Biosynthesis oF 4D-hydroxysphinganine by the rat

Biochmicu

et Biophysics

Acta, 195 (1984) 411-416

411

Elsevier

BBA 51749

BIOSYNTHESJS EN BLOC MICHAEL

OF 4D-HYDROXYSPHINGANINE

INCORPORATION W. CROSSMAN

and CARLOS

intravenously

(phytosphingosine)

was

sphingomyelin-rich

fractions

of the these

(in

from intestine

fractions

standard

Sphinganine

addition

1402 S. Grand Bled, St Louis,

backbone; (Rat)

to

sphinganine

Radiolabeled and

and kidney. Identification

and conversion

Dnp-4D-hydroxy[3H]sphinganine

raphy with authentic

bases

4D-Hydroxysphinganine a sphingolipid long-chain and intestine. the biosynthesis sphingolipids and germ-free

on thin-layer

(phytosphingosine) is base found in glyco-

evidence

[l-3H]sphinganine

labeled

is

strongly

4D-hydroxysphinganine

tion of the C,,

sphinganine,

serinol,

2,4-di-

and sodium

borohydride

carbon

backbone.

We have previously

demon-

Materials

and Methods

of this base in renal and following

injection

rats of a putative

into

precursor,

4-Sphingenine, nitrofluorobenzene

[4,5-3H]sphinganine was first converted to (2,33H]palmitic acid and that this fatty acid was sub-

were

TO whom correspondence

pregnated

dnp,

thin-layer

0005.2760/84/$03.00

radio-

of mammalian

from Aldrich Tetradecanol,

*

to

utiliza-

been directly derived from the precursor (eg. direct hydroxylation), the results did not rule out that

Abbreviations:

suggesting

via direct

purchased from Sigma Chemical MO). 4D-Hydroxysphinganine

metabolized

(Dnp)

chromatog-

converted

[4,5-3H]sphinganine, [l]. Although in the previous study labeled 4D-hydroxysphinganine may have

sequently ganine.

and

following

and (2) detection of pentadecanol and Dnp-[ 3H]serinol. These results strongly can be derived (en the C,s carbon backbone of 4D-hydroxysphinganine

and sphingomyelins

kidney

glycolipid-

to their N-dinitrophenyl

by: (1) comigration

We now present

strated

in

tissues

that

sphingolipids

4Dhydroxysphinganine

4-sphingenine)

of these bases was achieved

of the long chain

was characterized

Introduction

intestinal

BACKBONE

*

with &?@h~O-DL-[I-3H]sphinganine.

detected

suggest that in mammalian bloc) from sphinganine.

normal

CARBON

Saint Louis Unrversity School of Medicine,

synthesis; Sphingolipid;

Rats were injected

derivatives.

RAT

May 4th, 1984)

Key words: Phytosphinganine

hydrolysis

SPHINGANINE

B. HIRSCHBERG

of Biochemistry

Edward A. Daisy Department MO 63104 (U.S.A.) (Received

OF THE

BY THE

into

4D-hydroxysphin-

TLC-borate,

borate-im-

purchased

from

Supelco,

Science Publishers

B.V.

Inc.

(Bellefonte,

PA). Sodium metaperiodate, sodium tetraborate decahydrate and solvents were purchased from Fisher Scientific Co. (St. Louis, MO). All solvent

chromatography Chemical

chromatography.

0 1984 Elsevier

Chemical Co. (Milwaukee, WI). nn-pentadecanol and n-hexadecanol

ratios are expressed by volume. Silicic acid (Unisil, 100-200

should be addressed.

dinitrophenyl;

were

Co. (St. Louis, was purchased

Co.

was

purchased

(Williamsport,

mesh) for column from PA).

