- Email: [email protected]
Lipid biosynthesis by human endometrium
Lipid biosynthesis .J.\MES
A.
NICHOLAS OXlahomu
MERRILL, T.
City.
IM.D. WERTHESSEN,
PH.D.
Oklahomu
1-I 1. hr A N endometrium undergoes (‘!chc changes in structure and
I~hcting
by human endometrium
striking function
the luteal phase of the cycle in the monke, uterus, and McClennan and Koets”’ found total lipids to be in highest concentration during the secretory and menstrual phases in human endumetrium. The histochemistry of the human endometrium has been studied by many workeTs6’ 15, 13. 20, 26-2s but with little emphasis placed on the lipid changes. McKay and associates’“. ‘* reported that the proliferative phase of the cycle was characterized, histochemically, by the presence of large amount:, of ribonucleoprotein and alkaline phosphatase activity and by small amounts of acid phosphatase activity, glycogen, and glycoprotein. The secretory phase was characterized by a reversal of this pattern. Page, Glendening and Parkinson”” rcviewing the histochemical and biochemical changes reported in the human endometrium classified them into four patterns: ( 1) stimulation by estrogen (@glucuronidase) ; (2 1 stimulation by estrogen withdrawal (fibrinolysins) ; (3) stimulation by progesterorle by es(fat, &cog-en) ; and (4) stimulation trogen and inhibition by progesterone (RNA, alkaline phosphatase) . Experimental data support a theory (A hormonal control of lipid biosynthesis in endometrium” 4, 8, 9, 12, I33 IS as well as other tissues.“o Borell’ described the influence of ovarian hormones on the metabolism of nucleic acids and phospholipids in the rabbit uterus. He demonstrated greater incorporation of radioactive phosphate into phospholipids during the luteal phase. Progesterone was shown to promote a greater uptake of phosphate than estrogen. This has not been found by subsequent workers. Emmelot and Bosch” used acetate-f-Cl4 to study lipid bio-
hormonal inff uence. The histoIogic
I’hangcs are weI1 documented’? and the chemical changes are being studied increasiljgjy.“* 12. 11;.18-21, 23, L’G-2SIn 1896, Westphal(‘rr”’ first described the presence of stainable lipids in human endometrium. Aschheim’ lound that 80 per cent of endometrium studied contained fats during the premenstrual phase. Froboesc’” found that fluctuations in II tcr-inc. lipids correlated well with protein I.lrar~ges in the uterus. Gillman” thoroughly described the stainable lipids in human endothroughout the menstrual cycle and greatest abundance of stainable iat between the twenty-first and the twentyfifth day of the cycle. Black, Heyns, and C;illrnan proposed that stainable lipids be used as the basis of a bioassay method for determining progesterone activity. He observed that an optimum balance between estrogen and progesterone was necessary in the human for stainable fat deposition to occur. may be as dependent on Lipid staining the physiochemical state of the lipid as it is on the concentration. Furthermore, lipids such as cholesterol do not take the Sudan vanDyke and Chen”’ stains.“’ However, found fofnl neutral fat to be highest during rnctrirrnr
tound
the
From the Department of @necology rind Obstetrics, Uniuersity of Oklahoma School of Medicine. Supported by United lleulth Service Grant
States Public No. HD 01188.
Presented at the Eighty-ninth Annual Meeting of the American Gynecological Society. Hot Springs, Virginia, June 2-4, 1966. 619
620
Merrill
synthesis
and
Werthessen
by the uterus of mice and found of acetate to choksterol and fatty acids was promoted by estrogen administration as compared to untreated control castrates, He was primarily interested in the rrlative effect of the estrogens ( ET, Ez, W upon uterine lipid biosynthesis. The greatcst cffe,-t was obsrrved with 17,&estradioI rind the least. with cstriol. Leathem’” found vssentially no change in the quantitative amount of choIestero1 in the immature mouse uterus folIowing estradiol and progesterone injection. Davis and Alden” studying rats, found that estrogen treatment of spayed animals caused a significant decrease in concentration of neutral fat and an increase in phospholipid concentration. Proqesterone did not increase the concentration of neutral fat or phospholipid, hut did increase the osmiophilic or stainable fat. Goswami, Kar, and Chowdhury’” studied the mouse uterus by quantitative chemical analysis of total lipids, triglycerides, sterol esters, free sterols. and phospholipids. They demonstrated a similar change in metabolism of lipids. The most conspicuous change was the increase in concentration of phospholipids during estrus. This was abolished by ol-ariectomy but restored by estrogen therapy. The restoration of phospholipid concentration was clearly antagonized by progesterone. Less conspicuous changes occurred in the concentration of cholesterol and sterol esters. Aizawa and Mueller’ e\xluated the effect of estrogens on lipid biosynthesis in the rat uterus using both P:‘” and (:‘f. Estrogen was shown to cause rapid acceleration of all pathways for lipid production measured. Estradiol stimulated incorporation of acetate-I-C?’ into fatty acids, cholesterol and other constituents of the nonsaponifiable fraction Of uterine segments. GorsI2.i and r\‘icolettc.‘” [Ising P:“. found jn x.il.0 RNA and phospholipid svnthesis increased in subcellular fractions of the rat uterus as a resolt of cstradiol treatment. When uteri were incubated in vitro the estrogen pfI’cc[ c,n l
conversion
Lipid
and delivered to the laboratory. Kepresentati\.r aliquots of the tissue were fixed in Bouin’s solution and prepared for histologic vsamination. Specimens of tissue were trimmed SO that no piece was more than 1 111111.wide. Because of the variability in cohesiveness and friability of endometrium, it has not been possible to use any of the de\ices available to prepare perfectly uniform standard-sized slices. The tissue was incubated in 10 ml. of the patient’s serum, obtained shortly prior to removal of the tissue, to which acetate-l-c” \vas added at the concentration of 0.1 millic,urirs of CL4 per liter. The patient’s serum leas used as the incubation medium because it could be expected to provide the tissue Lvith a close approximation of the hormonal 4mulation it had received in situ and to provide carrier lipids for the small amount of lipid available from the tissue. Thus macro, rather than micro, techniques could be used for the separation of the lipid classes. Incubation was maintained in a Dubinoff shaker for 4 hours under 5 per cent CO, ,/ 95 pt’r cent 0, at 37.5’ C. At the end of the incubation period, serum was decanted from the tissue and treated with 3 volumes of chloroform-methanol (2 to 1 \-. V.) . The tissue was rinsed with saline: placed on filter paper and the excess seru~n removed. A wet weight was obtained. The tissue was placed in a weighing flask, a few drops of chloroform-methanol added to stop synthesis and the flask and contents placed in a high vacuum desiccator until constant weight was reached and the total dry weight determined. Following this determination, the tissue was further extracted with chloroform-methanol by a modification of the method of Bottcher and associates.” ‘I‘he same drying procedure was used followinx lipid estraction to determine the dry, dcfatted weight. In some experiments, it was elected to study only the relative distribution of labeled acetate into various lipid classes, and weights \vere not determined. In these experiments, the total incubation system was frozen and
biosynthesis
621
the water removed by lyophilization. The dried powder and tissue obtained by this procedure were treated by the Bottcher cxtraction technique’ in the same fashion as the dried tissue. The lipid extracts from the tissue and the serum were combined to yield a greater quantity of lipids with which to work. The chloroform-methanol was evaporated to an oil, extracted with chloroform, and filtered. Removal of residual acetate was acconlplished by eyuilibratin~ the total chlorofornl extract against two changes of IO per cent glacial acetic acid iti water. The titer of C” was determined at this point for the total lipid fraction. The lipids were separated into four classes: phospholipids, sterol esters, triglycerides. and the remainder of the lipids containing frecb fatty acids, free sterols, and other unidentified lipids. Initially, the free sterols wert’ separated as a class. Except for the phospholipids, the separation was performed h) column chromatography and later by thinlayer chromatography. The chloroform extract was evaporated and extracted with dry acetone and then M&l 2 added to the acetone to completely precipitate the phospholipids. The insoluble material (phospholipids) was redissolved in chloroform-methanol and assayed to give the <:I” titer of the phospholipid fraction. ‘The acetone soluble fraction was transferred to hexane, placed on a chromatoKraphic column (packed with Silica gel G, held in suspension with hexane), eluted with hexane and increasing concentrations of ether, and then stripped with methanol. This procedure has been found to provide good separation of the sterol esters from the triglycerides.’ The chromatographic column technique employed was that described by (Irider and associates.: In later experiments, thin-layer plates made of Silica gel G were used and the separation accomplished by developing in 7 per cent ethyl ether in petroleum ether. In thin-layer analyses, charring with HzS() L was used to identify fractions.
