THE D-a-HYDROXY FATTY ACID NUTRITION OF LACTOBACILLUS CASE1 280-16”

BY MERRILL N. CAMIEN AND MAX S. DUNN

(From the Chemical Laboratory, University of California, Los Angeles, California)

(Received for publication, June 30, 1954)

Lactobacillus casei 280-16, described previously (I), is a n-lactic acid- requiring variant of strain No. 7469 of the American Type Culture Col- lection. The parent strain satisfies its need for n-lactic acid apparently through the racemization of L-lactic acid, an abundant fermentation prod- uct of both organisms (1). It was of interest to continue the investiga- tion of L. casei 280-16 because its unique nutritive requirement for n-lactic acid implies a previously unrecognized essential function for this compound. It is noteworthy that an ultrasensitive compound-microbiological assay method based on the use of this organism has been reported (2).

The present report is concerned with the nutritional activity of n-lactic acid homologues and analogues for L. casei 280-16 and with the apparent occurrence of some of these substances in natural materials.

EXPERIMENTAL

The assay procedure was essentially the same as that employed previ- ously (l), except that the medium was simplified by eliminating the acid- hydrolyzed casein, nn-tryptophan and n-cysteine and by increasing the final concentration of the trypsinized casein to 600 mg. per cent. A mini- mal autoclaving time (1 to 5 minutes at 15 pounds) was employed to steri- lize the assay tubes because prolonged heating produced an effect com- parable to that of introducing n-lactic acid into the assay medium (Fig. 1). Evidently this effect was the result of decomposition of glucose, since it was not apparent when the glucose was autoclaved in a separate solution and added aseptically to the previously autoclaved and cooled assay tubes. The initial pH of the medium was left unadjusted (approximately pH 6.8) unless otherwise noted, but it was observed that considerable change in the initial pH of the medium had a marked effect on both the absolute and relative responses to active substances (Fig. 2).

Preparation of Deaminated Amino Acids-Deaminated amino acids were employed as a source of oc-hydroxy acids in preliminary experiments, since relatively few of the latter mere initially available. 1 mmole of amino acid

* Paper 100. This work was aided by grants from Eli Lilly and Company, the Nutrition Foundation, Inc., and the University of California. The authors are in- debted to Miss Lynn Wyler for technical assistance.

593

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594 D-a-HYDROXY FATTY ACID NUTRITION

was dissolved in 5 ml. of 3 N hydrochloric acid, 5 ml. of 3 N sodium nitrite solution were cautiously added, and the mixture was allowed to stand over- night, at room temperature. It was assumed that the amino acid Ivas con- verted nearly quantitatively to the corresponding hydroxy acid by this procedure, and the resulting reaction mixture was assayed directly for growth-promoting activity. The relative growth-promoting potencies of various deaminated amino acids are given in Table I.

15

IO

30 60 FIG. 1 FIG. 2

FIG. 1. Response of L. casei 280-16 to D-lactic acid, with 1 minute (Curve A), 15 minutes (Curve B), and 34 minutes (Curve C) of autoclaving at 15 pounds to sterilize the assay tubes with the n-lactic acid-supplemented test medium. The values on the horizontal scale are the millimicromoles of D-lactic acid per ml. of medium. Those on the vertical scale are the titration values (ml. of 0.01 N sodium hydroxide per ml. of culture).

FIG. 2. Response of L. casei 280-16 to DL-lactic acid (Curves A, A’) and fl-phenyl- DL-lactic acid (Curves B, B’) in the unadjusted medium (Curves A, B) and in the same medium adjusted to pH 5.5 (Curves A’, B’). The values on the horizontal scale are the millimicromoles of DL acid per ml. of medium. Those on the vertical scale are the titration values (ml. of 0.01 N sodium hydroxide per ml. of culture).