Clarkson Pre-coated

412

silica-gel 60 F-254 (5 X 20 cm, 0.25 mm) TLC plates were purchased from Brinkman (Chicago, IL). Borate-impregnated silica-gel G TLC plates were prepared as described by Karlsson and Pascher [2]. Gas-liquid chromatography (GLC) was performed on a Hewlett-Packard 5711A chromatograph (equipped with a gas-effluent splitter) with silanized all-glass columns (6 ft X l/8 in i.d.) containing 3% OV-1 (on Gas Chrom Q 60/80 mesh) at a column temperature of 170 “C, (for long-chain alcohol analyses). Injection port and detector were operated at 200 ‘C. Gas-liquid radiochromatography was performed as described by Crossman and Hirschberg [l]. Samples for liquid scintillation were counted in a Searle Analytic 6893 spectrometer. [3H]Toluene was purchased from New England Nuclear (Boston, MA). Preparation of erythro-DL-[I - ‘H, Jsphinganine. [l-3H,]Sphinganine was synthesized as described by Crossman and Hirschberg [3]. The radiochemical purity of the free base was greater than 99% as judged by thin-layer radiochromatography (solvent-system, CHCl,/CH,OH/2 M NH,OH 40 : 10 : 1; R, 0.33). The radio- and stereochemical purity of the N-acetylated 0-TMS derivative of [l-3H,]sphinganine was also greater than 99% as determined by gas-liquid radiochromatography. The specific activity was 60 Ci/mol. Injection of labeled sphinganine into rats. A suspension of [l-3H]sphinganine (80 PCi) in sterile saline (0.9%, 4’C, 1.2 ml) containing 5% bovine serum albumin, was sonicated for three 30-s periods with a Heat Systems ultrasonics sonicator at an output setting of 7. Following sonication, 5 1.11 (7.3.10’ dpm) were removed and partitioned according to Folch et al. [4]. The CHCl,/CH,OH layer was examined by thin-layer radiochromatography (solvent system, CHClJ CH,OH/ 2M NH,OH, 40: 10: 1) in order to ascertain whether any decomposition of the labeled precursor had resulted from the sonication procedure. No labeled impurities were observed by the above criterion. Four male rats (Sprague-Dawley, 125-150 g, SASCO, Omaha, NB) were starved for 48 h before injection into the tail vein of each animal of 0.3 ml of the above solution. Animals were killed by decapitation 24 h after injection. Intestines and kidneys were removed, washed with cold saline, and lyophilized.

Extraction and purification of lipids. Lipids were extracted from the intestines (3.2 g dry wt.) and kidneys (0.9 g dry wt.) of four rats by the procedure described by Karlsson et al. [5]. The extract was washed according to Folch et al. [4] and the lower layer was taken to dryness under reduced pressure. Total lipids of intestine 24 h after injection of labeled sphinganine contained 5.4. lo6 dpm, and total lipids of kidney 24 h after injection contained 2.9. lo6 dpm. The total lipid fraction was applied (in CHCI,) to a silicic acid column (70 g, 30 X 2.8 cm) and eluted with 1.5 1 CHCl, followed by 2 1 CH,OH. The polar lipids from intestine contained 3.1. lo6 dpm while those of kidney contained 1.2 1 lo6 dpm. The lipids were then dissolved in CHCl,/CH,OH, 2 : 1 (100 ml) and subjected to mild alkaline hydrolysis (100 ml, 1 M CH,OH-KOH, 25 ‘C, 1 h). Subsequent workup [6] yielded a polar lipid fraction stable to mild alkali containing 9.1 . lo5 dpm for intestine and 5.9. lo5 dpm for kidney. These sphingolipidrich samples were applied to silicic acid columns (20 g, 15 X 2 cm) and fractionated as follows: fatty acid methyl esters (CHCI,, 500 ml), ceramide-rich fraction (CHCl,/CH,OH, 98 : 2, 500 ml), glycolipid-rich fraction (CHCl,/CH,OH, 3 : 2, 500 ml) and sphingomyelin-rich fraction (CHCl,/CH,OH, 1 : 4, 500 ml). The glycolipid- and sphingomyelinrich fractions accounted for approx. 90% and 50% of the radioactivity attributed to the polar mild alkali stable lipids from kidney and intestine, respectively. Column recoveries of the applied radioactivity were 85% for kidney and 100% for intestine polar mild alkali stable lipids, respectively. Isolation of long-chain bases and characterization of Dnp-4D-hydroxy[3H]sphinganine. Glycolipidand sphingomyelin-rich fractions were subjected to acidic methanolysis (1 M CH,OH-HCl (10 M H,O), 80°C, 16 h); the free long-chain bases were recovered from the hydrolysates as described by Carter and Hirschberg [6]. Following conversion to Dnp-long-chain base derivatives, Dnp-4D-hydroxy[ 3H]sphinganine was purified by thin-layer chromatography as described by Karlsson et al. [51. For further characterization of this latter derivative, 0.2 M NaIO, (0.1 ml) was added to a solution of Dnp-4D-hydroxy[ 3H]sphinganine in CHCl,/CH,OH, 2: 1 (1 ml). The solution was