622
Merrill
and Werthessen
After appropriatr corl,ections for \.oltmlt’, the (:‘I titer, expressed as counts per rninute, for the combined eluate classes (sterol and the other lipids esters, triglycerides, ) free sterols! fatty acids, etc. 1 ) were recorded. All C:” determinations were made on a Packard Tricarb Scintillation Gaunter. using “POP and POPOP”” dissolved in toluene as the scintillators. ‘4 spot test based on the LiebermannISurchard reaction \vas used to locate the stcrol esters and free cholesterol fractions in the column eluates. In the chromatoqraphic svstems e111p10yccl. thr free fatty acids. monoglycerides. and diglyceridcs contaminatal the. free stcrol fraction. ‘Ik!rcfore, the free sterols were precipitated with digitonin determined. In and the (:I’ incorporation those instances in bvhich C:” titer was sufficient to permit ultimatr purification and assay, carrier cholesterol was added. and a dibromination purification done also. The cholesterol ester fractions. which tend to romc ofl‘ the colmnn in a sin&. massi\,c front, wt’re hydrolyzed in ethanolic KOH and the cholesterol similarly precipitated. ‘I’he fatty acids from the hydrolysis were collected and assayed for C:” incorporation as well as the sterols. It was soon found that (1” incorporation into cholesterol was not usual under the conditions employed. This will be the subject of a later communication. However, because of this findinp. our procedure was altered so that the free cholesterol fraction was for the time neglected. We have used only a part of thr information which we have obtained. Most of the emphasis has been placed on three fractions: phospholipids. triglycerides, and sterol esters. These fractions constitute over 90 per cent of the fat in the body and are the most consistent with re,qard to their purity from one experiment to the other in the analytical procedures employed, The fraction containing free sterols is contaminated by as much as 20 per cent of the total (1” incorporation into lipids in some experiments, but it usually contained 5 to 10 per cent. The nature of these lipids reytlirc fur-
ther study. Free fatty acids \\‘c’rt’ 11c)t (It*termined or studied. From the point of assay of ttlcs chlorofolnlsoluble lipids the continued assay of (1” titer in the \,arious fractions permits an audit of the over-all efficknc\. Where column chromatography was employed, ~111 over-all recovcllr of 85 1)~” c~nI of the ot iginal titer. was considered nc~trrssary. I.%? tliiniltia! thin-layer chrorriatogral~h~~. thr t‘t*cov(‘ry acct~ptabl~~ haa Ilccn raised to %I pt’r cc-nt and is commonl! t,et~veetI 9i ,ttlci 100 per crnt. Resolution of thta pr,oblenl 01 ~neasurin~ (mused substrarc c,ontanlinatinr: a fraction has been 111~1 I)\, 1\\0 prt~cautionk ‘I‘ht- first d thcsr was this &)rouKh w,ashinq of tht. total lipid cstrac’l \\.itl! 10 ljet ( (‘I~I glacial acetic ac.id in water. .l’llt, second tla5 heen thr dissolving of a I’raction irl wctir :uzid, rhcn c>vaporating ttic itcc.tic- acid ,jiibt prior to co~inting and cctrnparinq qlic-11 .L washed sample’s count L\ ith #iti unt5~ral~.(l aliyuot. .4 difference 1)etwrt.n th<, (‘oulltb (‘.~IE br ascribed to unused aceta TV ot short-chairi Iattb- acids distillable in ac,catic- ‘I(‘1‘d urttic~t these conditions. Fl’hcX audit oi leach t‘x[Jc’!‘I. Itltant ( onsidcrrhd both salts 01 \.alut*s. ‘1%~ representative aliquots ol’ tissue fis~tl and prepared for histologic. tbsarnination M’t’re stained with heniatos\~lin .rnrJ tGti. Moreo\,er. the routine i!irii(.;ll p2lt~JOkW\ specinirria Lvert’ also revirwcd. Sornlal IYXIOrllc*triurn was dated according IO thy c’ritt.ri;r ol’ Noytas. Herti,g:. and Koch.” ‘l‘h(* hospital chart of each patient bvas c-a&u iI\- rc*\+k~c~cl for evidence of menstrual or ~~ndocrine al)normalitv. Only those patients \\,ithollt (,1i11ical abnormalitv and with c.loxt, ~ot~rrlatio~~ of the mrnstrual dating I)v l&t~,Io$c~ ;itl(I historical criteria were includc~tl cl\ rr0r-nlcr/t. Although Iriariy more spccimc$ns II~\Y~ bc~71 studied. detailed analyses for [his prelimirlar-\ r‘tq)ort includes 1.5 normal ~ndotnrtria. L specimetls of endometrial adetloc,Llr,c,inottl;l, ‘i specimens of l~ostmenopausal t~rldorrlrtriI,rtr. and 6 specimt~tls of dt~cid~la. Results
‘l.able I lists the incorporation of acetatcbI -( 1’ ’ into total lipid estracted i‘l,onr \,aric)It\ c*ntlometria and incorporation itlro phospf~o-
Lipid
biosynthesis
623
Table I. The Cl4 titer expressed as counts per minute per milligram dry, defatted weight for total lipid extract (TL), phospholipids (PL), sterol esters (SE), triglycerides (Tg3), and the remainder of the total lipid extract (NEL) in various endometrial specimens ____
c.p.m. X I02/mg. Histology Day 1 Day 7 Day 7 Day 9 Day 11 Day 13 Proliferative Day 17 Day 17 Day 18 Day 21 Day 21 Day 22 Day 22 Day 22 Day 26 Secretory Decidua, Decidua, Decidua, Decidua, Deridua, Decidua, Decidua, Atrophic Atrophic .4trophic Atrophic
(mean)
(mean) term term term term term term (mean) 6 weeks
(mean)
Anaplastic noma
adenocarci-
Carcinoma
in situ
7-L
PL
SE
Tg3
NEL
292.8 349. 321.7 208.3 103.6 ‘J33 7 ..--.. 249.6
16.7 81.1 72.9 107.6 36.2 68.6 63.9
2.66 21.1 1.7 23.4 19.1 8.14 12.7
6.99 56.1 56.9 5.0 16.9 15.2 26.2
266.5 J90.8 190.2 72.2 31.4 130.4 146.9
11.8 2.9 0.37 8.6 0.91 19.3 28.6 0.30 9.1
21.1 10.7 33.7 24.7 31.0 10.8 4.4 37.3 21.7
8.5 0.37 0.64 3.3 0.46 3.2 1 .3
20.0 21.1 46.1 37.6 13.0 5.5 41.7 10.9 24.5
157.1 93.6 117.6 117.6 121.5 33.3
67.2 40.8 81.5 85.8 79.4 70.9 __
16.1 6.6 3.2 3.2 5.4 6.9 -
28.5 24.9 15.2 22.9
5.2 10.0 3.3 6.2
4.9 5.1 1.0 3.7
343.3
37.4
96.5
61.5 35.0 80.9 74.2 45.4 38.8 76.0 90.6 49.1 61.3
34.7
-
lipids, triglycerides, and sterol esters. Lipid biosynthesis from acetate by human endometrium occurs at a very rapid rate. The incorporation, on a per gram basis, by proliferative endometrium and anaplastic adenocarcinoma is comparable to that found in human liver (225 c.p.m. per milligram) .” In the exploratory phases of this study, cholesterol was studied as derived from both the free sterol and the sterol ester eluates. The separation and purification procedures were designed to allow a high degree of purity. Eleven of 19 normal endometria had no C” count in purified cholesterol and 6 of 14 abnormal endometria showed no count.
_____
21.2
0.56 2 .3 34.4 14.8 17.5 16.8 21.6 21.0 0.49 1.1 1.9 1.2 17.6 0.78
39.8 31.5 -. 11.7 11.3 23.6 _17.8 8.7 9.0 11.8 191.8 12.2
Moreover, the free sterol fraction constituted a small part of the total incorporation into lipids (usually less than 5 to 10 per centj. Having found little or no C” in the free cholesterol fraction, the sterol ester fraction was analyzed to determine the portion of the ester molecule into which the Cl4 had been incorporated. In all cases studied, following hydrolysis of the sterol esters into sterol and fatty acid moieties, most, if not all, into of the acetate- 1 -Cl4 was incorporated the fatty acid portion. The trace of activity found in the sterol fraction remained in the mother liquor when the sterols were subjected to digitonin precipitation.
624
Merrill
and
Werthessen
Biosynthesis of the non-fatty acid portion and triglycerides from of phospholipids labeled acetate has not been specifically checked. However, based upon the mo!ecular structure of these moieties, it is reasonable to assume that the major portion of the newly synthesized triglyceride and phospholipid molecules that contained C” derived from acetate is the fatty acid portion. All of the lipids which are eluted after the triglycerides have not been specifically studied. These eluates account for a significant part of the total lipid extract. According to Clrider and associates7 these eluates contain free sterols, free fatty acids, and residual phospholipids. The free sterols were identified. Thus the terminal fraction should contain mostly free fatty acids, any phospholipids which failed to precipitate with acetone (less than 3 per cent by weightj and probably diglycerides and monoglycerides. These eluates contain substances which require identification and study. It is also essential to find out what other sterols, besides cholesterolJ are contained in the free sterol fraction. Figs. 1 through 5 show the distribution of C:” (expressed as r.p.m./mg. dry, defatted
350-
0
325L 3OOL0
0
TOTAL
weight) incorporated into lipids by 15 normal endometria at various times during the menstrual cycle. The proliferative phase is considrred as day one through day 15 and the secretory phase, day 16 through day 28. Incorporation of label into total lipids MU greater in the proliferative phase (mean 24,960 c.p.m. per milligran~ than during 6,130 c.p.111. the secretory phase (mean lxr milligram) (P = O.OU I ! (Fig. 1 : ~ Incur1)oration of label into l~hosl~holipids XV;IS treater during the proliferatkrcs phase ~,mt’an 6,385 c.p.m. per milligram ; than during tlte secretory phase (mean mm-910 r.pm. prr milligram) [ P = 0.001 ) t Fiy. 2 !, ‘T?ic~ irlcorporation of label into triglvceridrs bvas greater during the proliferative phase / nwm 2,618 r-.p.m. per milligram,; than during the secretory phase (mean r-r 230 C.~.III. pvt. milligram) (P =z 0.02) (Fig. 3 i, Incorporation of label into sterol esters showed ruucll \,ariation with no siqiificant chanqe
PHOSPHOLIPIDS
LIPIDS
275250-
E E” ,\ 0
_ 0
m-
III 2
4
6
I
I,
6
10 DAY
Fk. I. Cl4 incorporation by normal cndometrium.
I 12 OF
/
I
/
,
14 16 CYCLE
18
20
22
i4
in the total lipid extract (From Table I.\
I 26
1
21
4
c
6r
lb DAY
Fig. ‘2. Incorporation normal endometrium.
121
T----v*-.-4, 14 16 OF CYCLE
I6
20
22
of Cl4 into phospholipids (From Table I.!
24
26
by
Volume Number
96 5
Lipid
throughout the cycle although incorporation was greater during the secretory phase (mean = 2,170 c.p.m. per milligram) than during the proliferative phase (mean = 1,268 c.p.m. per milligram) (P = 0.2) (Fig. 4). Incorporation of label into the nonestcrified lipids was greater during the proliferative phase (mean = 14,690 c.p.m. per milligram) than during the secretory phase (mean = 2,449 c.p.m. per milligram) (P = 0.01) (Fig. 5). On the basis of total experience, including specimens in which weights were not recorded, the percentage of total lipid extract not accounted for by phospholipids, triglycerides, and sterol esters ranged from 91 to 14 per cent. The mean of 20 normal endometria in the proliferative phase was 60 per cent and the mean of 11 endometria in the secretory phase was 37 per cent (P = 0.001) (Table II) . Fig.- 6 indicates the incorporation into each fraction
40 169 40 56I
TRIGLYCERIDES
biosynthesis
625
during the proliferative and secretory phases. During the proliferative phase incorporation into nonesterified lipids exceeded the ester classes and incorporation into phospholipids exceeded the other two classes of esters. During the secretory phase incorporation was greater into esters than nonesters, and incorporation into sterol esters exceeded the other two classes of esters. Fig. 7 illustrates the activity in each lipid fraction for various types of endometria. Five specimens of decidua from term pregnancy were studied. They showed greater incorporation of label into the phospholipid fraction than was seen in nonpregnant endometria and less incorporation into nonesterified lipids. The degree of activity and distribution within the three ester classes was more similar to proliferative than secretory specimen of decidua endometrium. One from a 6 week gestation showed very little incorporation of Cl4 (Table I). Three specimens of postmenopausal endometrium were studied. The activity was low in all fractions (Table I). Two specimens of endometrial cancc.r were studied. One was anaplastic adenocarcinema with a hi,gh degree of activity and 40 STEROL
I
1 1 1
2
4
6
Fig. 3. Incorporation normal endometrium.
