrolysis of the sample improves the chromatogram by converting several of these to products whose peaks are eluted well clear of DAPA. Thus, the common amino acids cystine and methionine are oxidized to cysteic acid and methionine sulphone, respectively, these being eluted close to the buffer front (16 mins.). Lanthionine, cystathionine, djenkolic acid and tyrosine can produce products whose peaks are registered within the first 25 mins., while chlorotyrosine is eluted with the basic amino acids. Citrulline produces several peaks, the last of which is recorded after 44 minutes.

cQ)

0

Time in minutes Fig. 1. Chromatogram showing isolation of the 2,6-diaminopimelic acid (DAPA) peak. (Oxidized sheep faeces).

The estimation Table 3

Acid ninhydrin reaction with 2,6diaminopimelic acid. Relationship

between colour yield andreaction time

Reaction time minuter

8 9 10 11 12

Colour yield as percent of maximum colour yield

$4.7 97.1 99.0 100.0 99.1

of

2,6-diaminopimelic acid in digesta

227

The only problem of separation so far encountered has been caused by the presence in hydrolysates of Bacitracin of an acid ninhydrin positive component which is eluted very close to DAPA This component can be separated from D A P A on a longer column using a modification of the procedure of Petersen & Bernlohr (1970) (first buffer pH 3.28 instead of pH 3.10) but i t is not isolated from D A P A with the present short column tcchnique. A commercial sample of Bacitracin analyzed using the acid ninhydrin technique gave the low estimate of 0.038 g :‘DAPA equivalents” per 16 g nitrogen, a figure which is about 2 to 10 percent of the concentration of D A P A normally found in hydrolysates of duodenal digesta and faeces. This material does not appear to be present digest from the alnimentary canal of cattle, sheeps

in hydrolysates of microbes and ang pigs. Problems were initially encountered in the preparation of the acid ninhydrin reagent due to the fact that volume changes occurred on mixing the ortho-phosphoric acid solution with the glacial acetic acid. These difficulties were overcome by mixing the reagents on a weight basis under standardized conditions. T h e relationship between colour yield and boiling time is curvelinear (Table 3), the colour yield increasing with boiling time up to 11 minutes and decreasing thereafter. T h e length of the reaction coil was selected to give maximal colour yield. A linear relationship was established between the D A P A concentration and colour yield in the range 0-0.4 p moles DAPA, the molar extinction coefficient, EM410, for the DAPA-ninhydrin complex being 7.5 x 103 moles-1 cm-’. Triplicate estimates of 5 samples covering this range show the acid ninhydrin reaction to be very reproducible, the highest coefficient of variation obtained with the Vitatron-Philips combination being 2.4 O / o . As mentioned earlier, one of the main problems faced in measuring DAPA by the long column, p H 5.5 ninhydrin method is that its peak is small relative to that of its neighbouring peaks, valine and isoleucine (the inethionine peak is removed by (1970) technique oxidation). Furthermore, with the modified PETERSONBERNLOHR used a t this laboratory we have noted that DAPA is eluted on a plateau between t w o very small peaks, which can make it difficult to position the base-line accurately. Despite these difficulties a comparison of this method with the acid ninhydrin technique has shown very good agreement (Figure 2), the correlation coefficient between these methods applied to sheep faeces being 0.98. This acid ninhydrin method has been used a t this laboratory over a period of 3 years and has proved to be robust and reliable. Its main advantage lies in its higher specificity, which simplifies the chromatogram considerably. I n addition it is applicable to hydrolysates containing very little DAPA, because large volumes of hydrolysate can be applied to the column without adversely affecting the chromatogram. Using narrower columns and high quality resins it is possible to reduce the analysis time appreciably.

V. C. Mason and S.Bech-Andersen

228 2. t

y = 1.01x - 0 . 0 2 r = 0.98

S,,

s-Y

= 0.028

= 0,022

m

0

5

B C

.3

4

x s c

1.5

. i

s

a C

M 0

2

.4

1.0

C

M 22 4

--. 4

k

4

CI M

0.5

0.0

0.5

1.0

1.5

2 .b

g D A P A / l t i g n i t r o g e n (pH 5 . 5 ninhydrin method)

Fig. 2. The relationship between 2,6-diaminopimelic acid (DAPA) concentrations in hydrolysates of oxidized sheep faeces measured with acid ninhydrin and the ninhydrin reagent of MOORE& STEIN(1954) using short and long columns, respectively.

