EXTRACTION, ISOLATION, AND IDENTIFICATION HYDROLYTIC PRODUCTS OF TRIGLYCERIDE DIGESTION IN MAN

BY DAVID (From H. BLANKENHORN

AND

OF

EDWARD for

H.

AHRENS, Research,

JR.

the Hospital

of The Rockefeller Institute New York, New York) for publication, July

Medical

(Received

14, 1954)

Recent reports from various laboratories (l-9) indicate that the hydrolysis of triglycerides in the intestinal lumen proceeds from triglyceride + 1,2diglyceride -+ 2-monoglyceride and 1-monoglyceride, with liberation of 1 mole of fatty acid at each step. While much of the evidence in favor of this hypothesis has been gained through the use of analytical procedures of high order of specificity, in only two studies have the lower glycerides Mattson et al. (5) isolated and been isolated and conclusively identified. characterized monoglycerides from the intestinal contents of rats after test meals of fat. Kuhrt et al. (10) fed mixed natural foods to two human subjects; from intestinal contents aspirated at the ligament of Treitz, they isolated monoglycerides in high yield and proved the identity of this fraction conclusively. The pertinent information on fat digestion has been reviewed recently by BergstrGm and Borgstrijm (11). The present report describes the use of an improved intubation technique for sampling the contents of the human gut under physiological conditions and demonstrates the application of counter-current distribution to the extraction and isolation of the lipides contained in the aspirated samples. Two subjects have been fed balanced test meals containing defined triglycerides, and the various hydrolytic products of fat digestion have been isolated. Diglycerides as components of intestinal contents during fat digestion have been conclusively identified for the first time in man. Materials and Methods

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Extraction and Separation-Solvents were reagent grade, redistilled in glass. Preparative distributions were carried out in 1 liter flasks with phase volumes of 400 ml. Further separations were accomplished in a Craig all-glass, fully automatic, 200 cell distribution train with 10 ml. phase volumes (12). Solutes were recovered by low temperature vacuum distillation (13). The anion exchange resin, Amberlite IRA-400, was obtained in the basic form and was discharged with 20 per cent HCl in ethanol; it was extracted with ethanol-ether (1: 1) until free of residue which would partition itself into the upper phase of System A (Table I) ;
69

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TRIGLYCERIDE

DIGESTION

it was recharged with half saturated aqueous NaOH and washed until the wash water was neutral. Analyses-Gravimetric analyses were carried out as described by Craig et al. (14), with a semimicro balance accurate to ~0.01 mg. Fatty acids were weighed with the precautions previously described (15). Total cholesterol was determined by the method of Abel1 et al. (16) and lipide phosphorus by a modification of Stewart and Hendrys method (17). Pettenkofer tests were performed as described previously (18). Saponificabion equivalents were determined on weighed samples of approximately 10 mg., by saponifying with 0.1 N KOH for 16 hours at 80 in closed vessels and back-titrating samples and blanks with standard 0.1 N HCl. Infra-red spectra of specimens dried out of petroleum ether (30-60) on NaCl plates or in 1 per cent CS2 solutions were recorded with a Perkin-Elmer double beam recording spectrophotometer. Glyceride glycerol determinations were carried out by a modification of the method of Lambert and Neish (19) in which glycerol is oxidized in an acid medium at 20 for 5 minutes with periodic acid; the formaldehyde produced is measured by the method of MacFadyen (20). In order to free the glycerol for analysis a preliminary saponification step was performed in 9 volumes of ethanol with 1 volume of freshly prepared 0.7 N alcoholic KOH at 57 for 30 minutes. When carried out in 100 ml. volumetric flasks with the reagent quantities specified by Lambert and Neish, the limits of the method are 2 to 8 mg. of triglyceride, 1 to 4 mg. of diglyceride, and 0.6 to 2.4 mg. of monoglyceride, assuming an average fatty acid chain length of 18 carbons. These limits can be reduced four times without loss of accuracy by 4-fold reduction of the volumes of all reactants. Solvent blanks and a standard glycerol solution were included with each series of determinations. The glycerol standard was prepared from reagent grade glycerol, the concentration of which was determined by its specific gravity from the tables of Bosart and Snoddy (21) and checked by dichromate oxidation (22). Authentic samples of mono-, di-, and tripalmitin were shown to yield theoretical amounts of glycerol, f2.5 per cent. Reference Materials-Highly purified samples of 1-monopalmitin, 2monopalmitin, 1 ,3-dipalmitin, 1,2-dipalmitin, and tripalmitin (all synthetic) and the infra-red spectra of these materials and of authentic samples of mono-, di-, and triolein were kindly furnished by Dr. Willy Lange, The Procter and Gamble Company. From the same source a generous supply of synthetic triglyceride, primarily triolein, was procured for incorporation in test meals. Analysis of this fat by Dr. Lange showed an iodine value of 84.2, saponification equivalent of 291, and the following fatty acid composition: oleic 85.6, linoleic 3.0, linolenic 0.41, arachidonic