I
/
IO DAY
I ii
12 OF
I
16 CYCLE
of C’4 into (From Table
I
18
1
20
1
22
triglycerides I.)
1 26I
24
by
2
4 6
Fig. 4. Incorporation normal endometrium.
ESTERS
8
IO DAY
0
I2
14
OF
CYCLE
of Cl’ (From
I6
into Table
18
20
sterol I.)
22
24
esters
26
by
626
Merrill
and
Werthessen
one was a very well-differentiated adenocarcinema or carcinoma in situ with low total activity in lipids. The pattern of distribution of activity within the ester classes resembled secretory endometrium more than it did proliferative (Table I ). Since our findings sugaested to us that endometrium is able to convert acetate to fatty of acids and to esterify a significant portion trithese fatty acids into phospholipids, glycerides, and sterol esters, it was considered worthwhile i and greatly time-saving) to see if the fractional distribution into each of thrse classes changes as a function of the menstrual cycle. Table II lists the distribution ratio of lipid classes studied for 37 normal endometria. ‘The C” titer in each fraction considered, expressed in counts per minute, was divided by the surn of the fractions to provide the relative distribution of label into each of the three classes. By this procedure, no note is taken of the C:‘l incorporated into a number of other compounds, for example, free fatty acids and sterols. A shift in distribution ratios of the Cl4 incorporated into these three classes \vas seen,
NON-ESTERIFIED
a
However, the phospholipid \.alurs contrib uted a major variation from specimen to specimen and no statistically significant (litfcrence occurred. When only the relative incorporations into the sterol ester and the triglyceride fractions were considered (Fic. 7 i a statistically valid differcnct. was tl(monstrated. with a shift of incorporation toward sterol esters at the tirnr of ovulation mjIIICYU~ proliferative 0.38: mean secrrtory 0.77. P 0.001). These observations arr difficult IO interpret as they are nnt rcblatttd to \\cigtlt or total lipid extract. They do suqgest that a chan,qr in thr pattern of cstr*rification ma\ occur at the time of ovulation. I~ktailecl analvses of these and related results \\ill io~lll the basis of another communication. Comment
The results arc preliminary and upon too few cases to provide anything
based more
225k
0
LIPIDS
PROL
SEC
50 0 5
0
25
1 ]
8 , 2
, 4
, 6
/ 8
/ IO DAY
, 12 OF
,
,
14 I6 CYCLE
,
,
0 0 ,
/
0 ,
18
20
22
24
26
Fig. 5. Incorporation of Cl” into the nonesterified lipids (that portion of the total lipid extract not accounted for by phospholipids, sterol esters, and triglycerides) by normal endometrium. (From Table I.)
NORMAL ENDOMETRIUM
Fig. 6. Mean values for incorporation of Cl4 into the lipid classe: by normal endometrium during the proliferative and secretory phases. TL = total lipid extract; NEL = nonesterified lipids, PL z-z phospholipids. SE = sterol esters. 7‘g.T = triglycrrides. (From Table I.)
Lipid
PROLIFERATIVE
Fig. 7. Mean Table I.)
SECRETORY
values
for
incorporation
DECIDUA
of C 1.1 into
than leads for future investigation, The fact that human endometrium is capable of incorporating labeled acetate into lipids agrees with studiesI”> ‘25,26 showing this tissue to be metabolically active. The analysis of the sterol esters, the molecular structure of triglycerides and phospholipids, plus the large amount of count found in eluates not specifically analyzed but known principally to contain fatty acids suggest that the major lipid biosynthesis observed in these experiments was of fatty acids which are rapidly esterfied to a significant degree. Apparently, although cholesterol is present in the endometrium, little is synthesized from acetate. Since other tissues synthesize cholesterol at a rapid rate, this finding needs further clarification. It is quite probable that endometrium is able to synthesize cholesterol but that under the conditions employed it does not use acetate as a preferred substrate.
lipid
biosynthesis
ADENOCARCINOMA
ATROPHIC
classes
by various
627
endometria.
(From
This situation has been described for the brain.l’ Increased incorporation of label into total lipids and phospholipids during the proliferative phase probably reflects estrogen influence upon enzyme systems responsible for increased energy and synthetic activities of the growing cell. Nucleoprotein concentration and activity increase in human endornetrium to reach a peak at the end of the proliferative phase and early secretory phase.” Estrogen has been shown to cause an increase in cholesterol, fatty acid, and phospholipid synthesis in animal uteri.‘, a, ‘3 ” Gorski and Nicolette’” have shown that estrogen enters the endometrical cell and exerts early influence on the RNA synthesis by the nucleus. RNA then influences lipid synthesis. Similar investigation has shown that several metabolic pathways can be effected by estr0gen.l”) “1 26
628
Merrill
Day i 1 1 1
3 5 6 6 7 7 8 8 9 11 11 11 12 13 13 13 14 15 15 15 15 16 16 17 17 18 19 21 21 22 22 ‘2 22 26
and
Werthessen
NEL/?'L 0.91 0.44 0.39 0.74 0.71 0.71 0.34 0.68 0.30 0.69 0.66 0.77 0.59 0.41 0.72 0.80 0.45 0.53 0.86 0.39 0.43 0.44 0.33 0.60 n.5i 051 0.29 0.14 0.28 0.25 0.22
/
PL 0.63 0.52 0.09 0.65 0.24 0.07 0.16 0.03 K? 1 0.40 0.30 0.44 0.51 0.03 0.02 0.08
0.75 0.27 0.29 11.26 0.04 0.42 0.10 0.37 (0.003) n.zn 0.19 0.2 1 n.01 0.37 0.24 0.03 0.58 0.36 0.46 0.02 0.01
SE
I
O.l(l I). I I (1.23 0.1 7 O.L’7 0.5.5 0.14 O.-hi) O.-ill n .3 I Il.34 0.19 (I.26 0.36 0.08 0.45 0.119 0. I3 0.26 0.43 0.32 0.25 0.40 0.28 0.98 0.7L’ 0.51 0.77 0.97 o.:ii 0.67 0.9ti 0 3 i’ 0.38 0.2 I 0.30 0.98
The relatively high rate of incorporation into phospholipids found in term decidua probably reflects an estrogen effect and is compatible with rapid tissue growth. The contrary is characteristic of postmenopausal endometrium. The importance of phospholipids in the process of cell growth is well linown.’ 3 The difference in lipid biosynthesis between the anaplastic adenocarcinoma and the carcinoma in situ reflects the degree of cell growth. It has been reported that adeno-
j
Tg.9 I).?7 11.38 0.68 0. 18 0.49 1).38 0.7ll 11.53 l).-lO Ii.29 11.36 lr.:ci CI.L’Y 1l.h 1 l1.!?0
(I.47 lI.li li.hl Il.-l5 I).31 fI.G-1 n.:4:4 o..io 0.35 0.02 l).llll fl.Yll l).ll? fi.llL’ Ii.25 ll.ll9 11.01 0.11~ 0.26 ( 1, :i :4 0.6X 0.01
/
SE
i
'T@
0.28 Il.‘L’ 0.23 0.4X I I. 145 0.59 (I.16 O.-l6 l1.5ll 0.511 0.48 0.33 0.53 lJ.37 lI.llU 0.49 0.3-i
0.58 0.33 o.+-1 (1.4~4 O.-l-k (I.98 fl.!ll) 0.7 I
carcinoma of the endometrium has a histochemical pattern similar to that of secretor) endometrium.“* ?a Our observed patterns of esterification correspond to such findings. Because previous studies have dcmonstrated increased stainable fat and concentration of neutral fat during the secretor\ phase, our findings of drcreasccl inrorporation of acetate into total lipids, including triglycerides, during the secretory phase suggest that stainable lipids result from incorporation of lipids from the plasma. rather
Lipid
biosynthesis
629
Summary
c
c, I
Average Prol I I I 2 4 6
=.38 8
IO DAY
’ , 12 OF
, , 14 I6 CYCLE
Average Set = 77 , , , 1 1 I8 20 22 24 26
Fig. 8. The ratio distribution of Cl& between sterol esters and the triglycerides expressed as ratio of sterol ester Cl4 count per minute to sum of the count in the sterol esters and glycerides. (From Table II.)
the the the tri-
than synthesis. However, since there may bc a shift in the degree and the nature of esterification of lipids following ovulation, it is possible that synthesis of specific fatty acids, not readily available from the plasma, may occur and even be an important prerequisite for successful implantation. Analysis and definition of specific fatty acid synthesis is required to clarify this issue. Lipids contained within a ccl1 are likely to have several functions. Some of them are essential building blocks of the vital structure. Some are metabolites that are consumed to pro\;ide energy. Others may be present as eneqgy stores. The lipid portion (90 per cent phospholipid) of the mitrochondria is as vital to their function as is the protein portion.Ii Our studies suggest variations in lipid biosynthesis as a function of the menstrual cycle. Thus, it is important to pursue studies designed to codify the normal pattern of lipid biosynthesis in human endometriurn and to determine if deviations from an established pattern are related to clinical abnormalities of reproduction, such as infertility, abortion, and other pregnancy losses, as well as to malignancy.
Previous investigators have demonstratrd that lipid biosynthesis by animal endometrium is subject to the control of hormones. Chemical analyses and histochemical studies are available, but there are no previous studies of actual lipid biosynthesis by human endometrium. Fragments of human endometrium, obtained by hysterectomy, were incubated for 4 hours in the patient’s serum to which acetate-l-C” was added. Chloroform-methanol was used to terminate biosynthesis and begin extraction of lipids. Serum lipids served as carriers for the tissue lipids through a separation procedure which segregated phospholipids, sterol esters, triglycerides and the remainder of the total lipids. The incorporation of C?’ into each class was determined. The preliminary data suggest that lipid biosynthesis by human endometrium varies with time during the menstrual cycle and is characterized by greater incorporation of label into total lipids, phospholipids, and triglycerides during the proliferative phase than during the secretory phase, with no significant change in the incorporation of Cl’ into sterol esters throughout the cycle. The nonesterified lipids demonstrate greater synthesis during the proliferative phase than during the secretory phase. The major lipid synthesis is of fatty acids with prompt esterification. The proportion of total lipids esterified is greater during the secretory phase than during the proliferative phase. Moreover, the pattern of esterification may shift at thr time of ovulation, with a relative increase in sterol esters. This change reflects less variability in the proportional incorporation of label into the sterol esters and the triglyccrides than in the incorporation into phospholipids.