PuriJied ac-Hydroxy Acids and Related Compounds-m-or-Hydroxybutyric, m-cr-hydroxyvaleric, and m-a-hydroxycaproic acids were made available through the courtesy of Dr. Carl G. Baker and Dr. Alton Meister. Samples of m-or-hydroxypalmitic, m-ar-hydroxystearic, and m-ar-hydroxybehenic acids were kindly provided by Dr. A. C. Chibnall. The &phenyl-L-lactic and p-phenyl-m-lactic acids were the same as those described previously (3). D-Lactic, L-lactic, m-ar-hydroxy-a-methylbutyric, m-a-hydroxyca- prylic, m-ar-hydroxycapric, m-a-hydroxylauric, and m-cu-hydroxymyris- tic acids were prepared as described below. The remaining compounds were commercially available products. The relative growth-promoting potencies of all the materials tested are given in Table II.

D-Lactic acid was conveniently prepared by fermenting with Lacto-

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M. N. CAMIEN AND M. S. DUNN 595

bacillus delbrueckii (ATCC No. 9649), a mixture containing 5.4 per cent glucose, 3 per cent calcium carbonate, 0.5 per cent trypsinized casein, 0.5 per cent yeast extract (Difco), and 0.05 per cent autolyzed yeast (Difco) in distilled water. The glucose was dissolved and sterilized separately from the remaining constituents of the medium. The inoculated mixture was incubated for 10 days at 35”, steamed for 30 minutes at loo”, clarified by

TABLE I Relative Growth-Promoting Potencies of Deaminated Amino Acids*

Deaminated amino acid

nn-Alaninet m-or-Aminobutyric acidt rm-or-Aminocaprylic acidt oc-Amino-oc-ethylbutyric acid1 or-Aminoisobutyric acid$ nn-Aspartic acidi nn-Citrulline$ nn-Cystines nn-Ethionines L-Ethioninet nn-Glutamic acids Glycinell nn-Histidine$

Potency Deaminated amino acid Potency

100

66 215

20 3 0 0 0

31 28 0 0 0

nn-Isoleucinet 277 L-Isoleucinejj 7 nn-Leucinet 269 L-Leucine[/ 10 nn-Lysinei 0 nn-Methionines 28 n-Methioninet 14 DL-Norleucinet 253 nn-Norvalinet 195 nn-Omithines 0 nn-Serines 0 nn-Threonines 0 DL-Valinet 80 L-Valinell 7

* The amino acids were deaminated and tested as described in the text. The potency of 100 was assigned to deaminated rm-alanine, and the potency value for each of the remaining deaminated amino acids was obtained by multiplying by 100 the ratio between the molar concentrations of deaminated nn-alanine and test amino acid required to yield half maximal growth of L. casei 280-16.

t Possesses all four essential characteristics (see the text). $ Lacks two or more essential characteristics (see the text). 5 Lacks the second essential characteristic (see the text). [I Lacks the first essential characteristic (see the text).

passing (while still hot) through a Sharples supercentrifuge and a large Seitz filter, and concentrated under reduced pressure to one-fourth its initial volume. The calcium n-lactate which crystallized from the re- sulting solution upon standing in the cold was collected by filtration, re- crystallized twice from 50 per cent (by volume) acetone in water, and dried to constant weight at 70” in a vacuum oven. 105 gm. of purified anhydrous calcium n-lactate were obtained in this manner from 4 liters of fermented mixture. Calcium, found by ashing the product at 800-850” in the pres- ence of sulfuric acid and weighing the resulting anhydrous calcium sulfate,

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596 D-&HYDROXY FATTY ACID NUTRITION

was 18.43 per cent. That calculated for C6H100&a was 18.37 per cent. The specific rotation was [a] E5.” f5.32” f 0.05” in water at c = 4.95.

Calcium n-lactate was prepared by the same procedure, except that L. casei (ATCC No. 7469) was employed to ferment the mixture. Calcium found, 18.41 per cent; [LY]~~” -5.32” f 0.03” in water at c = 4.95.

nn-a-Hydroxy-cr-methylbutyric acid, nn-oc-hydroxycaprylic acid, DL-a~-

hydroxycapric acid, nn-a-hydroxylauric acid, and nn-cr-hydroxymyristic

TABLE II

Relative Potencies of PuriJied oc-Hqdroxy Acids and Related Compounds*

Compound

Acrylic acid Cinnamic acid Citric acid1 Crotonic acid Glycolic “ [j nn-&Hydroxybehenic acid7 nn-a-Hydroxybutyric “ t nn-@-Hydroxybutyric “ nn-a-Hydroxycapric acidt nn-a-Hydroxycaproic acidt nn-a-Hydroxycaprylic acidt a-Hydroxyisobutyric acid$ nn-a-Hydroxylauric acidt nn-a-Hydroxy-a-methylbutyric acid** on-a-Hydroxymyristic acidt nn+Hydroxypalmitic “ f nna-Hydroxystearic acid7

