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phonate (III) by acid hydrolysis (pH 1-2), treating the L-e-glyceryl-(2-bromoethyl)phosphonate (IV) in dimethylformamide with trimethylamine at 60-65C for 3 days, and isolating the L-e-glyceryI-(2-trimethylammoniumethyl)phosphonate as cadmium chloride complex (V). The preparation of compound V was carried out without isolating compounds II, III or IV in a pure state. The over-all yield of compound V was 37.5% o/ theory (calculated for acetone glycerol). Anal. Calcd. for [CsH2~O~NP]2[CdCI~]~ (1068.5) : C 17.98, H 4.15, N 2.62, P 5.80, Cd 31.56. Found: C I6.92, H 4.11, N 2.63, P 5.74, Cd 31.49, [~]o -0.6C in water (c 7). Vincinal-Glycol Titration: An aqueous solution of compound V was freed of cadmium chloride with potassium carbonate, and the amount of L-~-glyceryl- (2-trimethylammoniumethyl) phosphonate in the filtrate was ascertained by a phosphorus determination. An aliquot of the solution cont a i n i n g 0.0355 mmole of the monoester consumed 0.0350 mmole (98.5%) of periodic acid. Removal of the cadmium chloride moiety of compound V by treatment of its aqueous solution with a 1:1 mixture of Amberlites IR 45 and IRC 50 (H form) gave L-~-glyceryl-

(2-trimethylammoniumethyl) phosphonate (VI) in a yield of 80%. Over-all yield 30% based on acetone glycerol. The highly hygroscopic, glass-like material is soluble in water, methanol and 99% ethanol, but insoluble in chloroform, acetone, ether or benzene. [~]D 24 -1.5C in water (c 3.6). A 0.1 molar solution of compound VI in carbon dioxide-free water has a pH of 4.3 at 24C. Anal. Calcd.: for CsH220,NP (259.2): C 37.06, H 8.56, N 5.40, P 11.95. Found: C 37.13, H 8.61, N 5.42, P 11.99. Vicinal-Glycol Titration: 0.240 mmole of compound VI consumed 0.237 mmole (98.7%) of periodic acid. ACKNOWLEDGMENT Supported by grant (HE 08780-01) from the National Heart Institute, USPHS. ERIC BAER

Subdepartment of Synthetic Chemistry in Relation to Medical Research, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada [Received Nov. 28, 1966]

Effect of Phenazine Methosulfate on the Incorporation of C-Labeled Glucose into Lipids of Newborn Brain THE NEWBORN dog cerebral cortex, neuroI Nphysiologic function is minimal, possibly

due to the incomplete formation of lipid-con-

taining neuronal substructure such as myelin and nerve endings. In mature cortex, glucose, the primary energy source, is metabolized principally via glycolysis and the energy derived supports function. An alternate route for glucose metabolism, the hexose monophosphate pathway ( H M P ) , has previously been shown by McLean to be intimately associated with lipid biosynthesis in lactating mammary gland, where stimulation of glucose incorporation into lipids was paralleled by increased H M P activity (1). In this communication, data are presented to demonstrate that phenazine methosulfate, an artificial electron acceptor which stimulates H M P activity in newborn brain (2), inhibits the incorporation of l~C-glucose carbon into brain lipids. Dogs 1-3 days old were decapitated and slices of cerebral cortex (gray matter) were

prepared and incubated at 37C in standard Warburg vessels (see Table I for conditions). After 100 min. incubations, the reactions were stopped by quick-freezing at - 7 5 C (Cellosolvedry ice mixture). The frozen tissues were homogenized, extracted with chloroform/methanol (2/1), and the nonlipid contaminants removed on Sephadex G-25 (3). The individual phospholipids were determined after separation by two-dimensional t h i n - l a y e r chromatography (4). In Table I are recorded the relative amounts of ~C-glucose carbon incorporated into newborn brain lipids. Maximum activity occurred in the phospholipid fraction, while free fatty acids and cholesterol ester contained the lowest amount of isotope. No attempt was made to distinguish between net lipid synthesis and turnover because of the variation in individual lipid pools. With phenazine methosulfate a 4- 5-fold decrease was observed in the ~4Cincorporation into neutral lipids, whereas the

LIPIBS, VOL. 2, NO. 2

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SHORT COMMUNICATIONS

TABLE I Incorporation of x4C-Glucose [U.L.] into Newborn Brain Lipids a

TABLE II Incorporation of 14C-Glucose [U,L.] Into Newborn Brain Phosphoiipids

Control b Phenazine Methosulfatee (#c/dry gram) (#c/dry gram) Phospholipid Triglyceride Cholesterol Cholesterol ester Free fatty acids