REFERENCES
1.
Aizawa, Y., and Mueller, G. C.: J. Biol Chem. 236:381, 1961. 2. Aschheim. S.: Ztschr. Geburtsh u. GynHk. 77: 485, 1915.
3. 4. 5.
Black, J., Heyns, 0. S., and Gillman, J.: J. Clin. Endocrinol. 1: 547, 1941. Borell, U.: Acta endocrinol. 9: 141, 1952. Bottcher, C. J. F., Woodford, F. P., Van
6.
7.
8. 9. IO. 11. 12. 13. 14.
15. 16. 17.
Houte, E. B., and Van Gent, C. M.: Rec. d. trav. chim. d. Pays-Bas 78: 794. 1959. Cohen, S., Bitensky, L., Chayen, J., Cunningham, C. J., and Russell. J. K.: Lnncet 2: 56, 1964. Cridcr, E., Alaupovic, P., Hillsberry, J., Yen, C., and Bradford, R. H.: J. Lipid Res. 5: 479, 1964. Davis, J. S., and .4lden. R. H.: .4nat. Rec. 134: 725, 1959. Emmelot, T., and Bosch, I,.: Rec. d. trav. chim. d. Pays-Bas 73: 874. 1954. Froboese, C.: Vii-chow’s Arch. path, .4nat. u. Physiol. 250: 296. 1921. Gillman. J.: South African J. M. SC. 6: 59. 1941. Gorski, J., and Nicolctte, A.: Arch. Biochem. 103: 418, 1963. Goswami, A., Kar, A. B., and Chowdhury, S. R.: 1. Reurod. & Fertil. 6: 287. 1963. Green, “D. E.. and Fleischer, S.: Role of lipids in mitochondrial electron transfer and oxidative phosphorylation. in Frazer, A. C.. editor: Biochemical problems of lipids. New York. 1963, Elsevier Publishing Company, p. 325. Gross. S. J.: Ant. J, OUST. & C~YXEC. 88: 647, 1964. Hughes, E. C., Jacobs, R. D., and Rubulis, .4.: Anr. J. 0~s~. & GYN~X. 89: 59, 196-f. Kabara, J. J.: Brain cholesterol. The effect of its development on incorporation of acetate-2-H3 and glucose-U-C’ c% ill Himwich, W., and Himwich. H., editors: The Developing Brain. Progress in Brain Research, New York, 1964, Elsevier Publishin!: Company, vol. IX. p. 15.5.
18. 19.
2-l.
2.5.
26.
Leathem, J. H.: Ann. New York .4cad. SC 75: 463, 1959. McKay, D. G., Hertis. :\. T.. Bardxwil. M’. ii., and Velnrdo, J. T.: 0bst. & Gynec. 8: 22, 1956. McKay. D. G.. Hertig. :I. T.. Bardawil. M.. A.. and V’elardo. J, T.: Obst. iuc Gynec 8: I-&O, 1956. McClrnnan. C. E., and Koete, P.: W’esr. J. Surg. 61: 169. 1953. Noycs, R. W., Hertig. A. ‘I‘ , and Rock. J.: Fcrtil. c& Steril. 1: 3, 1950. P:igr. E. W., Glendening, M. B.. and Parkinson. I).: Aax. J. 0~s~. & GYNICL:. 62: llOt1. 1951. Pearse. A. G. E.: Histochcrnistry: Theoreti<..il and applied. ed. :‘. London. 1960. J, S: .4 Churchill. Ltd. Robertson. G. L., Ha~erman, D. D., Richardson. G. S., and Villee. (Z. 4.: Science 134: 1986, 1961. Saksenn, K. C., Arora, M. M.. Guptn. .J. (:.. :md Rangam. (:. M.: Indian J. M. St-, 19: 121. 1965. Stein, R. J., and Stuermrr, 1‘. M.: :Iht. ,J. OUST. 8.c GYXEC. 61: -11-1. 1951. Sturrrner, \‘. M., and Stein. R. J.: Ax. J. OUST. & GYSEC. 63: 359, 19.5”. v,anDykr. H. B., and Chrn. (;.: .2m. J, Anrt. 66: 411, 19.40. Werthessen, N. T.: CirrulJtion Rrs. 6: 759. 1958. Westphalen. I‘.: Arch. Gyniik. 52: 35. 189ti.
Discussion DK. Russ~1.1. R. IIF. AI.VAREZ, Philadelphia, Pennsylvania. This important paper of Drs. Mcrrill and Werthcsscn represents an approach to the study of lipid metabolism in the female through the incorporation of labeled acetate into three principal esterilied lipids. They report their resultant data in counts per minute, while oui serum data have been reported quantitatively in micromoles per milliliter and in weight per cent so that comparisons between our respective results are not presently possible. WC wonder, however, whether it is appropriate to incubate tissue in the patients’ sera and then attribute all lipid alteration as being characteristic of the tissue studied. The significant finding of their work is the demonstration of the ability of the endometrium not only to incorporate acetate-l-Cl4 but also to convert this fatty acid (in miniature) to
higher fatty acids and then to esterify theni into phospholipids and cholesterol esters. In our own evaluation of each lipid fraction in growth, COIIsistent alterations in the major lipid classes occur whether the growth be of trophoblastic or nl’crplastic origin. Increases in total lipid, triglycerides, total ester, and free cholesterol, free fatty acids, total phospholipids, and of each phosphatide subfraction tend to occur both in pregnanny and in gynecologic mali,~nancy. Wht seems to he most signifirant, however, is not so much the variation or even the direction of the major lipid fractions themselv~es, but the relationship of the fatty acid composition of each group of esters in the normal nonpregnant, the normally pregnant patient and in the patient with gynecologic cancer of various primary sites. The authors found similarities of radioisotope uptake in cancerous endometrium and in normal
Volrlme Number
96 5
preovulatory endometrium. The greater incorporation of labeled acetate into phospholipid fractions of decidua complements the observations noted by us in normal pregnancy. In our patients with endometrial carcinoma and in those with normal pregnancy, the serum lipid patterns of each group behave similarly. Both groups of patients demonstrate a general reduction of unsaturated fatty acids, particularly linoleic arid in most lipid groups but usually associated with an elevation of palmitic acid, especially in the phosphatidyl esters. WI, believe some of the alterations in lipid synthesis of active endometrium (whether it be cyclic proliferation and growth, pregnancy, or cancer) must he endocrinologic and, thus, constitute a reflection of the energy requirements necessary to initiate a metabolic mechanism for stress and growth. It seems reasonable to cogitate that the frequent combination of endometrial carcinoma, obesity, and diabetes may well reprcsent the endpoint of abnormal hyperlipemic states occurring prior to the appearance of clinical diahetes and even indicate the likelihood that latent diabetes is latent only as regards carhohydrate metabolism, but how can it be so limited? Therefore, we would raise for consideration the thesis that clinical or laboratory evidence of hyperlipemia, or of diahctes, or of a family history of these states constitutes sufficient justification to study prospectively the potential lipid alterations, continued and repeated investigations in search of evidence of carbohydrate intolerance and an awareness of a lipogenic-cancer rrlationship. Drs. Merrill and Werthcssen have convinced us of the nerd to expand further our invcstigations of lipid metabolism not only in serum and in obviously pregnant and malignant tissue, but also in cyrlic and decidual endometrium and in normal gynrcologic structures capablr of the development of potential malignanry. LVith data from both our departments expressed in comparative and quantitatablc terms, \~e look forward to the devclopmrnt of a better understanding of the mechanisms responsible for the changes noted, not only in normal uteroovarian physiology, but in cancer as well. DR. NICHOLAS T. WERTHESSEN,” Oklahoma City, Oklahoma. An instrument is now being developed which in the near future will permit a tremendous reduction of the staff necessary to *By
invitation.
Lipid
biosynthesis
631
do this type of study, and our hope is that it will be possible to show changes in enzymatic structure within the cell with greater ease than is possible now. DR. J. DONALD WOODRUFF, Baltimore, Maryland. I gather from your presentation, Dr. Mrrrill, that estrogenic activity in the endometrium was correlated directly with lipid metabolism CYccpt for sterol esters, and that activity in adenocarcinoma, in turn, was correlated with that of the estrogenically stimulated endometrium. Tllis we have felt in the past to bc true, although there have bren dissenters to this thesis. I would like to know if, in reality this relationship cxistq, since I get a different point of view from our conclusions, nameI)that the patterns in adenocarcinoma simulate those found in the secretory cndomctrium rather than those of the proliferative phase. DR. SAUL 8. GUSBERG, New York, New York. I would like to make a comment related to that of Dr. Woodruff. As I looked at this material of Dr. Merrill, so beautifully investigated, it seemcsd that the lipid biosynthesis did not necessarily correlate with steroid function because the anaplastic adenocarcinoma-the high growth-phase carcinoma-showed more lipid synthesis than did the differentiated one which might be expectcxd to be more hormone-dependent. Thus it \~oultl, in this instance, appear related to growth phrnomena rather than hormonal ones. DR. MERRILL (Closing). So far as the qursticjn of adenocarcinoma is concerned and its lipid synthesis, there was a distinct difference in the rate of biosynthesis observed in the well-diff(*rentiated tumor as compared to the anaplastic. The latter showed a higher rate of incorpol-ation of label, hut the pattern of cstrrifiration of lipids was the same in both. The pattern LV:IS one of greater incorporation of the label into stcrol esters than into the other esters. This KLS similar to the pattern of esterification found in normal secretory endometrium. Although the degree of activity, i.e., the total amount of lipid synthesis, was much greater for anaplastic carcinoma than for any of the normal nonpregnant endometriumspand thus resembled proliferati1.c rndometrium-the pattern of estcrifiration W;IS similar to secrctor)l endomctrium. This rorrcspends to th
632
Merrill
and
Werthessen
stimulation. One might wish to say that this is evidence that adenocarcinoma is the end stage of a progesterone stimulation situation. All I can say is that this is the pattern we found. A question was asked about abnormal lipid and glucose metabolism. I am sure that this would reflect itself in an abnormal pattern of synthesis of incubated tissues. This has been demonstrated in animals. The activity of incubated tissues can be altered if the animal has an abnormal nutritional or metabolic situation.