413

allowed

to stand at room temperature

in the dark. Following

oxidation,

for 90 min

the reaction

mix-

ture was applied to a small silica gel column (1 g), further eluted with CHCl,/CH,OH 2 : 1 (10 ml)

shown

to

labeled

palmitic

yield

labeled

ethanolamine

phatidylcholine

and sphingomyelin)

reutilized

long-chain

of NaBH,

pear as a more suitable precursor

the

in O.lM NaOH

reaction

reaction HCl

vessel

was

was stopped

(0.2

ml).

was extracted combined

The

for long-chain

aqueous

ice

analyzed

for

the

of 6 M the

and analyzed

Dnp-[3H]serinol

NH,OH

synthesis.

Therefore,

incorporation

Distribution

(solvent

by

system,

40 : 10 : 1; R,

of radioactivity

Approximately

0.4).

and kidney,

of [4,S3H]sphinganine

[l].

whether

4D-hydroxysphinganine

rectly

labeled from

the

into of

was demonstrated

and Hirschberg

This

injected

previ-

rats by Crossman

study did not establish precursor

to detect en bloc carbon

arose di-

follawing

fractions.

Table

of radioactivity The

radioactivity fractions

associated

had a mobility

bases

were detected

The radioactivity

associated

lin-rich

had a mobility

fractions

CH,OH/conc.

NH,OH,

the

with

No labeled in this

the

on TLC

65 : 25 : 4) consistent

and diglycosylceramides.

long-chain

in intestine

within the various lipid

with free

fraction.

with the sphingomyeon TLC(CHCl,/

15 : 10 : 3) consistent

with

bovine brain sphingomyelins.

or indirectly Isolation of Dnp-4D-hydroxy[3H]sphinganine Glycolipidand sphingomyelin-rich

[l-3H[-

from intestine

TABLE

of

I summarizes

through metabolic reutilization of labeled palmitic acid, derived from the precursor. Catabolism of or [I-‘4C]sphinganines

backbone

injection

was recovered

respectively.

distribution

mono-

and germ-free

acid

would ap-

3.1% and 1.6% of the total ad-

radioactivity

(CHCl,/CH,OH/H,O,

ously in normal

fatty

[I - -‘H]sphinganine ministered

Results and Discussion

4D-hydroxysphinganine

and was not or

[l- 3 Hlsphinganine

of the sphinganine

glycolipid-rich

The conversion

bases

into 4D-hydroxysphinganine.

radiochro-

methanolic-soluble

radiochromatography

for

solution (8 ml);

by gas-liquid

aqueous

CHCl,/CH,OH/2M

and

methanolic

were concentrated

The

were

thin-layer

on

by the slow addition

alcohols

matography.

pres-

(0.2 ml); after 30 min, placed

three times with hexane

extracts

products

under reduced

un-

ethanola-

mine was found to be distributed within the phospholipid pool (phosphatidylethanolamine, phos-

sure. The reaction products in CH,OH (1 ml), were reduced by addition of a 10% (w/v) solution

and the solvent was removed

and

acid [7,8]. The labeled

in rats

has

been

and kidney were hydrolyzed

fractions and the

I

DISTRIBUTION OF RADIOACTIVITY [l-‘H,]SPHINGANINE

IN LIPID

FRACTIONS

OF INTESTINE

AND KIDNEY

24 h AFTER

INJECTION

OF

Total lipids were extracted from lyophilized intestines and kidneys by repeated CHCl,/CH,OH (2 : 1) extraction. The lipid extract was fractionated into the various components by silicic acid column chromatography as described in Methods. Recovery of radioactivity is calculated as a percentage of the radioactivity recovered from the lipid extract. Aqueous Folch (polar mild alkali stable lipids, PMAL) is described as water-soluble radioactivity released from polar lipids following mild alkaline hydrolysis and CHC1,/CH30H/H,0 (8 : 4 : 3) partition. Concerning the sphingomyelin-rich fraction, both the long-chain base and choline moieties of sphingomyelin may contain 3H. Lipid fraction