3.21 1.30 0.91 0.55 0.20

1.62 0.30 0.16 0.10 0.05

aResults are expressed as averages of three experiments. bCounting yield ( % ) ---#c in total lipids/300 mg fresh brain ~C ~4C-glucose added/flask x 100 Counting yields: Control, 1.98%; phenazme methosulfate, 0.72%, ePhenazine methosulfate concentration: [1 x 10-4M]. The incubation mixture (200-300 nag fresh weight of brain in 2.0 ml of Krebs-Ringer-bicarbonate buffer, p H 7.4) contained 10 r e of a4C-glucose [U.L.]; substrate 5 mM cold glucose. Gas phase was 95% oxygen-5% carbon dioxide. The lipids were chromatographed by one-dimensional TLC in 4/1 chloroform/methanol with 1% acetic acid on plate spread with silicic acid-magnesium silicate (9/1). Authentic standards were used for identifications. Developed plates were exposed to iodine vapors and the silicic acid phospholipids suctioned into 20 ml scintillation vials. The silicic acid-lipid mixtures were then counted ira a liquid scintillation spectrometer.

phospholipid counts were reduced by one half. Phosphatidic acid had the highest specific activity among the phospholipids (Table II) but was of relatively small pool size. Phosphatidyl choline and phosphatidyl ethanolamine were the largest constituents in this fraction based upon phosphorus analysis. Although comprising the bulk of the total radioisotope content, these phospholipids have specific activities only 20-30% of phosphatidic acid in control tissue. In the presence of phenazinc methosulfate, there occurred a 5-fold decrease in isotope content in the major phospholipid components, a difference not observed in their tissue concentrations. Since such change resulted in a fall in specific activity, we interpret this to indicate a phenazine methosulfate inhibition of turnover rather than new synthesis. Phenazine methosulfate stimulates the direct oxidation of glucose in newborn brain but inhibits the incorporation of 14C-glucose carbon into brain lipids. The observation that phosphatidic acid maintains a relatively constant and high specific activity, even when phospholipid turnover is generally decreased, suggests that either phosphatidic acid formation precedes the site affected by phenazine methosulfate or that compartmentation renders it inaccessible. Current studies in this laboratory also indicate concomitant decreases in the inLIPIDS,

VOL. 2, No. 2

Phenazine Methosulfate

Control

#mole P Phosphatidic acid Phosphatidyl serine Phosphatidyl choline Phosphatidyl Ethanolamine Phosphatidyl inositol + sphingomyelin Total sample % Recovery

#mole P m#C S.A.

m a c S.A. a

dry g dry g

dry g dry g

2.2 57.3 26.0 26.1 80.2 3.1 124.6 917.1 7.4

2.4 62.9 26.2 23.8 56.7 2.4 118.1 227.8 1.9

56.7 327.9

5.8

53.3

97.1

1.8

25.0 150.0

6.0

23.6

63.9

2.7

234.6 265.0

221.2 280.5 88.6%

78,8%

a Specific activities are expressed as m,~C/#mole phosphorus. Phospholipids were separated using chloroform/ methanol/30% aqueous ammonia 65/35/5 followed by chloroform~acetone~methanol/acetic acid/water 5/2/1/1/0.5 and the phosphorus of each spot determined (4). The lipid samples for radioisotope assay were detected on duplicate plates with iodine vapor and the silicic acid-lipid mixtures collected and counted as noted in the legend for Table I.

corporation of ~C-glucose carbon into brain nucleic acid and protein with phenazine methosulfate. Therefore, the effects reported here may not be specific for inhibition of lipid metabolism but reflect a general action of phenazine methosulfate on processes requiring an adequate energy supply. THOMAS E. DUFFY 1 J O H N J. O ' N E I L L

Department of Cell Biology and Pharmacology University of Maryland School of Medicine Baltimore, Maryland A. N. SIAKOTOS Medical Research Laboratory Edgewood Arsenal, M a r y l a n d ACKNOWLEDGMENT Supported by USPHS Grant No. MH-05317-06. aPredoctoral Fellow. National Medical Sciences (T. E. Duffy).

Institute

of

General

REFERENCES 1. McLean, P,, Biochim. Biophys. Acta 57, 620 (1962). 2. O'Neill, J. J., and T. E. Duffy, Life Sciences, 5,'1849 (1966). 3. Siakotos, A. N. and G. Rouser, JAOCS, 42, 913 (1965). 4. Rouser, G., A. N. Siakotos and S. Fleischer, Lipids 1, 85-86 (1966).

[Received Sept. 9, 1966]