Determination of available lysine in foods

ANALYTICAL

BIOCHEMISTRY

21, 382-460

Determination L. BLOM, Central Laboratory

of

(1967)

Available

P. HENDRICKS, Staatsmijnen/DSM, Received

March

Lysine AND

in Foods

J. CARIS

Geleen, The Netherlands 23, 1967

In the past ten years the problem of the availability of lysine in all kinds of processed food has been studied by many investigators. AS several authors have treated the matter rather thoroughly-Carpenter (l), Erbersdobler and Zucker (2)) Rao, Carter, and Frampton (3), and Schwerdtfeger (4), it seems superfluous to give an extensive review of the literature. It is generally accepted now that part of the lysine in foods can become “unavailable” by reactions of the (free) c-NH, group with reactive components of the food, i.e., with aldehydes (Maillard reaction). If the relative moisture content of the atmosphere is rather high (7OoJo), or the moisture content of the sample exceeds certain limits, these reactions may take place at temperatures as low as 3040°C. It is especially the reducing pentoses and hexoses which play a part in such reactions. Also, autoxidized fats may give rise to low-temperature reactions. On the other hand, the availability may be decreased by high-temperature reactions (140150”), which cause a strong denaturation of the protein (internal cross-linking?). Part of the lysine that has participated in the above-mentioned reactions is set free by hydrolysis with hydrochloric acid and is therefore determined by chemical methods. However, this part is not accessible to the proteolytic enzymes and therefore not available. The most common chemical method for measuring lysine availability is based on the Sanger reaction. The reagent, fluorodinitrobenzene (FDNB), reacts with the free <-NH, group in the protein to yield a dinitrophenyl (DNP) derivative (c-N-DNP protein). After hydrolysis, the c-DNP-lysine is determined quantitatively on the assumption that the value found corresponds with the amount of lysine that is physiologically available. Indeed, the studies of Boyne, Carpenter, and Woodham (5), Carpenter (6), Mann, Carter, Frampton, Watts, and Johnson 382

AVAILABLE

f7), Martinez shown that a found in this proteins more

LYSINE

IX

383

FOODS

and Frampton (8), and Carpenter and Ellinger (9) have good correlation exists between the “available lysine” value way and the growth response of young animals fed with or less deficient in lysine.

I. METHODS

FOR

DETERMINATION

OF

e-DNP-LYSINE

The most common met,hod, based on the work of Carpenter et al. (1, 9, lo), may .be summarized as follows. After extraction of other DNP derivatives with ether, the r-DNP-lysine is converted to its methylchloroformate derivative, which may be extracted with ether. By measuring the difference in absorbance before and after this extraction, the corresponding lysine content can be calculated. For protein-rich foods a correction factor of I .09 is proposed because, after hydrolysis, added r-DNP-lysine is recovered to only 92%. It is questionable, however, whether this is a good estimate of the loss of the e-DNP-lysine set free during hydrolysis of the vN-DNP protein. More severe difficulties are encountered with this method when it is applied to foods that are rich in carbohydrates (1). Losses of 30%, and in special cases even of 70%, are reported (11)) but there is some contradiction in the literature on this subject (2). On the other hand, the accuracy is said to be adversely influenced by formation of humin-like products (4). Recently, methods were published in which use was made of a chromatographic separation procedure to make the determination more specific (3, 4). After a few preliminary experiments, the authors were convinced that application of the method to a wide variety of foodstuffs made it necessary to use such a. chromatographic separation procedure in order to eliminate the large number of by-products which might influence the quantitative measurement of t.he c-N-DNP-lysine. For this measurement three different detection methods were tried. (1) Direct photometric measurement of the yellow color. (2) Photometric measurement of the reaction product with ninhydrin. 1.3) Polarographic measurement based on reduction of the nitro groups at the dropping mercury electrode (12). In the majority of cases t.he “blocked lysine” was also determined, i.e., the lysine set free by hydrolysis from a FDNB-treated sample or, in other words, the lysine which somehow was masked against reaction with dinitrofluorobenzene. As far as is known to the authors, the measurement of this value is mentioned only once (3). The difference between total lysine and blocked lysine might also be considered as available lysine.