A.
NICHOLAS OXlahomu
MERRILL, T.
City.
IM.D. WERTHESSEN,
PH.D.
Oklahomu
1-I 1. hr A N endometrium undergoes (‘!chc changes in structure and
I~hcting
by human endometrium
striking function
the luteal phase of the cycle in the monke, uterus, and McClennan and Koets”’ found total lipids to be in highest concentration during the secretory and menstrual phases in human endumetrium. The histochemistry of the human endometrium has been studied by many workeTs6’ 15, 13. 20, 26-2s but with little emphasis placed on the lipid changes. McKay and associates’“. ‘* reported that the proliferative phase of the cycle was characterized, histochemically, by the presence of large amount:, of ribonucleoprotein and alkaline phosphatase activity and by small amounts of acid phosphatase activity, glycogen, and glycoprotein. The secretory phase was characterized by a reversal of this pattern. Page, Glendening and Parkinson”” rcviewing the histochemical and biochemical changes reported in the human endometrium classified them into four patterns: ( 1) stimulation by estrogen (@glucuronidase) ; (2 1 stimulation by estrogen withdrawal (fibrinolysins) ; (3) stimulation by progesterorle by es(fat, &cog-en) ; and (4) stimulation trogen and inhibition by progesterone (RNA, alkaline phosphatase) . Experimental data support a theory (A hormonal control of lipid biosynthesis in endometrium” 4, 8, 9, 12, I33 IS as well as other tissues.“o Borell’ described the influence of ovarian hormones on the metabolism of nucleic acids and phospholipids in the rabbit uterus. He demonstrated greater incorporation of radioactive phosphate into phospholipids during the luteal phase. Progesterone was shown to promote a greater uptake of phosphate than estrogen. This has not been found by subsequent workers. Emmelot and Bosch” used acetate-f-Cl4 to study lipid bio-
hormonal inff uence. The histoIogic
I’hangcs are weI1 documented’? and the chemical changes are being studied increasiljgjy.“* 12. 11;.18-21, 23, L’G-2SIn 1896, Westphal(‘rr”’ first described the presence of stainable lipids in human endometrium. Aschheim’ lound that 80 per cent of endometrium studied contained fats during the premenstrual phase. Froboesc’” found that fluctuations in II tcr-inc. lipids correlated well with protein I.lrar~ges in the uterus. Gillman” thoroughly described the stainable lipids in human endothroughout the menstrual cycle and greatest abundance of stainable iat between the twenty-first and the twentyfifth day of the cycle. Black, Heyns, and C;illrnan proposed that stainable lipids be used as the basis of a bioassay method for determining progesterone activity. He observed that an optimum balance between estrogen and progesterone was necessary in the human for stainable fat deposition to occur. may be as dependent on Lipid staining the physiochemical state of the lipid as it is on the concentration. Furthermore, lipids such as cholesterol do not take the Sudan vanDyke and Chen”’ stains.“’ However, found fofnl neutral fat to be highest during rnctrirrnr
tound
the
From the Department of @necology rind Obstetrics, Uniuersity of Oklahoma School of Medicine. Supported by United lleulth Service Grant
States Public No. HD 01188.
Presented at the Eighty-ninth Annual Meeting of the American Gynecological Society. Hot Springs, Virginia, June 2-4, 1966. 619
620
Merrill
synthesis
and
Werthessen
by the uterus of mice and found of acetate to choksterol and fatty acids was promoted by estrogen administration as compared to untreated control castrates, He was primarily interested in the rrlative effect of the estrogens ( ET, Ez, W upon uterine lipid biosynthesis. The greatcst cffe,-t was obsrrved with 17,&estradioI rind the least. with cstriol. Leathem’” found vssentially no change in the quantitative amount of choIestero1 in the immature mouse uterus folIowing estradiol and progesterone injection. Davis and Alden” studying rats, found that estrogen treatment of spayed animals caused a significant decrease in concentration of neutral fat and an increase in phospholipid concentration. Proqesterone did not increase the concentration of neutral fat or phospholipid, hut did increase the osmiophilic or stainable fat. Goswami, Kar, and Chowdhury’” studied the mouse uterus by quantitative chemical analysis of total lipids, triglycerides, sterol esters, free sterols. and phospholipids. They demonstrated a similar change in metabolism of lipids. The most conspicuous change was the increase in concentration of phospholipids during estrus. This was abolished by ol-ariectomy but restored by estrogen therapy. The restoration of phospholipid concentration was clearly antagonized by progesterone. Less conspicuous changes occurred in the concentration of cholesterol and sterol esters. Aizawa and Mueller’ e\xluated the effect of estrogens on lipid biosynthesis in the rat uterus using both P:‘” and (:‘f. Estrogen was shown to cause rapid acceleration of all pathways for lipid production measured. Estradiol stimulated incorporation of acetate-I-C?’ into fatty acids, cholesterol and other constituents of the nonsaponifiable fraction Of uterine segments. GorsI2.i and r\‘icolettc.‘” [Ising P:“. found jn x.il.0 RNA and phospholipid svnthesis increased in subcellular fractions of the rat uterus as a resolt of cstradiol treatment. When uteri were incubated in vitro the estrogen pfI’cc[ c,n l
conversion
Lipid
and delivered to the laboratory. Kepresentati\.r aliquots of the tissue were fixed in Bouin’s solution and prepared for histologic vsamination. Specimens of tissue were trimmed SO that no piece was more than 1 111111.wide. Because of the variability in cohesiveness and friability of endometrium, it has not been possible to use any of the de\ices available to prepare perfectly uniform standard-sized slices. The tissue was incubated in 10 ml. of the patient’s serum, obtained shortly prior to removal of the tissue, to which acetate-l-c” \vas added at the concentration of 0.1 millic,urirs of CL4 per liter. The patient’s serum leas used as the incubation medium because it could be expected to provide the tissue Lvith a close approximation of the hormonal 4mulation it had received in situ and to provide carrier lipids for the small amount of lipid available from the tissue. Thus macro, rather than micro, techniques could be used for the separation of the lipid classes. Incubation was maintained in a Dubinoff shaker for 4 hours under 5 per cent CO, ,/ 95 pt’r cent 0, at 37.5’ C. At the end of the incubation period, serum was decanted from the tissue and treated with 3 volumes of chloroform-methanol (2 to 1 \-. V.) . The tissue was rinsed with saline: placed on filter paper and the excess seru~n removed. A wet weight was obtained. The tissue was placed in a weighing flask, a few drops of chloroform-methanol added to stop synthesis and the flask and contents placed in a high vacuum desiccator until constant weight was reached and the total dry weight determined. Following this determination, the tissue was further extracted with chloroform-methanol by a modification of the method of Bottcher and associates.” ‘I‘he same drying procedure was used followinx lipid estraction to determine the dry, dcfatted weight. In some experiments, it was elected to study only the relative distribution of labeled acetate into various lipid classes, and weights \vere not determined. In these experiments, the total incubation system was frozen and
biosynthesis
621
the water removed by lyophilization. The dried powder and tissue obtained by this procedure were treated by the Bottcher cxtraction technique’ in the same fashion as the dried tissue. The lipid extracts from the tissue and the serum were combined to yield a greater quantity of lipids with which to work. The chloroform-methanol was evaporated to an oil, extracted with chloroform, and filtered. Removal of residual acetate was acconlplished by eyuilibratin~ the total chlorofornl extract against two changes of IO per cent glacial acetic acid iti water. The titer of C” was determined at this point for the total lipid fraction. The lipids were separated into four classes: phospholipids, sterol esters, triglycerides. and the remainder of the lipids containing frecb fatty acids, free sterols, and other unidentified lipids. Initially, the free sterols wert’ separated as a class. Except for the phospholipids, the separation was performed h) column chromatography and later by thinlayer chromatography. The chloroform extract was evaporated and extracted with dry acetone and then M&l 2 added to the acetone to completely precipitate the phospholipids. The insoluble material (phospholipids) was redissolved in chloroform-methanol and assayed to give the <:I” titer of the phospholipid fraction. ‘The acetone soluble fraction was transferred to hexane, placed on a chromatoKraphic column (packed with Silica gel G, held in suspension with hexane), eluted with hexane and increasing concentrations of ether, and then stripped with methanol. This procedure has been found to provide good separation of the sterol esters from the triglycerides.’ The chromatographic column technique employed was that described by (Irider and associates.: In later experiments, thin-layer plates made of Silica gel G were used and the separation accomplished by developing in 7 per cent ethyl ether in petroleum ether. In thin-layer analyses, charring with HzS() L was used to identify fractions.