Total lipid extract Polar lipids Aqueous Folch (PMAL) Polar, mild alkaline stable lipids Ceramide-rich Glycolipid-rich Sphingomyelin-rich

Intestine

Kidney

‘Hdpm(xl0~‘)

Recovery

3Hdpm(X10-5)

Recovery

54.0 31.0 20.3 9.1 3.9 2.1 2.4

100 51 38 17 7 4 4

29.0 12.0 4.3 5.9 0.3 2.1 1.8

100 41 15 20 1 9 6

414 ORIGIN

FRONT

CH3(CHd,2-CH2-CH-CH-CH-C3H20H dH

dH

iH

Dnp- [I-3H,]4D-

NO2 :I 0

hydroxysphlnganlne

NO2 3H DPM

1450

10400

5700

Fig. 1. Preparative TLC (silica gel G impregnated with 2.5% Na,B,O. lOH,O) of Dnp-long-chain bases from intestinal glycolipids 24 h after injection with [1-3H]sphinganine. Solvent system: n-C,H,,/CHCl,/CH,OH, 50:50: 15; A, Dnp-4Dhydroxysphinganine; B, Dnp-4-sphingenine; C, Dnpsphinganine. Compounds were eluted from the silica gel with CH,OH (20 ml) and an aliquot of each sample was taken for liquid scintillation counting.

long-chain bases were recovered from the hydrolysate as previously described [6]. The mixture of free long-chain bases was converted quantitatively to their N-Dnp derivatives and purified by preparative thin-layer chromatography on borate-impregnated silica gel. Fig. 1 shows the TLC-borate separation of a mixture of Dnp-long-chain bases obtained from an intestinal glycolipid-rich fraction 24 h after injection of [1-3H]sphinganine. Intense yellow bands were visible for compounds comigrating with authentic Dnp-4D-hydroxysphinganine (A) and Dnp4-sphingenine (B). No yellow band was observed for Dnp-sphinganine (C) because sphinganine is relatively low in abundance in this mammalian tissue. Table II shows the radioactivity in different long-chain bases derived from Dnp-sphinganine, Dnp4-sphingenine and Dnp-4D-hydroxysphinganine (from glycolipid- and sphingomyelin-rich fractions of intestine

I. Na104 2.NaBH4 1 HOH2C-CH-C3H20H

+

CH3(CH2),3CH20H

iH 3 0 NO2

pentadecanol

ND2

Dnp- [I-3Hdserlnol Fig. 2. Reactivity of Dnp-4D-hydroxy[3H]sphinganine with sodium metaperiodate followed by reduction with sodium borohydride.

and kidney, 24 h after injection of [lThe glycolipid-rich fraction 3H]sphinganine). showed a 1.6- to 3.0-fold higher incorporation of labeled long-chain bases than the sphingomyelinrich fraction. Interestingly, this latter fraction contained higher percentages of endogenously synthesized bases (i.e., 4-sphingenine and 4-D-hydroxysphinganine) as compared to glycolipids. Characterization of Dnp-4D-hydroxy[-‘H]sphinganine The Dnp-4D-hydroxy[ 3H]sphinganine samples, obtained from glycolipid- and sphingomyelin-rich fractions 24 h after injection of [l-3H]sphinganine,

TABLE II DISTRIBUTION OF RADIOACTIVITY IN Dnp-LONG-CHAIN h AFTER INJECTION OF [1-3H]SPHINGANINE

BASES OF INTESTINE

AND KIDNEY

SPHINGOLIPIDS

24

The long-chain bases were recovered in the acid hydrolysates of glycolipid- and sphingomyelin-rich fraction, converted to dinitrophenyl derivatives and separated by borate-impregnated thin-layer chromatography as described in Methods. Percentages of the total radioactivity recovered within the long-chain bases as listed above are in parentheses. Long-chain base

Radioactivity (3H dpm X lo-‘) Kidney

Intestine

Sphinganine CSphingenine 4D-Hydroxysphinganine

Glycolipid

Sphingomyelin

Glycolipid

Sphingomyelin

5.7 (32) 10.4 (59) 1.5 (9)

1.2 (20) 3.1 (64) 0.9 (16)

5.0 (44) 5.9 (52) 0.5 (4)

1.6 (23) 4.1(68) 0.6 (9)

415

were combined oxidation tion.

and

followed

We reasoned

isolated

then

subjected

that if the labeled

from TLC-borate

(containing

one

to

reaction in

be

able

products,

Fig.