384

BLOM,

HENDRICKS,

II.

CARIS

EXPERIMENTAL

1. Apparatus SEPARATION

AND

and Methods

METHODS

(a) Chromatography on polyamide powder was used for separating the DNP derivative of lysine from products which might interfere with the detection. The nylon 6 prepared by Steuerle’s method (13) proved to yield columns which were mechanically unstable. The resistance of these columns changed continuously. Therefore, so-called “metallization powder” was tried.l Columns made from this material remained stable for several months. Recently also nylon powder D.C. supplied by Woelm2 for thin-layer chromatography was tried. The impression gained so far is that this material is also suitable. Initially a column length of 20 cm was used (50°C, buffer pH 5.3, flow rate 40 gm/hr). However, for carbohydrate-rich products the separation power was insufficient. Therefore 100 cm columns were chosen. At 50” with a buffer of pH 3.00 and a flow rate of 150 gm/hr the separation was satisfactory. As in the other chromatographic procedures mentioned here, the buffer was deaerated before use by passing it over Pt wool in a U-tube at about 100”. This method, described earlier by Spackman, Stein, and Moore (14)) proved to be very satisfactory. (b) Chromatographic separation on Amber&e C.G. 1&J was the other method used. On this column both lysine and c-DNP-lysine could be separated from components which might interfere with their determination. Lysine has an acceptable retention volume on such a column (40 ml), but for r-DNP-lysine (100 ml) the method is not very attractive. Moreover the separation of arginine from the lysine derivative is incomplete, which adversely affects the results. The experimental conditions were as follows: column length 17 cm, diameter 0.9 cm, temperature 5O”C, and particle diameter of ion-exchange resin 27-31 pi. The eluent was a citrate buffer of pH 5.28 and the flow rate was 40 ml/hr. This separation method was always used in combination with the ninhydrin detection method. DETECTION

METHODS

(a) DNP derivatives are reducible at the dropping mercury electrode. By passing the eluent from the chromatographic column through a polarographic cell and continuously measuring the diffusion current, one obtains a chromatopolarogram. Figure 1 shows the complete apparatus. Figures ’ A product used for whirl-sintering * M. Woelm, Eschwege, Germany.

from

Metallisation

Ltd.,

England.

AVAILABLE

LYSINE

IN

ac~tr!c

385

FOODS

ocld

( shock

ItrOtQ

absorber

3-woy-z
oscor

tof Ion stopcock

buffor

/-

tta

QlQCtrOdQ

(Pt

)

rh NoOH -02N mercury

connectcon 50’ water

D -9 3

to both

washlnq bottle SlntQrQ-d-glass a) with water

rQsQrvolr

‘+

WI filter

QlQCtrOdQ

b) empty Fm.

1.

1A and 1B give some details. The eluent from the column first passes through the oxygen desorber in which the liquid is propelled by a stream of moistened, oxygen-free nitrogen. In this way, interfering traces of oxygen are completely removed (15). The liquid level at B can be

rubber

C SIDE

VIEW FIG.

1-k.

stopper

386

BLOM,

HENDRICKS,

AND

CARIS

mm 8mm o.d. 16 mm o.d.

effluent 1

S13&

I;

“r’ Ji

iA

FIG. 1B.

adjusted by moving D in the desired direction. The polarographic cell is thermostated. ‘Consumed” mercury is continuously removed by suction (Fig. 1A) to hold the level in C constant. Figure 2 shows the electrical circuit of the homemade polarograph. A Radiometer polarograph (type PO 3 m) also gave very satisfact’ory results. However, the speed of the recorder paper had to be diminished. The recorder paper was rolled on a 2V

(stabilized

)’

cl

I

PolarographicCQII

18KI-I

u hI

2OObF

Paper-capacitor

18K a)KO~stO~tspa~nu~gs~uQllQ

Type (same

N 6/2 time GMBH

JOQnS DuSSQldOrf

rectifier)

n

(no

t0

Klpp (Delft (MIcrograph

FIQ. 2.