- t? B Lz -

Compound

0 nn-cr-Hydroxyvaleric acidt 0 or-Ketobutyric acid 0 L-Lactic acid11 0 nn-Lactic acid+ 0 D-Lactic acidt 0 nn-Lactic acid ethyl ester

75 nn-Malic acids 0 nn-Mandelic acid1

143 meso-Tartaric acids 133 Oleic acid 140 &Phenyl-n-lactic acid/j

0 p-Phenyl-nn-lactic acid1 154 &(p-Chlorophenyl)-nn-lactic acids

0 Phenylpyruvic acid 46 Pyruvic acid

0 Stearic acid 0 nn-Tartaric acidi

252 0 0

100 200

2 0

127 0 0 0

61 0 8 0 0 0

* The potency of 100 was assigned to nn-lactic acid, and the relative potencies of the other compounds were calculated as in Table I. See Table I for an explanation of the symbols other than those listed below.

7 Lacks the third essential characteristic (see the text). ** Lacks the fourth essential characteristic (see the text).

acid were prepared by a-bromination of the corresponding acids, followed by hydrolysis of the resulting a-bromo acids with sodium hydroxide solu- tion. 50 gm. of each acid were refluxed with bromine in 120 per cent excess of the required amount and in the presence of red phosphorus. Heating at 100” overnight was sufficient to complete the reaction with the lower mem- bers of the series, but the lauric and myristic acid reaction mixtures were heated 2 more days at IOO”, and the latter mixture was heated additionally at 110-130” overnight. The resulting ol-bromo acids were isolated from the reaction mixtures and purified by double distillation under reduced pres-

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M. N. C.iMIEN AND M. S. DRiN 597

sure. The cY-bromolauric acid and cr-bromomyristic acid were white crys- talline solids at room temperature, whereas the lower members of the series were water-white oily liquids. Hydrolysis to the corresponding cr-hydroxy acid was accomplished by dissolving the cr-bromo acid in an equimolar amount of standardized N sodium hydroxide, heating the resulting solution nearly to boiling, and adding a second equimolar amount of N sodium hy- droxide at a rate sufficient t.o keep t,he mixture slightly alkaline to bromo- thymol blue. Complete hydrolysis was insured by adding a 10 per cent excess of N sodium hydroxide and refluxing the mixture overnight, The hydrolysate was saturated with ether and the calculated amount of stand- ardized 6 N hydrochloric acid was added to neutralize the excess sodium hy- droxide and to convert the oc-hydroxy acid salt to the free acid. The mix- ture was extracted with consecutive portions of ether until the acidity remaining in the aqueous phase was negligible, 100 ml. of chloroform were added to the combined ether extracts, and the solvent mixture was evap- orated to dryness at room temperature in a stream of air. The residual crystalline ol-hydroxy acid Teas recrystallized twice from chloroform. The equivalent weight of the purified compound was determined by titrating it in alcohol solution with standardized 0.1 N sodium hydroxide to the end-point of phenolphthalein indicator. The equivalent weights were as follows (formula weights are given in parentheses) : m-a-hydroxy-a-methyl- butyric acid 118.1 (llS.l), nn-a-hydroxycaprylic acid 160.8 (160.2), DL-CY-

hydroxycapric acid 188.7 (188.3), nn-a-hydroxylauric acid 216.4 (216.3), and m,-ol-hydroxymyristic acid 242.5 (244.4). The over-all yields of twice recrystallized products were 21, 21, 14, 12, and 27 per cent, respectively, of the theoretical amounts.

