The assay method for CEF is being reported in detail elsewhere. In short, the sample is homogenized with water and saponified with alcoholic KOH. The unsaponifiable portion is extracted with petroleum ether and chromatographed first on an alumina column. The einate obtained with 25% ethyl ether in petroleum ether, following prior elution with petroleum ether and 5% ethyl ether in petroleum ether, is concentrated and chromatographed on 500 thin Inver silica gel plates with 3% ethyl ether in petroleum ether. The silica gel in The area of H 0.80-100 is removed and eluted with ethyl ether, the solvent removed, and the residue is redissolved in isooctane for gas chromatography. We used an F & M Model 400 gas chromatograph with an electron capture detector and a U-tube column, 3 ft X 6 mm (o.d.) X 4 mm (i.d.), packed with

• Supported by UBPHS Grant EF 00305. Contribution 858 from Dept. of Nutrition and Food Relence, Massachusetts Inst. of Technology.

14. ( Campbell and L. Friedman in press, J. AOAC, "Chemical Assay and Isolation of Chick Edema Factor,"

1% SE-30 on ANAKROM ABS (Analabs). The operating conditions were: temperatures, column oven 180°C, EC detector 200°C, flash heater 240°C; gas flows, helium carrier gas, 60 cc/min; argon, 10% methane purge gas 180 cc/min. The limit of detection by this method is approximately 0.2 ppb, assuming a sample size of 5 g and a sensitivity of the EC detector of 5 X 10-" g.

The so-called CEF components can be seen as a gas chromatographic pattern of peaks shown in Fig. 1. This pattern is that of a highly concentrated material isolated from the toxic unsap. It is similar to a material isolated by Yartzoff et al and kindly supplied to us by Firestone (3). We have numbered the peaks 1 through 8 as shown. Peak 4a was not seen in the Firestone preparation.§

Results and discussion. Table II shows the effects upon body weights, feed conversion, and feed consumption for the rats. The results for the vital organ weights and HPF volume are shown in Table III. There was no apparent gross pathology in either species with the exception of HPF and some ascites and subcutaneous edema in the chicks.

While only the chicks develop hydropericardium, apparently both are equally sensitive to liver weight increase, as shown in Table III. In the case where each species was maintained on CEF for 6 days, (Trials II and III), the rats

TABLE III.-ORGAN WEIGHTS EXPRESSED AS PERCENT OF BODY WEIGHTS

(9 μg/kg/day) showed an increase over controls of 21% while the chicks (10 μg/kg/day) showed an increase of 15%. (The increase in liver weight in the chicks was not due to moisture or fat.)

Also, in Table III, neither heart nor spleen weights in either species are significantly affected. In rats, there appears to be a slight increase in kidney weights, though not statistically significant, and a highly significant increase of 50% in adrenal weights for animals on the high level. Whether this adrenal weight increase is simply a non-specific stress effect from intubation is not known, although it would seem that this cannot be entirely responsible, since the high level group showed an increase of nearly twice that of the low level group.

Some other observations which have been made on previously studied birds in this laboratory are of interest. Hematocrit values are depressed. Of a total of 43 birds, we have observed that, with an average of 0.08 ml HPF in control

The chromatogram for the Firestone preparation is presented elsewhere.

FIGURE 1-Toxic CEF components found in unsap used in this study.
FIGURE 2-Rat liver extract showing 2 CEF peaks.

Fietse 3-Rat feces extract showing CEF peaks, with altered Nos. 4a and 7.
FIGURE 4-Chick liver extract showing 2 CEF peaks. (Large peak just before
No. 4 found to be contaminant leached from liner of sample vial.)

birds (considered normal), there was a packed cell volume of 33.0%, while for diseased birds having 0.73 mi HPF, the packed cells volume was 27.9

FIGURE 5-Chick feces extract showing CEF peaks, with altered Nos. 4a and 7.