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0.04, saturated 6.5, trans acids 11.8 per cent. It contained no free fatty acids or non-saponifiable material. Corn oil (Mazola, Corn Products Refining Company), used in other feeding tests, was stated by the manufacturer (courtesy of Dr. A. R. Baldwin) to cont,ain 1.9 per cent nonsaponifiables and 98.1 per cent glycerides, with fatty acids as follows: linoleic 56.2, oleic 30.1, palmitic 9.9, stearic 2.9, and others 0.9 per cent. Its iodine value was 125 and the saponification equivalent 293. Intubation and Aspiration-Two male subjects, aged 27 and 43 years, who presented no clinical or radiologic evidences of gastrointestinal abnormalities, were intubated for periods of 24 and 6 days, respectively, during which time they had no discomfort or gastrointestinal symptoms and slept well. Throughout the study period they were fed a balanced maintenance diet (protein 15, plant fat 40, and carbohydrate 45 per cent of calories). An intubation technique was designed which minimized disturbances in intestinal function during hydrolysis and aspiration of gut contents. Plastic tubing (Pharmaseal K-30, 2.1 mm. outer diameter) was passed into the stomach through the nose by the Cantor technique (23) and was permitted to progress freely until it appeared at the anus. As further tubing progressed through the anus, it was cut off to a length convenient for taping over the sacrum. Solid foods aided in the transintestinal progress of the tube. The tube was then occluded by silk sutures, and three or four small aspiration holes were made proximal to the occlusion. As this portion of the tube approached the desired point of aspiration, oral feedings were changed from solids to a diet in which all nourishment was derived from a liquid formula. The advantages of complete formula feeding in metabolic studies are discussed elsewhere (24). The calories and composition remained unchanged in amount; fat was furnished by corn oil, protein by a salt- and fat-poor milk protein product (Lesofac, Wyeth), and carbohydrate by dextrose. Total salt intake was brought up to 6 and 4 gm. per day in the two patients by addition to the formula. The total daily intake of formula in each case was 2250 ml., divided into three portions fed 4 hours apart during the day, and water was permitted by mouth ad Zibitum. The subject,s were conditioned to complete formula feeding for 16 and 2 days, respectively, prior to the test. When the holes in the tube had reached the desired level in the intestinal tract, the test meal was fed by mouth, 50 ml. every 5 minutes for 55 and 70 minutes, respectively. The intestinal contents were aspirated by suction (Gomco thermotic drainage pump, 10 cm. of mercury pressure) for 4 hours. In one case the test meal was the same as the formula fed during the conditioning period, while in the other case synthetic triolein was substituted isocalorically for corn oil in the test meal and also in the

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TRIGLYCERIDE

DIGESTION

preceding feeding. Specimens were aspirated into packed in dry ice and thus were frozen within a few gut lumen. The specimens were stored at -20 position of the tube in the intestine was checked after completion of aspiration. Results

1 liter Tygon bottles seconds of leaving the until analyzed. The by x-ray examination