Acknowledgements W e wish to express our thanks to Miss A. KARLSSON and Messrs. J. HANSEN and N. 0. ALBERTSEN for their interest and technical assistance in this project.

Summary

A rapid semi-automated method is described for the estimation of 2,6-diaminopimelic acid (DAPA) in hydrolysates of animal digesta and faeces, using the acid ninhydrin reaction. This colour reaction is more specific than the p H 5.5 ninhydrin reaction normally used for amino acid analysis, and permits the determination of DAPA with

The estimation of 2,6-diaminopimelic acid in digesta

229

short columns. T h e method is robust and has been in continuous use a t this laboratory over the last 3 years. Zusammenfassung Bestimmung von 2,6-Diaminopimelinsiure in Darminhalt und Kot mit Hilfe des SiureNinh ydrin-Reagenz Eine schnelle semi-automatische Methode zur Bestimmung von 2,6-Diaminopimelinsaure (DAPA) in Hydrolysaten aus dem Darmkanal und dem K o t von Tieren bei Anwendung der Saure-Ninhydrin-Reaktion wird beschrieben. Die Farbreaktion ist spezifischer als die bei Aminosaureanalysen gewohnlich benutzte pH 5,s NinhydrinReaktion und ermoglicht die Isolation von DAPA an kurzen Saulen. Die Methode ist robust und in den letzten drei Jahren standig am hiesigen Laboratorium benutzt worden. References 1 . CHINARD, F. P., 1952: J. biol. Chem. 199. 91. - 2. EL-SHAZLY, K.; HUNGATE, R. E., 1966: Appl. Microbiol. 14, 27. - 3. FUJIWARA, T.; AKABORI, S., 1954: J. &em. SOC.Japan (Pure Chem. Sec.) 75. 990. - 4. HUTTON,K.; BAILEY, F. J.; ANNISON, E. F., 1971: Br. J. Nutr. 25, 165. - 5. IBRAHIM, E. A.; INGALLS, J. R.; BRAGG,D. B., 1970: Can. J. anim. Sci. 50, 397. 6. MASON,V. C., 1963: “Amino acids in nutrition.” Thesis. KVL. Copenhagen. - 7. MASON, V. C., 1969: J. agric. Sci. (Camb.) 73, 99. - 8. MASON,V. C.; WHITE,F., 1971: J. agric. Sci. (Camb.) 17, 91. - 9. MOORE,S., 1963: J. biol. Chem. 238, 235. - 10. MOORE,S.; STEIN, D . H.; STEIN,W., 1958: W. H., 1954: J. biol. Chem. 211, 907. - 11. MOORE,S.; SPACKMAN, Anal. Chem. 30, 1185. - 12. PETERSON, D. E.; BERNLOHR, R. W., 1970: Anal. Uiochem. 33, 238. - 13. SEN, N . P.; SOMERS, E.; O’BRIEN,R. C., 1969: Anal. Biochem. 28, 345. - 14. SYNGE,R. L. M., 1953: J. gen. Microbiol. 9, 407. - 15. VIRTANEN, A. I., 1967: Neth. Milk & Dairy J. 21, 223. - 16. WEIDNER, K.; EGGUM,B. O., 1966: Acta agric. Scand.16, 115. 17. WELLER,R. A.; GRAY,F. V.; PILGRIM, A. F., 1958: Br. J. Nurr. 12, 421. - 18. WORK, E., 1957: Biochem. J. 67. 416. - 19. WORK,E.; DEWEY,D. L., 1953: J. gen. Microbiol. 9. 394.

Anscbrij? der Autoren: Department of Animal Physiology and Biochemistry, Rolighedsvej 25, 1958 Copenhagen V, Denmark