622
Merrill
and Werthessen
After appropriatr corl,ections for \.oltmlt’, the (:‘I titer, expressed as counts per rninute, for the combined eluate classes (sterol and the other lipids esters, triglycerides, ) free sterols! fatty acids, etc. 1 ) were recorded. All C:” determinations were made on a Packard Tricarb Scintillation Gaunter. using “POP and POPOP”” dissolved in toluene as the scintillators. ‘4 spot test based on the LiebermannISurchard reaction \vas used to locate the stcrol esters and free cholesterol fractions in the column eluates. In the chromatoqraphic svstems e111p10yccl. thr free fatty acids. monoglycerides. and diglyceridcs contaminatal the. free stcrol fraction. ‘Ik!rcfore, the free sterols were precipitated with digitonin determined. In and the (:I’ incorporation those instances in bvhich C:” titer was sufficient to permit ultimatr purification and assay, carrier cholesterol was added. and a dibromination purification done also. The cholesterol ester fractions. which tend to romc ofl‘ the colmnn in a sin&. massi\,c front, wt’re hydrolyzed in ethanolic KOH and the cholesterol similarly precipitated. ‘I’he fatty acids from the hydrolysis were collected and assayed for C:” incorporation as well as the sterols. It was soon found that (1” incorporation into cholesterol was not usual under the conditions employed. This will be the subject of a later communication. However, because of this findinp. our procedure was altered so that the free cholesterol fraction was for the time neglected. We have used only a part of thr information which we have obtained. Most of the emphasis has been placed on three fractions: phospholipids. triglycerides, and sterol esters. These fractions constitute over 90 per cent of the fat in the body and are the most consistent with re,qard to their purity from one experiment to the other in the analytical procedures employed, The fraction containing free sterols is contaminated by as much as 20 per cent of the total (1” incorporation into lipids in some experiments, but it usually contained 5 to 10 per cent. The nature of these lipids reytlirc fur-
ther study. Free fatty acids \\‘c’rt’ 11c)t (It*termined or studied. From the point of assay of ttlcs chlorofolnlsoluble lipids the continued assay of (1” titer in the \,arious fractions permits an audit of the over-all efficknc\. Where column chromatography was employed, ~111 over-all recovcllr of 85 1)~” c~nI of the ot iginal titer. was considered nc~trrssary. I.%? tliiniltia! thin-layer chrorriatogral~h~~. thr t‘t*cov(‘ry acct~ptabl~~ haa Ilccn raised to %I pt’r cc-nt and is commonl! t,et~veetI 9i ,ttlci 100 per crnt. Resolution of thta pr,oblenl 01 ~neasurin~ (mused substrarc c,ontanlinatinr: a fraction has been 111~1 I)\, 1\\0 prt~cautionk ‘I‘ht- first d thcsr was this &)rouKh w,ashinq of tht. total lipid cstrac’l \\.itl! 10 ljet ( (‘I~I glacial acetic ac.id in water. .l’llt, second tla5 heen thr dissolving of a I’raction irl wctir :uzid, rhcn c>vaporating ttic itcc.tic- acid ,jiibt prior to co~inting and cctrnparinq qlic-11 .L washed sample’s count L\ ith #iti unt5~ral~.(l aliyuot. .4 difference 1)etwrt.n th<, (‘oulltb (‘.~IE br ascribed to unused aceta TV ot short-chairi Iattb- acids distillable in ac,catic- ‘I(‘1‘d urttic~t these conditions. Fl’hcX audit oi leach t‘x[Jc’!‘I. Itltant ( onsidcrrhd both salts 01 \.alut*s. ‘1%~ representative aliquots ol’ tissue fis~tl and prepared for histologic. tbsarnination M’t’re stained with heniatos\~lin .rnrJ tGti. Moreo\,er. the routine i!irii(.;ll p2lt~JOkW\ specinirria Lvert’ also revirwcd. Sornlal IYXIOrllc*triurn was dated according IO thy c’ritt.ri;r ol’ Noytas. Herti,g:. and Koch.” ‘l‘h(* hospital chart of each patient bvas c-a&u iI\- rc*\+k~c~cl for evidence of menstrual or ~~ndocrine al)normalitv. Only those patients \\,ithollt (,1i11ical abnormalitv and with c.loxt, ~ot~rrlatio~~ of the mrnstrual dating I)v l&t~,Io$c~ ;itl(I historical criteria were includc~tl cl\ rr0r-nlcr/t. Although Iriariy more spccimc$ns II~\Y~ bc~71 studied. detailed analyses for [his prelimirlar-\ r‘tq)ort includes 1.5 normal ~ndotnrtria. L specimetls of endometrial adetloc,Llr,c,inottl;l, ‘i specimens of l~ostmenopausal t~rldorrlrtriI,rtr. and 6 specimt~tls of dt~cid~la. Results
‘l.able I lists the incorporation of acetatcbI -( 1’ ’ into total lipid estracted i‘l,onr \,aric)It\ c*ntlometria and incorporation itlro phospf~o-
Lipid
biosynthesis
623
Table I. The Cl4 titer expressed as counts per minute per milligram dry, defatted weight for total lipid extract (TL), phospholipids (PL), sterol esters (SE), triglycerides (Tg3), and the remainder of the total lipid extract (NEL) in various endometrial specimens ____
c.p.m. X I02/mg. Histology Day 1 Day 7 Day 7 Day 9 Day 11 Day 13 Proliferative Day 17 Day 17 Day 18 Day 21 Day 21 Day 22 Day 22 Day 22 Day 26 Secretory Decidua, Decidua, Decidua, Decidua, Deridua, Decidua, Decidua, Atrophic Atrophic .4trophic Atrophic
(mean)
(mean) term term term term term term (mean) 6 weeks
(mean)
Anaplastic noma
adenocarci-
Carcinoma
in situ
7-L
PL
SE
Tg3
NEL
292.8 349. 321.7 208.3 103.6 ‘J33 7 ..--.. 249.6
16.7 81.1 72.9 107.6 36.2 68.6 63.9
2.66 21.1 1.7 23.4 19.1 8.14 12.7
6.99 56.1 56.9 5.0 16.9 15.2 26.2
266.5 J90.8 190.2 72.2 31.4 130.4 146.9
11.8 2.9 0.37 8.6 0.91 19.3 28.6 0.30 9.1
21.1 10.7 33.7 24.7 31.0 10.8 4.4 37.3 21.7
8.5 0.37 0.64 3.3 0.46 3.2 1 .3
20.0 21.1 46.1 37.6 13.0 5.5 41.7 10.9 24.5
157.1 93.6 117.6 117.6 121.5 33.3
67.2 40.8 81.5 85.8 79.4 70.9 __
16.1 6.6 3.2 3.2 5.4 6.9 -
28.5 24.9 15.2 22.9
5.2 10.0 3.3 6.2
4.9 5.1 1.0 3.7
343.3
37.4
96.5
61.5 35.0 80.9 74.2 45.4 38.8 76.0 90.6 49.1 61.3
34.7
-
lipids, triglycerides, and sterol esters. Lipid biosynthesis from acetate by human endometrium occurs at a very rapid rate. The incorporation, on a per gram basis, by proliferative endometrium and anaplastic adenocarcinoma is comparable to that found in human liver (225 c.p.m. per milligram) .” In the exploratory phases of this study, cholesterol was studied as derived from both the free sterol and the sterol ester eluates. The separation and purification procedures were designed to allow a high degree of purity. Eleven of 19 normal endometria had no C” count in purified cholesterol and 6 of 14 abnormal endometria showed no count.
_____
21.2
0.56 2 .3 34.4 14.8 17.5 16.8 21.6 21.0 0.49 1.1 1.9 1.2 17.6 0.78
39.8 31.5 -. 11.7 11.3 23.6 _17.8 8.7 9.0 11.8 191.8 12.2
Moreover, the free sterol fraction constituted a small part of the total incorporation into lipids (usually less than 5 to 10 per centj. Having found little or no C” in the free cholesterol fraction, the sterol ester fraction was analyzed to determine the portion of the ester molecule into which the Cl4 had been incorporated. In all cases studied, following hydrolysis of the sterol esters into sterol and fatty acid moieties, most, if not all, into of the acetate- 1 -Cl4 was incorporated the fatty acid portion. The trace of activity found in the sterol fraction remained in the mother liquor when the sterols were subjected to digitonin precipitation.
624
Merrill
and
Werthessen
Biosynthesis of the non-fatty acid portion and triglycerides from of phospholipids labeled acetate has not been specifically checked. However, based upon the mo!ecular structure of these moieties, it is reasonable to assume that the major portion of the newly synthesized triglyceride and phospholipid molecules that contained C” derived from acetate is the fatty acid portion. All of the lipids which are eluted after the triglycerides have not been specifically studied. These eluates account for a significant part of the total lipid extract. According to Clrider and associates7 these eluates contain free sterols, free fatty acids, and residual phospholipids. The free sterols were identified. Thus the terminal fraction should contain mostly free fatty acids, any phospholipids which failed to precipitate with acetone (less than 3 per cent by weightj and probably diglycerides and monoglycerides. These eluates contain substances which require identification and study. It is also essential to find out what other sterols, besides cholesterolJ are contained in the free sterol fraction. Figs. 1 through 5 show the distribution of C:” (expressed as r.p.m./mg. dry, defatted
350-
0
325L 3OOL0
0
TOTAL
weight) incorporated into lipids by 15 normal endometria at various times during the menstrual cycle. The proliferative phase is considrred as day one through day 15 and the secretory phase, day 16 through day 28. Incorporation of label into total lipids MU greater in the proliferative phase (mean 24,960 c.p.m. per milligran~ than during 6,130 c.p.111. the secretory phase (mean lxr milligram) (P = O.OU I ! (Fig. 1 : ~ Incur1)oration of label into l~hosl~holipids XV;IS treater during the proliferatkrcs phase ~,mt’an 6,385 c.p.m. per milligram ; than during tlte secretory phase (mean mm-910 r.pm. prr milligram) [ P = 0.001 ) t Fiy. 2 !, ‘T?ic~ irlcorporation of label into triglvceridrs bvas greater during the proliferative phase / nwm 2,618 r-.p.m. per milligram,; than during the secretory phase (mean r-r 230 C.~.III. pvt. milligram) (P =z 0.02) (Fig. 3 i, Incorporation of label into sterol esters showed ruucll \,ariation with no siqiificant chanqe
PHOSPHOLIPIDS
LIPIDS
275250-
E E” ,\ 0
_ 0
m-
III 2
4
6
I
I,
6
10 DAY
Fk. I. Cl4 incorporation by normal cndometrium.
I 12 OF
/
I
/
,
14 16 CYCLE
18
20
22
i4
in the total lipid extract (From Table I.\
I 26
1
21
4
c
6r
lb DAY
Fig. ‘2. Incorporation normal endometrium.
121
T----v*-.-4, 14 16 OF CYCLE
I6
20
22
of Cl4 into phospholipids (From Table I.!
24
26
by
Volume Number
96 5
Lipid
throughout the cycle although incorporation was greater during the secretory phase (mean = 2,170 c.p.m. per milligram) than during the proliferative phase (mean = 1,268 c.p.m. per milligram) (P = 0.2) (Fig. 4). Incorporation of label into the nonestcrified lipids was greater during the proliferative phase (mean = 14,690 c.p.m. per milligram) than during the secretory phase (mean = 2,449 c.p.m. per milligram) (P = 0.01) (Fig. 5). On the basis of total experience, including specimens in which weights were not recorded, the percentage of total lipid extract not accounted for by phospholipids, triglycerides, and sterol esters ranged from 91 to 14 per cent. The mean of 20 normal endometria in the proliferative phase was 60 per cent and the mean of 11 endometria in the secretory phase was 37 per cent (P =
40 169 40 56I
TRIGLYCERIDES
biosynthesis
625
during the proliferative and secretory phases. During the proliferative phase incorporation into nonesterified lipids exceeded the ester classes and incorporation into phospholipids exceeded the other two classes of esters. During the secretory phase incorporation was greater into esters than nonesters, and incorporation into sterol esters exceeded the other two classes of esters. Fig. 7 illustrates the activity in each lipid fraction for various types of endometria. Five specimens of decidua from term pregnancy were studied. They showed greater incorporation of label into the phospholipid fraction than was seen in nonpregnant endometria and less incorporation into nonesterified lipids. The degree of activity and distribution within the three ester classes was more similar to proliferative than secretory specimen of decidua endometrium. One from a 6 week gestation showed very little incorporation of Cl4 (Table I). Three specimens of postmenopausal endometrium were studied. The activity was low in all fractions (Table I). Two specimens of endometrial cancc.r were studied. One was anaplastic adenocarcinema with a hi,gh degree of activity and 40 STEROL
I
1 1 1
2
4
6
Fig. 3. Incorporation normal endometrium.