Extraction of Lipicles from Intestinal Contents-A two phase solvent extraction system was sought which would leave water-soluble non-lipide materials of an intestinal sample in the lower aqueous phase while extracting lipides quantitatively into the upper organic phase. A system composed of water, ethanol, ether, and heptane was found satisfactory for this purpose (System A, Table I). The sample of intestinal contents, acidified with hydrochloric acid to pH 3.5, was mixed successively with equal volumes of ethanol, of ether, and of heptane. This mixture, which settled into two phases of equal volume after equilibration, was treated as the first cell in a five cell distribution train. The lower phases in the other four cells were derived from a large batch of System A, previously equilibrated. 8 successive volumes of the upper phase of this system were carried stepwise across the five lower phases and were pooled. Milk protein, intestinal juice protein, glucose, and dextrose remained in the lower phases in the first two cells, while monopalmitin added to intestinal juice was quantitatively recovered in the pooled upper phases. Table I indicates the partition ratios in System A of some of the substances which might be anticipated in intestinal juice. It is apparent that the long chain fatty acids, glycerides, sterols, and sterol esters would be completely extracted from non-lipide materials in the lower phases. Only the most non-polar unconjugated bile acids would be extracted. In a further application of this preparative distribution procedure, the anion exchange resin, Amberlite IRA-400, was added in the basic form to the lower phases of Cells 4 and 5 prior to the distribution (20 gm. of wet resin per 100 ml. of the lower phase). As the extract phase was passed over these two raffinate phases, fatty acids were trapped by the resin and held in the lower phases, and the remaining lipides passed into the extract pool. The presence of the resin in Cells 4 and 5 did not appear to alter the partition ratios of monopalmitin or triolein significantly, since in pilot experiments these compounds passed into the extract pool quantitatively. To recover the acids from the resin, concentrated HCl in ethanol was added as displacer, and the organic acids then migrated back into the upper phase. During this displacement ethyl esters have sometimes been formed, and recovery has not, always been quantitative. Mattson and
1 Personal communication.

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Borgstrom have also had this experience. Discharging the resin with acetic acid in ether is stated by Borgstrom (25) to overcome these difficulties, but his suggestion has not been evaluated in this study.
Purtitio~n Ratios* o.f Various
TABLE I Cwmpounds in Five Distribution System A

Solvent

Systems

for

Counter-Current

Compound

System 0.231

system

system

System

.................. ..................... ..................... Monoolein ...................... Diolein ......................... Triolein. ........................ Corn oil monoglyceride .......... < diglyceride. ............ < triglyceride. ........... Laurie acid. .................... Stearic acid ..................... Palmitic acid. .................. Oleic acid ....................... Linolenic acid., ................. ................... Ethyl oleate. Cholesterol. .................... < palmitate ........... Glycocholic acid ................ Cholic acid. .................... Deoxycholic acid ................ Lithocholic acid ................. Mixed phosphatides from human serum ......................... Dextrose, glucose, milk protein, intestinal juice protein ........

Monopalmitin. Dipalmitin. Tripalmitin

11.5

0.06 1

2.20

.I

>20 >20

1.52t 8.06t 0.16t 1.217 1o.st

5.4 >20

>20 >20

0.16 12.5

>20
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0.07 't 1.7.F ;t 10.81 0.41 0.76

6.30 1.90

>20 >20
>20 >20 <0.05 0.09 0.70 5.5 0.60 <0.05 1.09t 8.511<0.05 <0.05 0.07 >20

1.87 1.9c 't 7.1a 'I 3.23

<0.05 <0.05

3.40 2.50 2.20 3.52 >20 <0.05

<O.OE 1 <O.OE 1 O.OE 1

<0.05

0.24

* Partition ratios (concentration in upper phase)/(concentration in lower phase) were determined in equal phase volumes of previously equilibrated solvents. t Ratios calculated from multiple transfer counter-current distributions. System A, heptane, ethyl ether, ethanol (95 per cent), water (equal volumes) ; System B, heptane, methyl Cellosolve, isoamyl alcohol (60:40:2 volumes) ; System C, heptane, acetonitrile, methanol, glacial acetic acid (4:l:l:l volumes, see also (15)); System D, heptane, ethyl ether, ethanol (95 per cent), 0.2 N NaOH (equal volumes) ; System E, heptane, glacial acetic acid, water (100:97.5:2.5 volumes, see also (18)).