Butter Fat Fatty Acid Mixture-Crude butter fat was isolated from fresh cream as a yellow oil by high speed centrifugation. The oil was filtered at about 40”, dissolved in 2 volumes of anhydrous ether, and precipitated from this solvent as a white granular solid by chilling the mixture to - 10”. The precipitated fat was furt,her purified by two reprecipitations from absolute ethanol. The granular product was washed thoroughly with cold distilled water and dried at room temperature in a vacuum desiccator over anhy- drous calcium sulfate. It was assumed that the product would have a lower content of unsaturated fatty acids (desirable since these acids are often markedly inhibitory) and a higher content of hydroxy acids than the original material. The pure white fat melted to yield a water-white oily liquid which \vas hydrolyzed by refluxing it (w&h mechanical stirring) for 20 hours wi-ith 15 volumes of A x hydrochloric acid. The layer of fatty acids (and possibly unhydrolyzed fat) which separated on standing was washed several times by shaking with hot water. The white waxy product (which solidified on the surface of the last wash water upon stand-

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598 D-a-HYDROXY FATTY ACID NUTRITION

ing at room temperature) was removed, blotted dry with filter paper, granulated, and stored in a refrigerator. Immediately before use an ali& quot of the fatty acid mixture was dissolved by suspending it in hot water and adding sufficient sodium hydroxide to form a neutral (to bromo- thymol blue) solution.

Wool WUJ: Hydrolysate-U. S. P. lanolin (3.2 gm.) was refluxed for 20 hours with 50 ml. of a 4 per cent solution of potassium hydroxide in abso- lute ethanol. The resulting mixture was diluted to 200 ml. with mater and was homogenously suspended by shaking before aliquots for testing were removed. This rigorous alkaline hydrolysis procedure was employed be- cause wool wax is notably refractory to mild hydrolysis conditions. It is recognized, however, that the cr-hydroxy fatty acids may have been racem- iced by this treatment.

RESULTS AND DISCUSSION

The growth-promoting potencies of the deaminated amino acids (Table I) were consistent with those of the purified cr-hydroxy acids and related compounds (Table II). It may be inferred from the results (Tables I and II) that the following characteristics are essential (under the stipulated conditions) to the growth-promoting activity of cr-hydroxy acids for L. casei 280-16: (1) asymmetry and D configuration, (2) absence of substitu- ents (excepting phenyl) other than the cr-hydroxyl and ar-carboxyl groups, (3) chain length of 14 or less carbon atoms, and (4) presence of an CY- hydrogen atom. The cr-hydroxy acids in Tables I and II, possessing all four of the essential characteristics, were active, whereas the other oc-hy- droxy acids lacking one or more of these characteristics were essentially inactive (relative potency less than 32).l

It is evident (Table II) that CX, P-unsaturated acids (acrylic, cinnamic, and crotonic acids), p-hydroxy acids (P-hydroxybutyric acid), and ar-keto acids (phenylpyruvic,2 pyruvic, and cr-ketobutyric acids) corresponding to active cw-hydroxy acids do not promote growth of L. casei 280-16.

That amino acids will not replace u-a-hydroxy acids for L. casei 280-16 has been indicated by the extremely low activity of DL- and n-alanines (1) and of other m-amino acids (unpublished data). It is of interest that, con- versely, the n-alanine requirement of L. casei in a vitamin BG-free me-

1 It is noteworthy that the purified cr-hydroxy acids lacking essential characteris- tics (Table II) were in all cases entirely inactive, whereas the deaminated amino acids theoretically lacking essential characteristics (Table I) often were slightly active. It seems likely, therefore, that these activities may have been due to side products of deamination.

2 The slight activity of phenylpyruvic acid has been discounted because of the probability that this product contained active impurities.

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M. N. CAMIEN AND M. S. DUNN 599

dium (4) was not alleviated by n-lactic acid (Fig. 3), although the response to n-alanine was apparently stimulated by the n-lactic acid supplement.

40 80

FIG. 3. Response of L. casei to n-alanine in the medium described below (Curve A) and in the same medium supplemented with 1 pmole of n-lactic acid per ml. (Curve B). The medium was the same as that described in the text, except that vitamin Be was omitted and a vitamin-free trypsinized casein preparation was substituted for the N-Z-Case. The values on the horizontal scale are the millimicromoles of D-

alanine per ml. of medium. Those on the vertical scale are the titration values (ml. of 0.01 N sodium hydroxide per ml. of culture).