QlQCtrOlytlC

capacitor

4PF

rQCOrdQr

-Holland) BD

1 )

I)

AVAILABLE

LYSINE

IN

387

FOODS

16 mm dial connected to the axis of a synchronized motor rotating at a speed of 12 rph. Polarographic measurements normally are carried out in nonmoving liquids. Therefore the question arose whether the signal would be independent of the flow rate in the cell. If so, the product of peak area and flow rate should be constant at a given concentration. From Table 1 TABLE 1 Relation between Flow Rate (200 pg of -DNP lysine Flow

rate

(F). ml/bra

Peak

29.9

60.0 86.8 127.1 152.9 192.8 269.4 a Determined

by weighing

and Peak HCl.H&)

Area

zwtx (A’), cm2

FXS

100.5 49.9 34.75 23.7 19.55 15.65 11.65

3005 2993 3016 3011 2991 3013 3133

the effluent.

it appears that this holds for flow rates varying from 20 to 200 ml/hr. Only at the highest flow rate does there seem to be some deviation from true linearity. There proved to exist a linear relationship between the concentration of c-DNP-lysine and the area under the recorded peak over a wide range of concentrations, as shown in Table 2. During these experiRelat,ion

between

TA4BLE 2 Concentration (at 150 ml/hr)

and

Peak

Area -

Weight

of a-DNP-lysine.HCl.HzO,

10 20 40 100 200 400 1000

pg

Sensitivity

of polarograph

5 10 20 50 100 200 500

Peak

area, cm2

22.2 22.2 22.4 21.8 21.6 22.0 21.8

ments the sensitivity of the polarograph was altered to equalize the measured areas. Of course, any deviations from linearity of the sensitivity regulator influence the results. (b) The yellow color of c-DNP-lysine shows a maximum absorption at 364 I-+ However, 0-DNP-tyrosine may interfere at this wavelength; therefore, measuring at 440 rnp was recommended (3, 4). It was found that at this wavelength a linear relationship between concentration and

388

BLOM,

HENDRICKS,

AND

CARIS

extinction exists only within certain limits of concentration. For this reason and because no disturbing influence of 0-DNP-tyrosine was ‘observed during their investigation the authors are of the opinion that measuring at 364 rnp is to be preferred. DINITROPHENYLATION

AND

HYDROLYSIS

Several methods were tried for the conversion of the free amino groups of the proteins to the dinitrophenyl derivative. The reaction medium was about the same in all experiments: 8 ml of a saturated solution of sodium hydrogen carbonate plus 12 ml of an alcoholic solution of dinitrofluorobenzene. Variation of the reaction temperature from 25 to 75°C had no influence on t,he results. A reaction period of 5 hr at 40” was sufficient in all cases, ;Yhile on the other hand standing overnight did no harm. Ultimately a procedure was chosen (see Part IV below) which is to a large extent identical with that of Portugal et al. (16). There is one important, alteration, which will be discussed below. 2. Results COMPARISON

OF

RESULTS

FQCND

BY

DIFFERENT

DETECTION

METHODS

A comparison was made between the results obtained with the photometric and the polarographic methods (Table 3). Samples of different protein cont.ents were chosen. The photometric method used was almost identical with that of Schwerdtfeger (4). A chromatographic column of greater length proved desirable, however. With regard to the anaIysis of products of low protein content the authors prefer the polarographic method as being more sensitive. An additional advantage is the ease with which the sensitivity can be varied over a wide range. From Table 3 it

Comparison

TABLE 3 of Photometric and Polarographic Photometric method, weight y. found

0.80,0.81 2.23,2.26 4.08,4.07 4.40,4.47 4.51,4.52 4.52,4.73 4.65,4.70 5.35,5.67 6.12,6.13

Detection Polarographic method. weight y. found

0.78,0.80 2.12,2.16 3.99,4.04 4.60,4.62 4.52,4.59 4.53,4.66 4.17,4.58 5.64,5.90 6.30,6.40,6.41,6.55

AVAILABLE

LYSINE

IN

389

iFQOD5

appears, that both methods have a rather good duplicability the agreement between them is very satisfactory. INFLUENCE AND