Also, we observed that whereas control birds will show disappearance of a given amount of an I.V. injected dose of T-1824 dye (Evans Blue) from their vascular system of approximately 1% min. poisoned birds will show a disappearance of approximately 2 min, supporting the observation of Allen (4), that the permeability of the vascular wall appears to be increased.

Distribution of the CEP in animal dody.—Of the rat tissues and samples examined. CEF was detected only in the liver and feces. Fig. 2 through 5 show typical chromatograms of purified extracts of feces and liver of both species.

Control animals not receiving CEF did not show these peaks. These chromatograms show that only peaks 4 (or 4a?) and 7 were present in the liver. In the fecal extracts all peaks were found, with the exception that instead of peaks 4a and 7 showing their original retention times, these were slightly increased in each case and have been designated 4a′ and 7'.

These retention times increases in the fecal components were measured by noting their retention times in relation to their neighbors, as shown in Table IV. Whether the 2 components of the liver are the products of components 4 and 7 or the original unaltered substances cannot be accurately determined from these chromatograms, since the rest of the CEF chromatographic pattern is missing. It may be concluded, however, that there is a selective absorption of peaks 4 (or 4a) and 7, with metabolism by the liver and excretion into the intestine.

It appears, on the basis of the tissues analyzed, that the liver is the target organ. Furthermore, the pattern of CEF chromatographic peaks presented here, Nos. 4 and 7 are the key components. We have succeeded in partially separating the CEF components such that 4a and 7 crystallized together, indicating a certain chemical as well as physiological similarity. There is no way of differentiating 4 from 4a with regard to its absorption and metabolism on the basis of the available evidence.

Summary. Unsaponifiable matter isolated from a toxic fat and containing an estimated 10 ppm chick edema factor (CEF) was force-fed daily to adult rats at levels of 2.0 cc and 1.0 cc/kg body weight/day in 2 studies of 14 and 6 days, respectively. Feed consumption, body weight, and digestibility were depressed. Heart and spleen weights were unaffected, kidney weights seemed to be slightly increased, and adrenal and liver weights were significantly increased. In the chick, typical hydropericardium, ascites, and subcutaneous edema were observed. There were no significant changes in heart or spleen weights. Liver weights were significantly increased. The rat was as sensitive as the chick to CEF according to increase in liver weight. Of the 8 or 9 CEF components shown to be in this unsap, 2 (Nos. 4 and 7) were found to be absorbed and located in the liver, while the other components were not

TABLE IV.-CHANGES IN CHROMATOGRAM RETENTION TIMES OF CEF PEAK NOS. 4a AND 7

2 Includes feces of 11 individual rats and the composite feces of 2 groups of 6 chicks each. 3 The deviation includes the total range of values.

detected. In place of Nos. 4 and 7, there were 2 new peaks in the feces with slightly increased retention times. This suggests that the 2 active CEF components are metabolized in the liver and excreted into the intestine via the bile, both in the chick and the rat. No CEF-like material was found in kidneys, adrenals, or urine.

STUDIES ON THE METABOLISM OF CHICK EDEMA FACTOR:
DISTRIBUTION IN CHICK TISSUES

D. Firestone, G. R. Higginbotham, D. F. Flick and J. Ress

The distribution of chick edema factor in chick tissues following consumption of rations containing toxic fat has been of considerable interest. Chick

1 Harman, R. E., Davis. G. E., Ott, W. H., Brink. N. G., Kuehl, F. A., J. Am. Oil Chem. Soc., 1960, v82, 2078.

2 Wootton, J. C.. Courchene. W. L., J. Agric. Food Chem., 1964, v12, 94.

3 Yartzoff, A.. Firestone, D., Banes, D., Horwitz, W., Friedman, L., Nesheim, S., J. Am. Oil Chem. Soc., 1961, v38, 60.

Allen, J. R., Ph. D. Thesis, 1961, Univ. of Wisconsin.

5 Hogben, C. A. M., J. Lab. Clin. Med., 1964, v64, 815. Received January 13, 1966. P.S.E.B.M., 1966, v121.

edema factor was detected 11 chicker tissues by electron capture gas chromaTOSTANET ANČ at estimation of the concentration in various tissues was made by encimation of gas chromatographic response.