TABLE III

or-Hyldroxy Acid Concentrations Yielding Half Maximal Growth in Tween .bO- Supplemented Media

I Tween 40 supplement, mg. per cent

Test acid 0 I

4 I

16

Concentration of test acid yielding half maximal growth, m~moles per ml.

nL-Lactic acid. nL-a-Hydroxyvaleric acid.. nL-a-Hydroxycaproic “ nL-a-Hydroxycaprylic “ . nL-oc-Hydroxycapric “ nL-cu-Hydroxylauric “ nL-oc-Hydroxymyristic acid, .

. 71.0 25.2 35.0 36.1 8.7 12.6 24.7 5.3 7.3 23.5 4.9 6.8 49.7 8.7 10.4 46.0 7.0 9.4

153 10.6 10.6

The relative potencies of the straight chain cu-hydroxy acids (Table II) generally increased with increase in chain length up to 8 carbon atoms, and decreased with further increases until complete inactivity was reached with the CIG acid. Markedly elevated activities, particularly of the longer chain acids, were observed in media supplemented with Tween 403 (Table

3 Atlas Powder Company product.

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600 D-a-HYDROXY FATTY ACID NUTRITION

III), and it may be calculated that the 4 mg. per cent supplement of this re- agent resulted in 2.8, 4.1-, 4.7-, 4.8-, 5.7-, 6.6-, and 14.4-fold increases in activity, respectively, for the Cs through CM acids as listed in Table III. These results are in accord with the following hypotheses: (a) The permea- bility of the bacterial cell membrane is progressively less for a-hydroxy acids of increasing chain length, (b) the permeability of the membrane is increased in media supplemented with Tween 40, and (c) the intrinsic ac- tivities of a-hydroxy acids increase w&h increasing chain length up to and beyond 8 carbon atoms (whereas the observed activities, being influenced by permeability, fall off with increasing chain lengths above 8 carbon atoms). Hence the essential metabolites derived from the nutritionally active CY- hydroxy acids may be long chain a-hydroxy acids such as those occurring in mammalian cerebrosides, and it is of interest that brain cerebronic acid has recently been shown to be of the D configuration (5).

The slope of the m-ac-hydroxymyristic acid response curve was positive with either low or high concentrations of this nutrient in media supple- mented with Tween 40, but was negative with intermediate concentrations (Fig. 4). It seems likely that growth at low concentrations of DL-CC-

hydroxymyristic acid was largely that of a secondary mutant characterized by increased permeability to longer chain hydroxy acids, that growth at high concentrations was largely that of the unaltered L. casei 280-16, and that growth at intermediate concentrations was mixed. That DL-cr-hy- droxymyristic acid is the highest nutritionally active member of the homol- ogous series (Table II) may account for these exceptional results (not observed with other Lu-hydroxy acids).

Hydrolyzed butter fat exhibited marked nutritional activity for L. casei 280-16, a definite response being evident with less than 7 y of hydrolysate per ml. of culture (Curve A, Fig. 5). The inhibition with more than 27 b of hydrolysate per ml. was thought to be due to oleic acid and related un- saturated fatty acids. An increased concentration of butter fat hydroly- sate was required for either growth stimulation or inhibition in media supplemented with Tween 40 (Curves B and C, Fig. 5). The explanation is not evident but it seems likely that soaps of the commonly occurring fatty acids present in the butter fat hydrolysate may have influenced the permeabi1it.y of the bacterial cell membrane. That such fatty acids are otherwise inactive was suggested by the inactivity of oleic and stearic acids (Table II).

Apparently at least 2.8 per cent of bhe butter fat hydrolysate consisted of n-a-hydroxy acids since the lowest concentrat’ion of a purified a-hydroxy acid yielding half maximal growt,h was 4.9 mpmoles of m-oc-hydroxyca- prylic acid (equivalent to 0.39 y of the D acid) per ml. (Table III), whereas approximately the same growth was obtained with 14 y of hydrolysate per

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M. N. CAMIEN AND M. S. DUNN 601

ml. (Curve A, Fig. 5). It is of interest in this connection that an un- identified optically active isomer of monohydroxypalmitic acid has been isolated from butter fat (6), although it does not seem likely that this com- pound contributed to the activity observed for butter fat hydrolysate (since nn-a-hydroxypalmitic acid was inactive (Table II)).