ON OTHER

RESULTS

OF

SAMPLE

TYPE,

SAMPLE

and that

WEIGHT,

FACTORS

The literature reports significant losses of c-DNP-lysine during acid hydrolysis of samples rich in carbohydrates.3 It has even been said that there is hardly any point in using the method with carbohydrate-rich samples because of the large variations in the correction factors to he applied. The most common method for determination of these factors is based on measurement of the recovery of added &DNP-lysine; this method has been criticized by Erbersdobler and Zucker, who stress that added c-DNP-lysine may be much more subjected to undesirable side reactions than the “built-in molecule.” In contrast to the above view some authors (Erbersdobler et aZ., Rao et al.) find hardly any destruction of a-DNP-lysine during hydrolysis of carbohydrate-rich samples. There are several possible causes for the discrepancies mentioned. The destruction of r-DXP-lysine is strongly influenced by: (I) The type of carbohydrate present. (2) Pretreatment of the carbohydrate: after reaction with FDNB the destructive power of the carbohydrate is considerably reduced. (3) Sample weight-the higher the sample weight, the larger the deviation from the exact value. It will be shown that the influence of sample weight cannot be ascribed to consumption of too much FDNB by carbohydrates, or other sample components, and hence not to incomplete reaction of the e-NH2 group. (4) Amount of dinitrophenol or other reaction products of FDNB present during hydrolysis. This point will also be illustrated below. On the other hand it is most probable that the E-DNP groups present in a material which contains carbohydrate-like structures are much more subjected to destruction than groups which are not in the same material. The present authors systematically studied the influence of sample weight on the results and found that a very distinct relationship exists between amount of sample taken and magnifude of the correction to be applied. This relationship is shown in Table 4 and in Figures 3 and 4. All results were obtained by the polarographic method. From this one may conclude that the most exact method for determining the available lysine content is undoubtedly the “extrapolation method,” which involves doing the analysis at least three times with ‘Some authors even report destruction of rDNP+sine under hydrolysis con&tions in the absenceof any organicmaterial. The authorscould not, confirm this.

390

BLOM,

Correlation

AND

CARIS

TABLE 4 between Sample Weight and per cent Available Sample

Milk replacer K

Milk replacer B

Concentrated

HENDRICKS,

mixed food

Casein Glutin

Sample

weight, m!z

Albumin Barley Fish meal

Maize

Peanut oil meal (P.O.M.)

Blocked found,

lysine $4,

115

1.75

0.06

1.69

0.08

1.60 1.42

0.07

1100 107 309 505 1013 106 305 500 1007 133 525 1046

119 108

1.20 1.17 1.10 1.00 0.90

0.08 0.11 0.14 0.13 0.13 0.12

2.60 2.45 2.40

0.25 0.23 0.26

2.00 7.12

0.29

6.82 5.77

0.22 0.24

1.08 0.98 0.79 5.21 7.39

545 1048 114 471

6.44 5.10

998

4.52

200 600

0.39

1000 100 203 362 TO6 868 1114 106 330

987 Soybean oil meal (S.B.O.M.)

lysine 70

205 315 705

525 1026

Lactalbumin

Available found,

Lysine Found

171 305

1490 177 317 1425

4.98

0.31 0.26 4.65 4.60 4.56 4.02 3.85 3.74 0.25 0.22

0.18 2.21 2.10 1.55 1.46 1.26 1 .Ol

0.13

0.09 0.52 0.54 0.55

0.90 0.99 0.70 0.73 0.02

0.12

0.16

-

AVAILABLE

LYSINE

IN

391

FOODS

% 5.00 0

Albumin

? 4.00

FishmQal

:iz.

SoybQan 011. mQal PQOnUt 011.rnQOl * Barley 0.20

1 100

1 400

I r 800 -Sample

M~IZQ , 1200

1600 weight

(mg)

FIG. 3.

rather different sample weights and determining the value for the zero sample weight by graphic extrapolation. This being a rather laborious procedure, it became desirable to improve it. First of all, attempts were made at reducing the effect of sample weight by addition of larger amounts of reagent FDNB. This had a favorable effect indeed (Fig. 5), as appears from the much weaker slope of the lines. As the percentage of “blocked lysine” found was independent of the amount of reagent (see Table 4), the above effect could not be ascribed to a more complete reaction of free amino groups with the reagent. This supported the hypothesis that the loss of c-N-DNP-lysine might be due to reduction of the nitro groups, which reaction is counteracted by the presence of other compounds containing nitro groups, e.g., hydrolysis products from dini-

392

BLOM,

100

HER‘DRICKS,

300

AXD

500 -----9Sample FIG.