The upper in the phases

capacity of IRA-400 to trap fatty acids and bile acids out of the phase of System A has been explored. Solvents and resin were used same proportions as described above 5 ml. of the upper and lower with 1 gm. of resin (wet weight). Increasing amounts of fatty

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TIUGLYCEI~IUE

DIGESTIOK

acids or non-polar bile acids were dissolved in the mixture, and after equilibration the upper phase was dried to determine it.s residue. It was found that 1 gm. of wet resin, prepared and used as described, trapped a maximum of 120 mg. of palmitic acid. On the other hand, the most nonpolar bile acid, lithocholic, partitioned itself between upper and lower phases with a K of 1.75, in contrast to K 5.5 when no resin was included in the lower phase. When 3 times as much resin was used, the K was lowered to 0.22. It is apparent that under these conditions the resin is not sufficiently strong to attract lithocholic acid entirely into the lower phase. However, three cells containing 60 gm. of IRA-400 per 100 ml. of the lower phase of System A would trap 99 per cent of any lithocholic acid present in the extract. Separation of Mono-, Di-, and Tripalmitin-A synthetic mixture of the three glycerides of palmitic acid was cleanly separated into its three components in the system heptane 60 parts, methyl Cellosolve 40 parts, isoamyl alcohol 2 parts (System B, Table I) after 213 transfers (Fig. 1). There was only slight skewing of the three curves, and glyceride glycerol analyses calculated in terms of the corresponding glyceride checked with the weight values at various points on the three curves within the limits of accuracy of the glycerol procedure. Homogeneity of Test Triglycerides-The homogeneity of the corn oil and synthetic triolein to be used in the feeding experiments was tested by distribution of the two fats in System B. Both triglycerides appeared as single symmetrical peaks with K values of 9.5 and 7.7, respectively, and with slightly broader than theoretical curves. Glyceride glycerol analyses throughout the remainder of the distribution train were negative, denoting the absence of lower glycerides in these triglyceride samples. Non-glyceride contaminants of corn oil (1.9 per cent) were found to be more polar than the triglyceride itself; several incompletely separated bands with extremely small amounts of solute had K values of 0.6 to 2.2. The triolein sample was also distributed in System A over the resin IRA-400. Nothing was recovered from the resin when it was discharged and extracted, denoting the absence of free fatty acids. The triolein was recovered in 100 per cent yield from the pooled extracts and distributed in System B. Only one peak of solute was demonstrated. It was concluded that the extraction procedure, exposure to resin, and subsequent recovery of solute out of the solvents of System A had not produced hydrolysis of triglyceride to lower glycerides. Isolation of Lower Glycerides in Intestinal Digestion of Corn Oil-Patient 1 was fed 25 gm. of corn oil homogenized in 550 ml. of balanced formula, and over a 4 hour period 180 ml. of intestinal juice were aspirated at a point 17 inches below the ligament of Treitz in a small bowel measuring

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112 inches. The frozen specimen was rapidly thawed and brought to pH 3.5 with HCl, and the entire specimen was distributed in System A. IRA400 was not used in this distribution. Mixed solutes recovered from pooled extracts weighed 1324 mg. and included 34 mg. of cholesterol and 0.73 mg. of phosphorus. The solute mixture was distributed in System C (Table I). After 195 The band with transfers gravimetric analysis showed three major bands. K = 0.074, when data for glyceride glycerol and weight were compared, was considered to be a Cl*-monoglyceride. The band with K = 10.8 was calculated similarly to be C&triglyceride. The solute recovered from
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0
FIG.

20

40

60

) 80 100 120 140 16( Tube number

mixture of mono-, di-,

180 200
and tripalmitin in

System

1. Distribution B.

of a synthetic

these two bands weighed 167 and 45 mg., respectively; further identification was not attempted. The middle and major bands had two peaks with K = 1 and 1.5. The mixed solute in this band weighed 899 mg. on recovery. The K values and other analyses indicated a mixture of C18diglyceride, free fatty acids, bile acids, and cholesterol. Therefore, the mixture was separated by eleven transfers in System D (Table I) into non-acidic and acidic fractions, weighing on recovery 155 and 757 mg., respectively. The non-acidic fraction was taken up in ethanol-ether and cholesterol was removed with digitonin. Petroleum ether (3:1), (30-60) was added to the filtrate, and excess digitonin was removed by washing repeatedly with water. The solute recovered from petroleum ether was an oil (110 mg.), having a saponification equivalent of 316. Glyceride glycerol analyses were consistent wit,h the postulation that