I
/
IO DAY
I ii
12 OF
I
16 CYCLE
of C’4 into (From Table
I
18
1
20
1
22
triglycerides I.)
1 26I
24
by
2
4 6
Fig. 4. Incorporation normal endometrium.
ESTERS
8
IO DAY
0
I2
14
OF
CYCLE
of Cl’ (From
I6
into Table
18
20
sterol I.)
22
24
esters
26
by
626
Merrill
and
Werthessen
one was a very well-differentiated adenocarcinema or carcinoma in situ with low total activity in lipids. The pattern of distribution of activity within the ester classes resembled secretory endometrium more than it did proliferative (Table I ). Since our findings sugaested to us that endometrium is able to convert acetate to fatty of acids and to esterify a significant portion trithese fatty acids into phospholipids, glycerides, and sterol esters, it was considered worthwhile i and greatly time-saving) to see if the fractional distribution into each of thrse classes changes as a function of the menstrual cycle. Table II lists the distribution ratio of lipid classes studied for 37 normal endometria. ‘The C” titer in each fraction considered, expressed in counts per minute, was divided by the surn of the fractions to provide the relative distribution of label into each of the three classes. By this procedure, no note is taken of the C:‘l incorporated into a number of other compounds, for example, free fatty acids and sterols. A shift in distribution ratios of the Cl4 incorporated into these three classes \vas seen,
NON-ESTERIFIED
a
However, the phospholipid \.alurs contrib uted a major variation from specimen to specimen and no statistically significant (litfcrence occurred. When only the relative incorporations into the sterol ester and the triglyceride fractions were considered (Fic. 7 i a statistically valid differcnct. was tl(monstrated. with a shift of incorporation toward sterol esters at the tirnr of ovulation mjIIICYU~ proliferative 0.38: mean secrrtory 0.77. P 0.001). These observations arr difficult IO interpret as they are nnt rcblatttd to \\cigtlt or total lipid extract. They do suqgest that a chan,qr in thr pattern of cstr*rification ma\ occur at the time of ovulation. I~ktailecl analvses of these and related results \\ill io~lll the basis of another communication. Comment
The results arc preliminary and upon too few cases to provide anything
based more
225k
0
LIPIDS
PROL
SEC
50 0 5
0
25
1 ]
8 , 2
, 4
, 6
/ 8
/ IO DAY
, 12 OF
,
,
14 I6 CYCLE
,
,
0 0 ,
/
0 ,
18
20
22
24
26
Fig. 5. Incorporation of Cl” into the nonesterified lipids (that portion of the total lipid extract not accounted for by phospholipids, sterol esters, and triglycerides) by normal endometrium. (From Table I.)
NORMAL ENDOMETRIUM
Fig. 6. Mean values for incorporation of Cl4 into the lipid classe: by normal endometrium during the proliferative and secretory phases. TL = total lipid extract; NEL = nonesterified lipids, PL z-z phospholipids. SE = sterol esters. 7‘g.T = triglycrrides. (From Table I.)
Lipid
PROLIFERATIVE
Fig. 7. Mean Table I.)
SECRETORY
values
for
incorporation
DECIDUA
of C 1.1 into
than leads for future investigation, The fact that human endometrium is capable of incorporating labeled acetate into lipids agrees with studiesI”> ‘25,26 showing this tissue to be metabolically active. The analysis of the sterol esters, the molecular structure of triglycerides and phospholipids, plus the large amount of count found in eluates not specifically analyzed but known principally to contain fatty acids suggest that the major lipid biosynthesis observed in these experiments was of fatty acids which are rapidly esterfied to a significant degree. Apparently, although cholesterol is present in the endometrium, little is synthesized from acetate. Since other tissues synthesize cholesterol at a rapid rate, this finding needs further clarification. It is quite probable that endometrium is able to synthesize cholesterol but that under the conditions employed it does not use acetate as a preferred substrate.
lipid
biosynthesis
ADENOCARCINOMA
ATROPHIC
classes
by various
627
endometria.
(From
This situation has been described for the brain.l’ Increased incorporation of label into total lipids and phospholipids during the proliferative phase probably reflects estrogen influence upon enzyme systems responsible for increased energy and synthetic activities of the growing cell. Nucleoprotein concentration and activity increase in human endornetrium to reach a peak at the end of the proliferative phase and early secretory phase.” Estrogen has been shown to cause an increase in cholesterol, fatty acid, and phospholipid synthesis in animal uteri.‘, a, ‘3 ” Gorski and Nicolette’” have shown that estrogen enters the endometrical cell and exerts early influence on the RNA synthesis by the nucleus. RNA then influences lipid synthesis. Similar investigation has shown that several metabolic pathways can be effected by estr0gen.l”) “1 26
628
Merrill
Day i 1 1 1
3 5 6 6 7 7 8 8 9 11 11 11 12 13 13 13 14 15 15 15 15 16 16 17 17 18 19 21 21 22 22 ‘2 22 26
and
Werthessen
NEL/?'L 0.91 0.44 0.39 0.74 0.71 0.71 0.34 0.68 0.30 0.69 0.66 0.77 0.59 0.41 0.72 0.80 0.45 0.53 0.86 0.39 0.43 0.44 0.33 0.60 n.5i 051 0.29 0.14 0.28 0.25 0.22
/
PL 0.63 0.52 0.09 0.65 0.24 0.07 0.16 0.03 K? 1 0.40 0.30 0.44 0.51 0.03 0.02 0.08
0.75 0.27 0.29 11.26 0.04 0.42 0.10 0.37 (0.003) n.zn 0.19 0.2 1 n.01 0.37 0.24 0.03 0.58 0.36 0.46 0.02 0.01
SE
I
O.l(l I). I I (1.23 0.1 7 O.L’7 0.5.5 0.14 O.-hi) O.-ill n .3 I Il.34 0.19 (I.26 0.36 0.08 0.45 0.119 0. I3 0.26 0.43 0.32 0.25 0.40 0.28 0.98 0.7L’ 0.51 0.77 0.97 o.:ii 0.67 0.9ti 0 3 i’ 0.38 0.2 I 0.30 0.98
The relatively high rate of incorporation into phospholipids found in term decidua probably reflects an estrogen effect and is compatible with rapid tissue growth. The contrary is characteristic of postmenopausal endometrium. The importance of phospholipids in the process of cell growth is well linown.’ 3 The difference in lipid biosynthesis between the anaplastic adenocarcinoma and the carcinoma in situ reflects the degree of cell growth. It has been reported that adeno-
j
Tg.9 I).?7 11.38 0.68 0. 18 0.49 1).38 0.7ll 11.53 l).-lO Ii.29 11.36 lr.:ci CI.L’Y 1l.h 1 l1.!?0
(I.47 lI.li li.hl Il.-l5 I).31 fI.G-1 n.:4:4 o..io 0.35 0.02 l).llll fl.Yll l).ll? fi.llL’ Ii.25 ll.ll9 11.01 0.11~ 0.26 ( 1, :i :4 0.6X 0.01
/
SE
i
'T@
0.28 Il.‘L’ 0.23 0.4X I I. 145 0.59 (I.16 O.-l6 l1.5ll 0.511 0.48 0.33 0.53 lJ.37 lI.llU 0.49 0.3-i
0.58 0.33 o.+-1 (1.4~4 O.-l-k (I.98 fl.!ll) 0.7 I
carcinoma of the endometrium has a histochemical pattern similar to that of secretor) endometrium.“* ?a Our observed patterns of esterification correspond to such findings. Because previous studies have dcmonstrated increased stainable fat and concentration of neutral fat during the secretor\ phase, our findings of drcreasccl inrorporation of acetate into total lipids, including triglycerides, during the secretory phase suggest that stainable lipids result from incorporation of lipids from the plasma. rather
Lipid
biosynthesis
629
Summary
c
c, I
Average Prol I I I 2 4 6
=.38 8
IO DAY
’ , 12 OF
, , 14 I6 CYCLE
Average Set = 77 , , , 1 1 I8 20 22 24 26
Fig. 8. The ratio distribution of Cl& between sterol esters and the triglycerides expressed as ratio of sterol ester Cl4 count per minute to sum of the count in the sterol esters and glycerides. (From Table II.)
the the the tri-
than synthesis. However, since there may bc a shift in the degree and the nature of esterification of lipids following ovulation, it is possible that synthesis of specific fatty acids, not readily available from the plasma, may occur and even be an important prerequisite for successful implantation. Analysis and definition of specific fatty acid synthesis is required to clarify this issue. Lipids contained within a ccl1 are likely to have several functions. Some of them are essential building blocks of the vital structure. Some are metabolites that are consumed to pro\;ide energy. Others may be present as eneqgy stores. The lipid portion (90 per cent phospholipid) of the mitrochondria is as vital to their function as is the protein portion.Ii Our studies suggest variations in lipid biosynthesis as a function of the menstrual cycle. Thus, it is important to pursue studies designed to codify the normal pattern of lipid biosynthesis in human endometriurn and to determine if deviations from an established pattern are related to clinical abnormalities of reproduction, such as infertility, abortion, and other pregnancy losses, as well as to malignancy.
Previous investigators have demonstratrd that lipid biosynthesis by animal endometrium is subject to the control of hormones. Chemical analyses and histochemical studies are available, but there are no previous studies of actual lipid biosynthesis by human endometrium. Fragments of human endometrium, obtained by hysterectomy, were incubated for 4 hours in the patient’s serum to which acetate-l-C” was added. Chloroform-methanol was used to terminate biosynthesis and begin extraction of lipids. Serum lipids served as carriers for the tissue lipids through a separation procedure which segregated phospholipids, sterol esters, triglycerides and the remainder of the total lipids. The incorporation of C?’ into each class was determined. The preliminary data suggest that lipid biosynthesis by human endometrium varies with time during the menstrual cycle and is characterized by greater incorporation of label into total lipids, phospholipids, and triglycerides during the proliferative phase than during the secretory phase, with no significant change in the incorporation of Cl’ into sterol esters throughout the cycle. The nonesterified lipids demonstrate greater synthesis during the proliferative phase than during the secretory phase. The major lipid synthesis is of fatty acids with prompt esterification. The proportion of total lipids esterified is greater during the secretory phase than during the proliferative phase. Moreover, the pattern of esterification may shift at thr time of ovulation, with a relative increase in sterol esters. This change reflects less variability in the proportional incorporation of label into the sterol esters and the triglyccrides than in the incorporation into phospholipids.