CARIS

1000

weight

(mgl

4.

trofluorobenzene. Experimental evidence was gained by performing determinations in which dinitrophenol was added immediately before hydrolysis. From Figure 5 it appears that the slopes of the lines arc considerably reduced by this addition, i.e., to the extent, that addition of 1 gm of dinitrophenol renders the extrapolation procedure superfluous. For sample weights of the order of 100 mg, the correction now becomes negligible; for larger intakes a fixed correction factor can be applied. In this way the analytical procedure is very much simplified. Finally, a few experiments were carried out to check if the E-DNPlysine addition method would give similar results as the proposed procedure, even with carbohydrate-rich products. The agreement proved to be very satisfactory, both for samples of barley and fish meal, provided the c-DNP-lysine is added to the FDNB-treated sample. It, is questionable, however, if this is also valid for larger sample weights (>500 mg). The dinitrophenol addition procedure is preferred by the authors because of the much greater availability of this reagent. Moreover, a separate determinat,ion is superfluous in the majority of cases, as a relative correction of 1%/lo0 mg sample weight will do. A typical example of a chromatopolarogram is shown in Figure 6. It appears from this figure that a complete separation of r-DNP-lysine

AVAfLABLE

LYSIXE

IN

0.3

0

ml 1 .Oml

o

0.3

. 4.80-

393

FOODS F.D.N.B. F.D.N.B.

ml F.D.N.B. + 1 .Og 2.4.dlnitrophQnOl present during nydrOlySIS

4.202 flshmeal

Ava\loble lysine %

1; i

t

milk replacer

barley

'200

400

600

-Sample

weight

FIG.

FIG.

5.

6.

( mg)

394

BLOM,

HENDRICKS,

AND

CARIS

from DNP-tyrosine is achieved. This is in agreement with the literature (17). A number of accompanying peaks have not yet been identified (Table 4). III. DISCUSSION

The results show that, whatever the type of product, the amount of available lysine found decreasesmarkedly with increasing sample weight. The most surprising finding is that this decrease, expressed in relative amounts, is also practically independent of the type of product. In each case, a sample weight of 1500 mg corresponds to a decrease in “available lysine” of about one-third of the value found by extrapolation to zero weight (Figs. 3 and 4). So the differences between the correction factors found by various authors may be ascribed to the fact that, depending on the laboratory and on the type of sample (protein content!), different sample weights were used. It remains difficult to understand why the relative decay is almost independent of the type of sample, which suggests that the mechanism of this decay is identical both with protein-rich materials and with carbohydrate-rich products. A number of experiments were carried out to discover whether individual amino acids could be responsible for the lossesof a-DNP-lysine. From these experiments it may be concluded that none of the amino acids usually present in feedstuffs has a profound influence on the results of the determination of available lysine. Upon addition of a mixture of all these amino acids, each in an amount of 15 mg, to a solution of c-DNP-lysine, and subsequent normal “hydrolysis,” 99% of this compound was recovered, which is the same figure as that for c-DNP-lysine alone. Similar experiments with relatively large amounts (~100 mg) of tryptophan and cysteine gave the same results. Since only an effect of carbohydrates on the determination of available lysine has been proved, it might be thought that even in feedstuffs of animal origin the small concentrations of carbohydrates (e.g., from nucleic acids) present are responsible for the marked decrease at higher sample weights. If so, the similarity in behavior between feedstuffs from completely different origins may be purely accidental. From Table 5 it appears that in most casesthe sum of available lysine and blocked lysine is lower than the amount of total lysine. But for a few exceptions, the relative difference amounts to about 5%. The figures in the second column are determined by using separation method b (see above). At least three factors might be responsible for the differences between the figures of columns 4 and 3:

AVAILABLE

(1) mally (9) when (3)

LYSINE

IN

395

FOODS

The presence of free lysine in the original sample, which is noronly a fraction of the percentage under discussion however. Formation of di-DNP-lysine from lysine end groups, especially peptides are present. This point is under investigation. A Yoss of e-DNP-lysine” which is independent of sample size.