Fresh whole cream, employed as a source of emulsified unhydrolyzed butter fat, was nutritionally inactive for L. casei 280-16, indicating that this organism either cannot absorb the unhydrolyzed fat or cannot split

9

8

6

3 4

7 70 700 FIG. 4 FIG. 5

FIG. 4. Response of L. casei 280-16 to DL-o(-hydroxymyristic acid in the control medium (Curve A) and in the same medium supplemented with 4 mg. per cent (Curve B) and 16 mg. per cent (Curve C) of Tween 40. The values on the horizontal scale are the millimicromoles of DL-a-hydroxymyristic acid per ml. of medium. Those on the vertical scale are the titration values (ml. of 0.01 N sodium hydroxide per ml. of culture).

FIG. 5. Response of L. casei 280-16 to butter fat hydrolysate in the control medium (Curve A) and in the same medium supplemented with 4 mg. per cent (Curve B) and 16 mg. per cent (Curve C) of Tween 40. The values on the horizontal scale are the micrograms of hydrolysate per ml. of medium (plotted logarithmically). Those on the vertical scale are the titration values (ml. of 0.01 N sodium hydroxide per ml. of culture).

it. It may be noted that simple ester linkages are not split by L. casei 280-16, as evidenced by the inactivity of m-lactic acid ethyl ester (Table II).

The finding of specific growth-promoting activity for L. casei 280-16 in hydrolyzed butter fat is of interest because some research workers have long held that butter fat may possess a special nutritive value not found in certain vegetable fats. Results supporting this view (7) might be explained by the hypothesis that the n-cY-hydroxy fatty acid content of butter fat is responsible for the stimulation of rat growth by this product, but not by corn oil, in diets containing lactose. That growth was satisfactory in the

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602 D-a-HYDROXY FATTY ACID NUTRITION

absence of butter fat with diets containing carbohydrates other than lac- tose (7) may indicate that the lactose diet yielded an intestinal flora favor- ing the production of L-lactic acid, but little or no n-lactic acid which could serve as a precursor to the higher n-a-hydroxy fatty acids.

The response of L. casei 280-16 to lanolin hydrolysate (Fig. 6) was antici- pated because wool wax has long been noted for its unusual content of hydroxy acids and more specifically because the presence of ar-hydroxy- lauric and Lu-hydroxymyristic acids (nutritionally active, Tables II and III) in hydrolysates of this product has been established (8, 9). It seems likely that the a-hydroxy acids of wool wax are of the D series (5, 9), but alkaline hydrolysis (see “Experimental”) may have racemized these acids in the present experiments.

FIG. 6. Response of L. casei 280-16 to wool wax hydrolysate. The values on the horizontal scale are the mg. of hydrolyzed wool wax per ml. of medium. Those on the vertical scale are the titration values (ml. of 0.01 N sodium hydroxide per ml. of culture).

The mutant strain of L. casei employed in the present investigation appears to differ from its parent solely in its inability to produce n-lactic acid (1). It seems likely, therefore, that the metabolic fate of n-lactic acid produced by the normal L. casei strain is the same as that of the D-

lactic acid (or other n-a-hydroxy acid) supplied in the L. casei 280-16 growth medium. The present evidence suggests that n-lactic acid serves in L. casei, and probably in other species also, as a metabolic precursor to long chain n-a-hydroxy acids, and in this regard the following se- quence appears to be tentatively acceptable: glucose + pyruvic acid

L-lactic dehydrogennse lactic racemase ------t I,-lactic acid-----------+ u-lactic acid +

CH~(CH~).CHOHCOOH (intermediates of D configuration) + CHS(CHP).- CHOHCOOR (essential lipide, containing estcrified long chain n-a-hydroxy fatty acid). According to this scheme, L. casei 280-16 fails to grow in unsupplemented media because it lacks the racemase essential in the step

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M. N. CAMIEN AND M. S. DUNN 603

yielding n-lactic acid. Supplying n-lactic acid or some other absorbable (n-cr-hydroxy acid) intermediate permits growth. The essential lipide re- sulting from this metabolic sequence might be expected to be a cerebroside. It is of interest, therefore, that yeast cerebronic acid is dextrorotatory in pyridine and is essentially of the same composition4 as brain cerebronic acid (10). Hence, it is evidently of the D configuration (5). That the de- hydrogenase and racemase steps may occur separately as indicated in the proposed sequence has been shown by Kitahara et al. (11).