REFERENCES
1.
Aizawa, Y., and Mueller, G. C.: J. Biol Chem. 236:381, 1961. 2. Aschheim. S.: Ztschr. Geburtsh u. GynHk. 77: 485, 1915.
3. 4. 5.
Black, J., Heyns, 0. S., and Gillman, J.: J. Clin. Endocrinol. 1: 547, 1941. Borell, U.: Acta endocrinol. 9: 141, 1952. Bottcher, C. J. F., Woodford, F. P., Van
6.
7.
8. 9. IO. 11. 12. 13. 14.
15. 16. 17.
Houte, E. B., and Van Gent, C. M.: Rec. d. trav. chim. d. Pays-Bas 78: 794. 1959. Cohen, S., Bitensky, L., Chayen, J., Cunningham, C. J., and Russell. J. K.: Lnncet 2: 56, 1964. Cridcr, E., Alaupovic, P., Hillsberry, J., Yen, C., and Bradford, R. H.: J. Lipid Res. 5: 479, 1964. Davis, J. S., and .4lden. R. H.: .4nat. Rec. 134: 725, 1959. Emmelot, T., and Bosch, I,.: Rec. d. trav. chim. d. Pays-Bas 73: 874. 1954. Froboese, C.: Vii-chow’s Arch. path, .4nat. u. Physiol. 250: 296. 1921. Gillman. J.: South African J. M. SC. 6: 59. 1941. Gorski, J., and Nicolctte, A.: Arch. Biochem. 103: 418, 1963. Goswami, A., Kar, A. B., and Chowdhury, S. R.: 1. Reurod. & Fertil. 6: 287. 1963. Green, “D. E.. and Fleischer, S.: Role of lipids in mitochondrial electron transfer and oxidative phosphorylation. in Frazer, A. C.. editor: Biochemical problems of lipids. New York. 1963, Elsevier Publishing Company, p. 325. Gross. S. J.: Ant. J, OUST. & C~YXEC. 88: 647, 1964. Hughes, E. C., Jacobs, R. D., and Rubulis, .4.: Anr. J. 0~s~. & GYN~X. 89: 59, 196-f. Kabara, J. J.: Brain cholesterol. The effect of its development on incorporation of acetate-2-H3 and glucose-U-C’ c% ill Himwich, W., and Himwich. H., editors: The Developing Brain. Progress in Brain Research, New York, 1964, Elsevier Publishin!: Company, vol. IX. p. 15.5.
18. 19.
2-l.
2.5.
26.
Leathem, J. H.: Ann. New York .4cad. SC 75: 463, 1959. McKay, D. G., Hertis. :\. T.. Bardxwil. M’. ii., and Velnrdo, J. T.: 0bst. & Gynec. 8: 22, 1956. McKay. D. G.. Hertig. :I. T.. Bardawil. M.. A.. and V’elardo. J, T.: Obst. iuc Gynec 8: I-&O, 1956. McClrnnan. C. E., and Koete, P.: W’esr. J. Surg. 61: 169. 1953. Noycs, R. W., Hertig. A. ‘I‘ , and Rock. J.: Fcrtil. c& Steril. 1: 3, 1950. P:igr. E. W., Glendening, M. B.. and Parkinson. I).: Aax. J. 0~s~. & GYNICL:. 62: llOt1. 1951. Pearse. A. G. E.: Histochcrnistry: Theoreti<..il and applied. ed. :‘. London. 1960. J, S: .4 Churchill. Ltd. Robertson. G. L., Ha~erman, D. D., Richardson. G. S., and Villee. (Z. 4.: Science 134: 1986, 1961. Saksenn, K. C., Arora, M. M.. Guptn. .J. (:.. :md Rangam. (:. M.: Indian J. M. St-, 19: 121. 1965. Stein, R. J., and Stuermrr, 1‘. M.: :Iht. ,J. OUST. 8.c GYXEC. 61: -11-1. 1951. Sturrrner, \‘. M., and Stein. R. J.: Ax. J. OUST. & GYSEC. 63: 359, 19.5”. v,anDykr. H. B., and Chrn. (;.: .2m. J, Anrt. 66: 411, 19.40. Werthessen, N. T.: CirrulJtion Rrs. 6: 759. 1958. Westphalen. I‘.: Arch. Gyniik. 52: 35. 189ti.
Discussion DK. Russ~1.1. R. IIF. AI.VAREZ, Philadelphia, Pennsylvania. This important paper of Drs. Mcrrill and Werthcsscn represents an approach to the study of lipid metabolism in the female through the incorporation of labeled acetate into three principal esterilied lipids. They report their resultant data in counts per minute, while oui serum data have been reported quantitatively in micromoles per milliliter and in weight per cent so that comparisons between our respective results are not presently possible. WC wonder, however, whether it is appropriate to incubate tissue in the patients’ sera and then attribute all lipid alteration as being characteristic of the tissue studied. The significant finding of their work is the demonstration of the ability of the endometrium not only to incorporate acetate-l-Cl4 but also to convert this fatty acid (in miniature) to
higher fatty acids and then to esterify theni into phospholipids and cholesterol esters. In our own evaluation of each lipid fraction in growth, COIIsistent alterations in the major lipid classes occur whether the growth be of trophoblastic or nl’crplastic origin. Increases in total lipid, triglycerides, total ester, and free cholesterol, free fatty acids, total phospholipids, and of each phosphatide subfraction tend to occur both in pregnanny and in gynecologic mali,~nancy. Wht seems to he most signifirant, however, is not so much the variation or even the direction of the major lipid fractions themselv~es, but the relationship of the fatty acid composition of each group of esters in the normal nonpregnant, the normally pregnant patient and in the patient with gynecologic cancer of various primary sites. The authors found similarities of radioisotope uptake in cancerous endometrium and in normal
Volrlme Number
96 5
preovulatory endometrium. The greater incorporation of labeled acetate into phospholipid fractions of decidua complements the observations noted by us in normal pregnancy. In our patients with endometrial carcinoma and in those with normal pregnancy, the serum lipid patterns of each group behave similarly. Both groups of patients demonstrate a general reduction of unsaturated fatty acids, particularly linoleic arid in most lipid groups but usually associated with an elevation of palmitic acid, especially in the phosphatidyl esters. WI, believe some of the alterations in lipid synthesis of active endometrium (whether it be cyclic proliferation and growth, pregnancy, or cancer) must he endocrinologic and, thus, constitute a reflection of the energy requirements necessary to initiate a metabolic mechanism for stress and growth. It seems reasonable to cogitate that the frequent combination of endometrial carcinoma, obesity, and diabetes may well reprcsent the endpoint of abnormal hyperlipemic states occurring prior to the appearance of clinical diahetes and even indicate the likelihood that latent diabetes is latent only as regards carhohydrate metabolism, but how can it be so limited? Therefore, we would raise for consideration the thesis that clinical or laboratory evidence of hyperlipemia, or of diahctes, or of a family history of these states constitutes sufficient justification to study prospectively the potential lipid alterations, continued and repeated investigations in search of evidence of carbohydrate intolerance and an awareness of a lipogenic-cancer rrlationship. Drs. Merrill and Werthcssen have convinced us of the nerd to expand further our invcstigations of lipid metabolism not only in serum and in obviously pregnant and malignant tissue, but also in cyrlic and decidual endometrium and in normal gynrcologic structures capablr of the development of potential malignanry. LVith data from both our departments expressed in comparative and quantitatablc terms, \~e look forward to the devclopmrnt of a better understanding of the mechanisms responsible for the changes noted, not only in normal uteroovarian physiology, but in cancer as well. DR. NICHOLAS T. WERTHESSEN,” Oklahoma City, Oklahoma. An instrument is now being developed which in the near future will permit a tremendous reduction of the staff necessary to *By
invitation.
Lipid
biosynthesis
631
do this type of study, and our hope is that it will be possible to show changes in enzymatic structure within the cell with greater ease than is possible now. DR. J. DONALD WOODRUFF, Baltimore, Maryland. I gather from your presentation, Dr. Mrrrill, that estrogenic activity in the endometrium was correlated directly with lipid metabolism CYccpt for sterol esters, and that activity in adenocarcinoma, in turn, was correlated with that of the estrogenically stimulated endometrium. Tllis we have felt in the past to bc true, although there have bren dissenters to this thesis. I would like to know if, in reality this relationship cxistq, since I get a different point of view from our conclusions, nameI)that the patterns in adenocarcinoma simulate those found in the secretory cndomctrium rather than those of the proliferative phase. DR. SAUL 8. GUSBERG, New York, New York. I would like to make a comment related to that of Dr. Woodruff. As I looked at this material of Dr. Merrill, so beautifully investigated, it seemcsd that the lipid biosynthesis did not necessarily correlate with steroid function because the anaplastic adenocarcinoma-the high growth-phase carcinoma-showed more lipid synthesis than did the differentiated one which might be expectcxd to be more hormone-dependent. Thus it \~oultl, in this instance, appear related to growth phrnomena rather than hormonal ones. DR. MERRILL (Closing). So far as the qursticjn of adenocarcinoma is concerned and its lipid synthesis, there was a distinct difference in the rate of biosynthesis observed in the well-diff(*rentiated tumor as compared to the anaplastic. The latter showed a higher rate of incorpol-ation of label, hut the pattern of cstrrifiration of lipids was the same in both. The pattern LV:IS one of greater incorporation of the label into stcrol esters than into the other esters. This KLS similar to the pattern of esterification found in normal secretory endometrium. Although the degree of activity, i.e., the total amount of lipid synthesis, was much greater for anaplastic carcinoma than for any of the normal nonpregnant endometriumspand thus resembled proliferati1.c rndometrium-the pattern of estcrifiration W;IS similar to secrctor)l endomctrium. This rorrcspends to th
632
Merrill
and
Werthessen
stimulation. One might wish to say that this is evidence that adenocarcinoma is the end stage of a progesterone stimulation situation. All I can say is that this is the pattern we found. A question was asked about abnormal lipid and glucose metabolism. I am sure that this would reflect itself in an abnormal pattern of synthesis of incubated tissues. This has been demonstrated in animals. The activity of incubated tissues can be altered if the animal has an abnormal nutritional or metabolic situation.