Factor 3 might components during availability should, lysine and blocked

be due to decomposition catalyzed by other sample hydrolysis. If such a decomposition should occur the rather, be calculated as the difference between total lysine. However, the difference is only small and as

Comparison

TABLE 5 of Different Forms of Lysine Availgble,

Bloclzld,

Sample

Maize meal Glutin Milk replacer B Peanut oil meal (P.O.3I.I.) Milk replacer K Soybean oil meal (S.B.O.M.) Mixed food (concentrate) Fish meal Albumin Casein Lactalbumin

0.25 1.15 1 21 1.47 1.81 2.29 2.6i 4.82 5.27 7.54

8.39

0 0% 0.11 0.1s 0.16 (1.0X 0.12 0.26 0.72 0.94 0.23 0.54

in various Available blocked,

Foods + 70

Total, 70

A\-ailable, % of total

0. “i 1.26 I .34

0.2i

92

1 30

88

1 .4:3

1.63

1.66 2.01 2.3

85 90 90 ,(14 86 x2 89 95 96

1 89 2 41 2.9s 5.54 6.21 7.v 8.93

x11

5.x4 5.Y5 7.97 8.78

long as no certainty exists on this point it will be safer to use the directly determined figure, which is somewhat lower. It should be emphasized that among the samples used none was of inferior quality. Much lower figures for availability may be expected for products that have been heated more or less drastically or have been stored in very humid atmospheres. IV. STANDARD METHOD AVAILABLE LYSINE

FOR DETERMINATION IN FOOD PROTEINS

OF

1. Scope The method is intended for the determination of available lysine both in foods from animal origin and in foods rich in carbohydrates. 2. Outline of Method The sample is treated with l-fluoro-2,4-dinitrobenzene (FDNB) in a solution of sodium hydrogen carbonate. Available lysine is thus converted to r-2,4-dinitrophenyllysine (c-DNP-lysine) . After removal of excess reagent, the protein is hydrolyzed with 6 M hpdrochloria acid. Hydro-

396

BLOM,

HENDRICKS,

AND

CARIS

chloric acid is removed by evaporation in vacuum. The residue is subjected to a chromatographic separation on nylon powder to separate c-DNP-lysine from a number of interfering compounds. The detection is based on reduction of the nitro groups on a dropping mercury electrode in a polarographic cell. 3. Reagents Hydrochloric acid, 20.24% (w/w), constant-boiling mixture. Sodium hydroxide, 0.2 M Sodium cit’rate buffer, pH 3.00: 123 gm citric acid, 72 gm sodium hydroxide, 118 ml. hydrochloric acid (1.19) and 5 gm phenol in 5 liters. Nylon 6, particle size <60 ,JL.,obtained by sieving metallization powder for whirl sintering, or a product of Woelm, Eschwege, ‘(for chromatography.” Fluorodinitrobenzene, pure grade. Sodium hydrogen carbonate, saturated solut.ion. Ethyl alcohol, 96%. Diethyl ether. 2,4-Dinitrophenol. E-hr-2,4-Dinitrophenyllysine (British Drug Houses). Mercury, for polarography.

4. Sample Grind about 20 gm of sample to a particle product if necessary and mix carefully.

size GO.5 mm. Sieve the

5. Procedure PREPARATION

OF

APPARATUS

Heat tube K (Fig. 1) to 5O”C, open stopcock 1, suck some water into the tube to remove air from beneath the filter disc and close stopcock 1. Prepare a suspension of 1 vol nylon 6 in 2 vol citrate buffer. Transfer the suspension in portions to tube K as follows. Open stopcock 1 as soon as a 2 cm layer of nylon powder of the first portion has settled. Supply gas pressure at the top until the liquid level reaches the surface of the settled nylon. Transfer a second portion of suspension to the tube and remove the liquid as described ; repeat this process until the length of the chromatographic column is about 100 cm. Connect the column via ball joint S 19 with the pumps, ensuring that no air is present in the tubes. Connect the top of the column with the manometer and pass sodium hydroxide through the column until the effluent reacts alkaline. Turn stopcock 2 to connect the manometer with the atmosphere. Turn it again