Try summies consisting of homogenized tissues and parts from three groups of chickens, summited by the Invision of Nutrition, were examined for chick edems facte by a recently developed method One group had been fed a tation containing 89, of a reference Toxic fat. Another group had been fed a Tadio Contaming unsaporihanies enviem to 3% of the toxic reference fat. The last com reserved & ration free of toxic fat. The weights of tissue sampies ranged from 64 10 864 Crams. The samples were received in glass stoppered Erienmeyer fasts i ezhano..

1 Extract (Č Unse por:fiant matter from animal tissue (Modification of ADAC Offical Methać. 26.07.

ÚNENIILIVAT transer at Lizohohe solution of the homogenized tissues to a 24.90 round bitram flask väč echy Lizobial to give a final volume of 4 ni Fran tissue but not less that ặt mì. Add 2 mi KOH solution (3 + 2) per CLIL Of DISSTE.

Suplify by baling with, aceasing swirling or a steam bath for one hour under refux Lur candenser Transfer Lirabal song solution while still warm to SEPEZETOR US.ing Walet equivalent To Twice the volume of ethyl alcohol). Rinse SEÇƏLLİCEDOL flask with the same vacume of ethyl ether and transfer to sepaPetar. Shake vigorously, let invers segurate and clarify, breaking any emulsion by adding up 26 22 varmes if Liraha and swirling gently. Drain lower layer and pour ether layer through tig int & sevind separator containing water (2 1. tissue, but not less than 25 m. Make twr more extractions of soap soluDon with ethyl ether 8 mig tisseuse pouring edge with ethyl ether and Það rinsings to second seĢILDEDIO.

Butate combined ether extracts gerty with the 5-0 (violent shaking at this SCLFT ILAT CLuse troublesome enusions. Let larers separate and drain aqueous layer Wish with two additonal portions of H-0 (2 ml/g tissue), Shaking vigtevasky. Then wist echer solution two times with alternate portions 2 mg tissue of K00 and 5-0 H entision forms during washing, draiz as much of aqueous larer as possitie, leaving entision in separator with ether layer and proceed with DEX: Washing Wash final solution with H2O I wishings are dentral te zben;chalen

Transfer ether solution to erienmeyer, rinsing separator and its pouring edge with eben, göbing rinsings to men sizde. Try ether solution by adding aztpárvas N280, -1 £ £ tissue and swining vigtevasky ca 1 min. Let soluDie stand to min Treant echer seiztion tract class funnel containing pledges of pre-rinsed cotton in neck and bailing 25 er anhydrous Na,SO, into another erlenmeyer containing bolling chips. Wash first erlenmeyer with ether and transfer to second erienzeger

Evaporate most of solvent on steam bath and transfer to 100 ml (preweighed · extraction disk. Evaporate residual solvent on steam bath under N. Dry fask to constant weight and of tain weight of seponisable matter.

Proceed with fractionation of unsaponižable matter on alumina and cleanup of alumina fraction 3 as directed in method 1, ansiyning the residue by electron capture gas chromatography as directed.

RESULTS AND DISCUSSION

All residues except for liver samples were initially taken up in 100 ul of iso-octane and 5 u injected. The liver samples were taken up in 250 ul of isooctane and 1 tl injected Examination of the chromatograms indicated the presence of a small contaminant Ra 11.1 in the reagents used in the cleanup of the negative control samples.

Six out of 10 samples were "Toxic" from each group of chicks fed toxic fatty material The liver sample in each group was the most toxic", containing roughly 8% of the total toxicity as indicated in Table I. The terms "toxie" or "toxicity" as used here to describe results of gas chromatographic analyses refers to the amount of material in an individual tisse which produces characteristic peaks at Ra 12 and 22 resulting from feeding the toxic fat. Of the pooled adrenals, bone, brain, heart, intestine, kidney, liver, skeletal muscle, and testes that were examined, only the bone, heart, intestine, kidney,