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TRIGLYCERIDE

DIGESTION

this material was Cl*-diglyceride. The infra-red spectrum of this oil was not significantly different from that of authentic diolein. The acidic fraction from the distribution in System D was recovered into ether after acidification of the alkaline lower phase. It was distributed in System E (Table I) in order to separate the fatty acids from the more polar compounds. After eleven transfers there was clear-cut separation of a polar fraction in Tubes 0 to 2 (48 mg.) from the fatty acid fraction in Tubes 3 to 10 (710 mg.). The latter fraction was a colorless oil, volatile at 100 and 1 mm. pressure, which had an infra-red spectrum consistent with published spect$ra of C&unsaturated fatty acids (26). The small polar fraction gave a positive Pettenkofer reaction and may have included bile acids; it was not studied further. To summarize these experiments, after 25 gm. of corn oil were fed by mouth, 1300 mg. of mixed lipides were extracted from upper jejunal contents. Mono-, di-, and triglycerides were isolated in amounts of 167, 110, and 45 mg., respectively, together with 710 mg. of mixed fatty acids and 34 mg. of cholesterol. Isolation of Lower Glycerides in Intestinal Digestion of Triolein-Patient 2 was fed 40 gm. of synthetic triolein homogenized in 720 ml. of balanced formula, and over a 4 hour period 300 ml. of intestinal juice were aspirated at a point 30 inches below the ligament of Treitz in a small bowel measuring 90 inches. The frozen specimen was rapidly thawed and a 200 ml. aliquot was withdrawn; the pH was taken to 3.4 with HCI, and the entire specimen was distributed in System A over the anion exchange resin, IRA-400, 40 gm. per 400 ml. of lower phase. From the pooled extract 976 mg. of non-acidic lipides were recovered, including 60 mg. of cholesterol and 0.014 mg. of phosphorus. The solute mixture was distributed in System B, and after 195 transfers three well defined bands were demonstrated (Fig. 2). The material with K = 0.164 showed an excellent fit for monoolein when glyceride glycerol data were so calculated. 396 mg. of oil were recovered, with a saponification equivalent of 327. The infra-red spectrum of this oil was identical with that of authentic monoolein, except for the presence in the former of a band at 10.85 rnp due to trans acids. This band was also present in the synthetic triolein (11.8 per cent trans acids) fed to the patient. After crystallization from acetone at -4 and drying at 100 for 2 hours over PZ06 in high vacuum, elementary analyses were as follows: C 70.32, H 11.56, ash 2.75 per cent (theoretical for monoolein, C 70.8, H 11.3). The band with K 11.6 contained a small amount of esterified cholesterol equivalent8 to 25.5 mg. if calculated as cholesterol oleate. Excellent agreement was found between the total weight curve and the curve for

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glyceride glycerol calculated as triolein, when the 14 per cent contamination by esterified cholesterol was taken into consideration. 180 mg. of mixed solutes were recovered, which had an infra-red spectrum resembling that of the triolein originally fed to the patient. The intermediate band (K = 1.2) had a shoulder on the left due to a At other points on the curve the glycsmall amount of free cholesterol. eride glycerol-weight data supported the assumption that the main part of this band was diolein. The solute recovered from Tubes 78 to 130 this fraction (35 mg.) weighed 175 mg. The cholesterol contaminating The was removed with digitonin as described in the preceding section.
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20 40

60 80 100 120 140 160 180 200 Tube number

lipides (System extracted The B). from the intestinal K values of major contents bands are

FIG. 2. Distribution of non-acidic of Patient 2 after triolein feeding 0.164, 1.2, and 11.6 (left to right).

infra-red spectrum of the residual oil was identical with that of authentic diolein, except for the presence in the former of a band at 10.85 rnp due to trans acids, found also in the triolein fed to the patient. We were not successful in crystallizing this material, even at low temperatures. Subsequently the entire fraction was hydrogenated and then saponified, showing a saponification equivalent of 335. The fatty acids were recovered by ether extraction of the acidified saponification mixture and crystallized out of 95 per cent ethanol. After two crystallizations a melting point of 67.5 was obtained (theory 69.5). The X-benzylthiuronium salt of this material was prepared, which after two crystallizations melted at the temperature reported for this derivative of stearic acid (143) (27). The acids trapped by IRA-400 in the extraction of lipides from this aspirated specimen were freed from the resin by concentrated HCl in