AVAILABLE

LYSINE

IN

FOODS

397

to connect column and pump with the atmosphere. Connect the column with the buffer pump (stopcock 3) and pump 5 ml buffer through the tubes. Turn stopcock 2 to connect column with manometer and pass buffer through the column until the pH of the effluent is lowered to 3.00. Connect column and pump with the atmosphere; subsequently turn stopcock 3 in such a position that the exit of both pumps is closed. Turn stopcock 1 in a horizontal position ; connect the column by S 13 with desorber (see Note 1) and polarographic cell. REACTION

WITH

FLUORODINITROBENZENE

Weigh 0.1-l gm sample and put it into a 500 ml round-bottom flask. Add 8 ml sodium hydrogen carbonate solution, mix, and allow to stand for 1 min. Add 0.3 ml FDNB, dissolved in 12 ml ethyl alcohol. Heat the flask to 40°C for 5 hr; shake now and then. Evaporate the alcohol in a Rotavapor (manufacturer-W. Biichi, Flawil, Switzerland) at 40”. Extract the residue 3 times with 100 ml diethyl ether; remove the ether by decantation. Pass nitrogen through the flask at 40” to remove the residual ether. HYDROLYSIS

Add to the residue of the above treatment 1.00 gm 2,4-dinitrophenol and 250 ml HCl. Boil under reflux for 17 hr. Cool and extract 3 times wit,h 100 ml diethyl ether. Remove HCl at 40°C in a Rotavapor. Add citrate buffer to the residue and transfer the solution to a 50 ml measuring flask, make up to the mark with buffer and mix (= solution S) . CHROMATOGRAPHIC

SEPARATION

Open the column at S 19, remove most of the liquid on top of the column material, open stopcock 1, and press the liquid through the column until the liquid level has the same height as the column material. Close stopcock 1 and transfer a suitable amount (max. 2 ml) of solution S (= V ml) to the top of the column. Open stopcock 1 and press again until the same level is reached. Wash the wall of the tube 3 times with 0.5 ml portions of buffer, each time using nitrogen pressure. Release pressure and close stopcock 1. Fill the column up to S 19 with buffer and connect the column with the upper part of the apparatus. After pumping some citrate buffer through the tubing, turn stopcock 2 to connect column and manometer, open stopcock 1, and pass citrate buffer through the column (flow rate about 150 gm/hr) . DETERMINATION

OF

FLOW

RATE

When the flow rate is constant, collect the eluate in a beaker for a period of 10 min and determine the weight of effluent per hour.

398

BLOM,

POLAROGRAPHIC

HENDRICKS,

AND

CARIH

DETECTION

Pass nitrogen through the desorber (14) ; connect the electrodes with the polarograph. Apply a voltage of 1.0 V to the electrodes. Raise reservoir E (Fig. 1) until the mercury electrode delivers 5 drops/l0 set (see Note 2). Choose a suitable “damping” value and use a paper flow rate of 60 cm/hr. Determine the peak area by counting the number of squares (each square N 0.25 cm?), by the use of a planimeter or by any other suitable method. REGENERATION

OF

THE

COLUMN

Release pressure from the manometer, the pump, and the column by turning stopcock 2. Disconnect the column from the desorber at S 13 and pass sodium hydroxide through the column until a zone of at least 50 cm is filled with it. Again release pressure from manometer, column, and pump by turning stopcock 2. Subsequently pass citrate buffer through the column. 6. Standardization Dissolve 50 mg (= a @mole) DNP-lysine in citrate buffer in a 50 ml measuring flask, make up with buffer to the mark, and mix (solution I). Pipet 10 ml of solution I into a 50 ml measuring flask, make up with buffer to the mark, and mix (solution II). Then follow the procedure described above. Calculate the factor f at a sensitivity of 100 and a flow rate of 100 gm per hour by the formula: Vu x 104 ~mole/cm2 25OOSv number of ~molcs corresponding with one cm” peak area, weighed amount of DNP-lysine in @moles, amount of solution II taken in ml, area of peak in cm2, sensitivity of polarograph, and elution flow rate in gm/hr. f=

where:

f = a= I7 = 0 = S= ZI =

7. Cnlc&tiorl

Available where:

lysine in $& w/w =

OvSf x 146 10%

a = weight of sample in gm, 1’ = volume of sample solution taken in ml, and the other symbols have the same meaning as above.