2,

reduc-

compound

was in fact Dnp4D-hy-

droxy[ 3H]sphinganine should

to periodate

by sodium borohydride

according

unlabeled

‘H at C-l),

isolate

and

then

identify

to the reaction

pentadecanol

as

scheme

and

Dnp-

ated

with carbons

4-18

istered

precursor.

the possibility sphinganine

Therefore,

The possibility

nant of sphinganine)

tected

by

aqueous

gas-liquid

the

comigrated

with

The remaining to authentic

tained

in

the

were radio-

products

authentic

(lane

2),

(lane

1).

Dnp-serinol had a mobility

was detected

similar (lane 3).

comigrating

(lane

4).

portion the isotope

with

Although

(Fig. 2) does not specifically the result strongly

85%

aqueous

Dnp-4D-hydroxysphinganine

tritium to C-l, terminal

reaction

pentadecanol

procedure

by thin-layer

contained

radioactivity

radioactivity

authentic

The

products

as shown in Fig. 3. Approx.

radioactivity

methanolic-soluble

No

reaction

for Dnp-[ 3H]serinol

chromatography

as de-

radiochromatography.

methanolic-soluble

analyzed of

pentadecanol,

this

localize the

suggests that the

of 4D-hydroxysphinganine

con-

and that no tritium was associ-

rats

and

subsequently

separates

concentrated

as preparative from

in addition,

be purified

breaks

this long-chain

base

sources

was first isolated

has been

[12,13],

primarily

[18%20]

adenocarcinoma

because

it

from the silica gel. from

by Carter et al. [9]. Subsequently,

protozoa

sphingolipids,

clearly

4D-hydroxy-

4D-hydroxysphinganine

4D-Hydroxysphinganine plant glycolipids

intestine

thin-layer

by this procedure

down during elution

[lO,ll],

into the

by different

(used in the purification)

sphinganine

sphinganine;

(as a contami-

was actually injected

tissues is highly unlikely,

cannot

of an unknown

that a small amount

4D-hydroxysphinganine

chromatography

rule out

of 4D-hydroxy-

was a small quantity

contaminant. of labeled

with authentic

we cannot

that the precursor

mixture

comigrating

to pentade-

The amount of labeled 4D-hydroxysphinganine actually isolated was less than 0.1% of the admin-

[ ‘Hlserinol.

A hexane extraction of the reaction contained a single, non-radioactive peak

(converted

canal).

reported

as part

in yeast

of mammalian

from kidney [6,14-171

and

in

one

case

of

and

human

[21]. One of the most remarkable

of 4D-hydroxysphinganine

is the yeast,

ciferrii, which excretes large amounts of

Hansenula

tetraacetyl-4D-hydroxysphinganine medium.

Most

synthesis

of this long-chain

into

of our knowledge

about

the

the bio-

base comes from stud-

ies in this yeast. Green et al. [ll]

in a study in vivo

have shown that [9,10-3Hz]palmitic acid and [314C]serine were incorporated equally well into 4D-hydroxysphinganine. thetic

pathway

for

This the

C,,

suggests carbon

a biosynskeleton

of

4D-hydroxysphinganine similar (if not the same) to that proposed for sphinganine and 4-sphingenine

in mammals

ciferrii

[25-271

[22-241.

have

Other

suggested

may serve as the immediate droxysphinganine The Fig. 3. Thin-layer H,O

(9

and

: 1)

radiochromatographic

-soluble products

sodium

borohydride

hydroxy[3H]sphinganine

reduction

(obtained

ney sphingolipids

24 h after injection CHCl,/CH,OH/conc.

Lane

1, authentic

3, authentic pentadecanol.