It seems apparent from the preceding discussion that the cY-hydroxy acids of brain cerebronic acid, yeast cerebronic acid, wool wax, and butter fat and those with nutritional activity for L. casei 280-16 are of the D con- figuration. It may be suggested, therefore, that natural lipide a-hydroxy acids are predominantly of the D series.

SUMMARY

n-a-Hydroxy fatty acids of from 3 to 14 carbon chain length were nutri- tionally active in meeting an essential growth requirement of Lactobacillus casei 280-16. Corresponding acids of the L configuration were inactive, as were analogous DL acids containing hydroxyl, carboxyl, and certain other substituent groups (in addition to the cu-carboxyl and cu-hydroxyl). Hy- drolysates of butter fat and wool wax contained nutritionally active sub- stances, presumably n-or-hydroxy fatty acids. It was suggested that the n-cu-hydroxy fatty acids serve as metabolic precursors to long chain ~-a-

hydroxy acid components of essential lipides in L. casei and perhaps other species, and that naturally occurring lipide cr-hydroxy fatty acids are pre- dominantly of the D series.

Addendum-Horn and Pretorius (12) have demonstrated by direct comparison with the corresponding synthetic optically active a-hydroxy fatty acids that the wool wax or-hydroxy acids, cerebronic acid, and the 2-hydroxypentadecanoic acid derived from ustilic acid (13) are of the D configuration.

BIBLIOGRAPHY

1. Camien, M. N., and Dunn, M. S., J. Bid. Chem., 201,621 (1953). 2. Camien, M. N., and Dunn, M. S., Proc. Sot. Exp. Biol. and Med., 86, 177 (1954). 3. Eiduson, S., Camien, M. N., and Dunn, M. S., Arch. Biochem., 29, 302 (1950). 4. Holden, J. T., and Snell, E. E., J. Biol. Chem., 178, 799 (1949).

4 Yeast cerebronic acid is a mixture of 2-hydroxyhexacosanoic acid with less than 10 per cent 2-hydroxytetracosanoic acid, whereas brain cerebronic acid is a mixture of about 85 per cent 2-hydroxytetracosanoic acid and 15 per cent X-hydroxyhexa- cosanoic acid with, probably, a small amount of 2-hydroxydocosanoic acid (10). Mixtures such as these of closely homologous compounds of very long chain length have properties approaching those of a pure compound. The term cerebronic acid has been employed for convenience in the present discussion, even though it evi- dently does not refer to a true chemical entity.

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004 D-a-HYDROXY F.4TTY ACID NUTRITION

5. Mislow, K., and Bleicher, S., J. Am. Chem. Sot., 76, 2825 (1954). 6. Bosworth, A. W., and Helz, G. E., J. Biol. Chem., 112, 489 (1935-36). 7. Nutr. Rev., 2, 267 (1944). 8. Weitkamp, A. W., J. Am. Gem. Sot., 67, 447 (1945). 9. Horn, D. II. S., Hougen, F. W., von Rudloff, E., and Sutton, D. A., J. Chem.

sot., 177 (1954). 10. Chibnall, A. C., Piper, S. H., and Williams, E. F., Biochem. J., 65,707,711 (1953). 11. Kitahara, K., Obsyashi, A., and Fukui, S., Enzymologia, 16, 259 (1952). 12. Horn, D. H. S., and Pretorius, Y. Y., J. Chem. Sot., 1460 (1954). 13. Lemieux, R. U., Canad. J. Chem., 31, 396 (1953).

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Merrill N. Camien and Max S. DunnCASEI 280-16

NUTRITION OF LACTOBACILLUS -HYDROXY FATTY ACIDαTHE d-

1954, 211:593-604.J. Biol. Chem. 

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