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TRIGLYCERIDE

DIGESTION

ethanol. The recovered solute weighed 1506 mg. and gave a negative Pettenkofer reaction. The entire batch was distributed in System C (Table I), in which bile acids with very low K values would be clearly separated from long chain fatty acids (K of oleic acid = 1.9). Analysis after 195 transfers showed two bands at K 1.93 and K 7.1. There was no material with a low K, confirming the absence of bile acids. The highly non-polar solute (K 7.1) was subsequently identified as ethyl oleate on the basis of. its failure to bind base, the absence of glycerol, and its infra-red spectrum. The band at K 1.93 was identified as oleic acid with trans acid contamination on the basis of its K in this solvent system and its infra-red spectrum. Subsequent studies showed that the ethyl ester of oleic acid had been formed during the process of displacing it from the resin with HCl in ethanol, about 50 per cent of the acid having been converted to the ester in the above experiment. Thus, at least 1448 mg. of oleic acid must have been trapped on the resin in the original extraction-distribution in System A. Since we are not certain that recoveries of organic acids from the resin are quantitative under the conditions reported, this is a minimal figure. In summary of this experiment, when 40 gm. of synthetic triolein were fed by mouth, 1300 mg. of non-acidic lipides were extracted from midjejunal contents. From this were isolated 532 mg. of monoolein, 186 mg. of diolein, 186 mg. of triolein, 47 mg. of free cholesterol, and 33 mg. of cholesterol oleate. In addition, at least 1900 mg. of oleic acid were found.
DISCUSSION

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Separation of monoglycerides from diglycerides was reported by Zilch and Dutton (28) in the system, heptane-80 per cent ethanol, in twenty-four transfers. This procedure for isolating monoglycerides was adopted by Mattson et al. (5, 9), while Kuhrt et al. (10) used Skellysolve B-85 per cent methanol with similar effectiveness. The present report describes two solvent systems (B and C, Table I) in which excellent separation of the three glyceride classes from each other has been attained and a third system (E) in which monoglycerides are separated from higher glycerides in less than ten transfers. The isolation of each glyceride class from the complex mixture of lipides present in the intestinal lumen during fat digestion is, however, complicated by the fact that the distribution characteristics of long chain fatty acids are similar to those of long chain diglycerides. To overcome this problem, Borgstrom (29) admitted such mixtures to a column containing the anion exchanger, IRA-400, in order to adsorb the fatty acids. In our experiments IRA-400 was used for the same purpose as one phase of a three phase distribution. There are, however, unexplored hazards of sorption and desorption of trapped fatty

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acids from the resin, and the attainment of resin from which undesirable residues are not extractable is always uncertain. It seemed preferable and no more laborious to distribute the fatty acids along with the glycerides and at a later stage to separate the diglycerides from the fatty acids which overlap them in all systems studied so far. This can be accomplished quantitatively in eleven transfers in the alkaline System D (Table I). Whether isomerization of unstable lower glycerides (2-monoglycerides and 1,2-diglycerides) occurs during counter-current distribution, as Borgstrom (30) has found to occur on silicic acid columns, has not been investigated. Monoglycerides could be isolated from diglycerides and triglycerides without isomerization of monoglycerides by partition chromatography (30). In the present experiments diglycerides have been characterized by their partition ratios in three counter-current distributions, by correspondence of glyceride glycerol and weight data, by infra-red spectra, and by identification of the fatty acid after hydrogenation, saponification, and formation of a derivative. For the first time diglycerides have been demonstrated in human intestinal contents during triglyceride digestion. The findings confirm the results of experiments in vitro and in vivo by other workers (5, 7, 9, 25, 31) and are consistent with the current belief that diglycerides originate from triglycerides after loss of 1 mole of fatty acid. However, the possibility must be seriously considered that diglycerides may appear after resynthesis from monoglycerides, since Borgstram (25, 31) has demonstrated conclusively that synthetic as well as transesterification reactions take place in the intestinal contents of rats. The present experiments offer no data relevant to this issue. Whether the diglycerides isolated were the 1,2 or 1,3 isomers cannot be answered at present. In pilot experiments we have not been able to separate mixtures of these compounds by countercurrent distribution. Our results form an interesting contrast to those of Kuhrt et al. (lo), who reported 37 to 50 per cent of intestinal lipides to be monoglycerides when aspiration was carried out at the ligament of Treitz. Our experiments were made on aspirated samples recovered distal to the ligament of Treitz (15 and 33 per cent of the jejuno-ileal length) and showed that only 13 and 17 per cent of the lipides were monoglycerides, 9 and 6 per cent diglycerides, 4 and 6 per cent triglycerides, 58 and 60 per cent fatty acids, respectively. It seems probable that the discrepancies between the data of Kuhrt et al. and ours in human subjects will be resolved when repeated aspirations are made in a single subject at various locations in the gut, an objective which seems attainable with the present intubation technique.