Dnp-serinol;

of

oxidation Dnp-4D-

from rat intestine

Solvent

system:

analysis of CH,OH/

released after periodate

and kid-

with [l-3H]sphinganine). NH,OH,

lane 2, reaction

Dnp-4Ghydroxysphinganine;

40

products;

lane

: 10 : 1. lane

4, authentic

evidence

supports sphinganine

the

studies

that

precursor

in H.

sphinganine to 4D-hy-

via direct hydroxylation. presented en

carbon

bloc

in this paper incorporation

skeleton

backbone

strongly of

the

in the bio-

synthesis of 4D-hydroxysphinganine of rat intestinal and renal sphingolipids. The results presented support the hypothesis of a direct hydroxylation of sphinganine; however, they do not rule out that an unsaturated intermediate (i.e., 4-sphingenine), might serve as the immediate precursor to 4D-hy-

416

droxysphinganine in mammals via stereo-specific hydration. We are currently attempting to develop an in vitro system for the synthesis of 4D-hydroxysphinganine in order to ascertain what factor(s) regulate its synthesis. Acknowledgements

This work was supported by NIH grant NS12827 and GM 30365. CBH is a Faculty Research Awardee of the American Cancer Society. We thank Catherine Mack for expert typing. References Crossman, M.W. and Hirschberg, C.B. (1977) J. Biol. Chem. 252, 5815-5819 Karlsson, K.-A. and Pascher, 1. (1971) J. Lipid Res. 12, 466-472 Crossman, M.W. and Hirschberg, C.B. (1984) J. Lipid Res. 25, 729-737 Folch, J., Lees, M., and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509 Karlsson, K.-A., Samuelsson, B.E. and Steen, G.O. (1973) B&him. Biophys. Acta 316, 317-335 Carter, H.E. and Hirschberg, C.B. (1968) Biochemistry 7, 2296-2300 Stoffel, W. and Sticht, G. (1967) Hoppe-Seyler’s Z. Physiol. Chem. 348, 1345-1351 Stoffel, W. and Sticht, G. and LeKim, D. (1969) HoppeSeyler’s Z. Physiol. Chem. 350, 63-68 Carter, H.E., Celmer, W.D., Lands, W., Mueller, K.L. and Tomizawa, H.H. (1954) J. Biol. Chem. 206, 613-623

10 Stodola, F.M. and Wickerham, F. (1960) J. Biol. Chem. 235, 2584-2585 11 Greene, M.L., Kaneshiro, T. and Law, J.H. (1965) Biochim. Biophys. Acta 98, 582-588 12 Taketomi, T. (1961) Z. Allg. Mikrobiol. 1, 331-349 13 Carter, H.E., Garver, R.C. and Yu, R.K. (1966) Biochem. Biophys. Res. Commun. 22, 316-320 14 Karlsson, K.-A. (1964) Acta Chem. Stand. 18, 2397-2398 15 Karlsson, K.-A. and Martensson, E. (1968) Biochim. Biophys. Acta 152, 230-233 16 Karlsson, K.-A. and Steen, G.O. (1968) Biochim. Biophys. Acta 152, 798-800 17 Pure, K. and Keranen, A. (1969) Biochim. Biophys. Acta 187, 393-400 18 Okabe, K., Keenan, R.W. and Schmidt, G. (1968) Biochem. Biophys. Res. Commun. 31, 137-143 19 Yurkowski, M. and Walker, B.L. (1970) Biochim. Biophys. Acta 218, 378-380 20 Breimer, M.E., Karlsson, K.-A. and Samuelsson, B.E. (1974) Lipids 10, 17-19 21 Yang, H. and Hakomori, S. (1971) J. Biol. Chem. 246, 1192-1200 22 Stoffel, W., LeKim, D. and Sticht, G. (167) Hoppe-Seyler’s Z. Physiol. Chem. 348, 1570-1574 23 Stoffel, W., LeKim, D. and Sticht, G. (1968) Hoppe-Seyler’s Z. Physiol. Chem. 349, 664-670 24 Braun, P.E., Morrel, P. and Radin, N.S. (1970) J. Biol. Chem. 245, 335-341 25 Weiss, B. and Stiller, R.L. (1967) J. Biol. Chem. 242, 2903-2908 Z. Phys26 Stoffel, W. and Binczek, E. (1971) Hoppe-Seyler’s iol. Chem. 352, 1065-1072 27 Polito, A.J. and Sweeley, CC. (1971) J. Biol. Chem. 246, 4178-4187