AVAILABLE

LYSINE

Iii

399

FOODS

8. Notes 1. The desorber serves to remove any oxygen, which would interfere in the polarographic determination. Oxygen-free nitrogen, saturated with water and at a flow rate of 20 liters/hr, is used. 2. Regular control of the drop rate of the electrode is necessary. V. SUMMARY

1. Methods for the determination of available lysine in foodstuffs all based on reaction with fluorodinitrobenzene are discussed with special reference to the problem of analyzing carbohydrate-rich products. 2. A recording and very sensitive method, based on chromatographic separation and polarographic detection of the eluents, has been developed for the determination of available lysine. The results are compared with those obtained with a photometric method. 3. Experiments were carried out to prove an expected effect of sample weight on the results found in the determination of available lysine with the above method. It was shown that with the usual methods an extrapolation procedure (to zero sample weight) is necessary to overcome interferences by other sample components. 4. Experimental evidence was obtained to show that interferences in the determination of available lysine can be largely diminished by adding dinitrophenol before hydrolysis of the fluorodinitrobenzenetreated sample. 5. A comparison is made between available lysine and the difference between tot.al lysine and blocked lysine for a variety of samples. The latter figures were, with one exception, systematically higher. 6. A standard method for the determination of available lysine is described. ACKNOWLEDGMENT WC are discussions

indebted on this

to Dr. subject.

P. Slump

of the

C.I.V.O.

at Utrecht

for

some

interesting

REFERENCES 1. 2. 3. 4. 5.

CARPENTER, K. J., &o&em. J. 77, 604 (1960). ERBERSDOBLER, H., AND ZUCKER, H., 2. Tklphysiol. Tiererniihr. 19, 244 (1964). RAO, S. R., CARTER, F. L., AND FRAMPTON, V. L., Anal. Chem. 35, 1927 (1963). SCHWERDTFEGER, E., 2. Tierphysiol. Tiererniihr. 16, 262 (1961). BOYNE, H. W., CARPENTER, K. J., AND W~~DHAM, A. A., J. Sci. Food Agr. 12, 832 (1961). 6. CARPJZNTER, K. J., Proc. Nutr. Sot. Engl. Scot. 17, 91 (1958). 7. MANN, G. E., CARTER, F. L., FRAMPIDN, V. L., WATTS, A. B., AND JOHNSON, C., J. Am. Oil Chemists’ Sot. 39, 86 (1962). 8. M.+RTINEZ, W. H., AND FRAMPTON, V. L., .I. Agr. Food Chem. 6, 312 (1958).

400 9. 10. 11. 12. 13. 14. 15. 16.

17.

BLOM,

HENDRICKS,

AND

CARIS

CARPENTER, K. J., AND ELLINGER, G. M., Pouhy fki. 34, 1451 (1955). CARPENTER, K. J., AND ELLINCER, G. M., Biochem. J. 61, XI-XII (1955). CARPENTER, K. J., AND MARCH, B. E., Brit. J. Nutr. 15, 403 (1961). WAINTRAIJB, I. A., Biochimiya 28,661 (1963). STJDJERLE, H., AND HILLE, E., Biochem. Zjt. 331, 220 (1959). SPACRMAN, D. M., STEIN, W. H., AND MOORE, S., Anal. Chem. 30, 1190 (195%. BLAEDEL, W. J., AND TODD, J. W., Anal. Chem. 30, 1821 (1958). PORTUGAL, A. V., GREEN, R., AND SUTHERLAND, T. M., J. Chrmatog. 12, 183 (1963). HILLE, E., Biochem. Zjt. 333, 269 (1960).