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TRIGLYCERIDE

DIGESTION

SUMMARY

Methods by which lipides are quantitatively extracted from intestinal contents and further separated into fa.tty acid, bile acid, and mono-, di-, and triglyceride fractions are described. Samples of intestinal contents have been aspirated from two healthy human subjects after test meals containing defined fats, and the various products of fat hydrolysis have been isolated. Diglycerides have been positively identified for the first time as components of intestinal contents during fat digestion in human subjects. The authors gratefully acknowledge the invaluable Herbert Jaffe in interpretation of infra-red spectra.
BIBLIOGRAPHY

assistance

of Dr.
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1. Frazer, A. C., and Sammons, H. G., &o&em. J., 39, 122 (1945). 2. Desnuelle, P., Bull. Sot. chim. biol., 33, 909 (1951). 3. Reiser, R., Bryson, M. J., Carr, M. J., and Kuiken, K. A., J. Biol. Chem., 194, 131 (1952). 4. BorgstrGm, B., Acta them. &and., 7, 557 (1953). 5. Mattson, F. H., Benedict, J. H., Martin, J. B., and Beck, L. W., J. Nutr., 48, 335 (1952). 6. Schgnheyder, F., and Volqvartz, K., Biochim. et biophys. acta, 8, 407 (1952). 7. Desnuelle, P., and Constantin, M. J., Biochim. et biophys. acta, 9, 531 (1952). 8. Harris, R. S., Chamberlain, J. W., and Benedict, J. H., Federation Proc., 13, 525 (1954). 9. Mattson, F. H., Benedict, J. H., and Beck, L. W., J. Nutr., 62, 575 (1954). 10. Kuhrt, N. H., Welch, E. A., Blum, W. P., Perry, E. S., Weber, W. H., and Nasset, E. S., J. Am. Oil Chem. Sot., 29, 217 (1952). 11. Bergstrijm, S., and BorgstrGm, B., Acta sot. med. Upsaliensis, 58, 331 (1953). 12. Craig, L. C., Hausmann, W., Ahrens, E. H., Jr., and Harfenist, E. J., Anal. Chem., 23, 1236 (1951). 13. Craig, L. C., Gregory, J. D., and Hausmann, W., Anal. Chem., 22, 1462 (1950). 14. Craig, L. C., Hausmann, W., Ahrens, E. H., Jr., and Harfenist, E. J., Anal. Chem., 23, 1326 (1951). 15. Ahrens, E. H., Jr., and Craig, L. C., J. Biol. Chem., 195, 299 (1952). 16. Abell, L. L., Levy, B. B., Brodie, B. B., and Kendall, F. E., J. Biol. Chem., 196, 357 (1952). 17. Stewart, C. P., and Hendry, E. R., Biochem. .I., 29, 1683 (1935). 18. Ahrens, E. H., Jr., and Craig, L. C., J. Biol. Chem., 196, 763 (1952). 19. Lambert, M., and Neish, A. C., Co.nud. .I. Res., Sect. B, 28, 83 (1950). 20. MacFadyen, D. A., J. BioZ. Chem., 168, 107 (1945). 21. Lawrie, J. W., Glycerol and the glycols, American Chemical Societ,y monograph series, New York, 158 (1928). 22. Bragdon, J. H., J. RioZ. Chem., 190, 513 (1951). 23. Cantor, M. O., Intestinal intubation, Springfield, 122 (1949). 24. Ahrens, E. II., Jr., Dole, V. P., and Blankenhorn, D. H., Am. J. CZin. Nutr., 2, 336 (1954).

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