68 NVTRrnIOS RE\’IEWS [Vol. 28. No. 3

cated as being inadequate should be bene- the calorie gap may be aasteful or inefiec- ficial but the provision of protein concen- tive in preventing malnutrition in pre- trates or amino acids without correcting school children.

PROPERTIES OF MITOCHONDRIA FROM MAMMALIAN CELLS CULTURED ON LIPID-FREE MEDIA

In spite of the k k of linolale and omchidonote in mitochondrinl lipids. mitochondria /mm moue fibrobhts f L refisl pawn for seceml m a chemically defied. lipid-frw media did not diflrr in several properties from mitochmdria from eel& gnnm on m d i a supplemented with u m m or holeate. 7 % ~ eficimq o/ cuidative phatphq\btion urn rrduced, houewr--on obnenution in aRrrrmpnt uith results for mitmhmdria obtained from mtn deficient in ensentiof fafty acids.

In spite of extensive studies on the me- tabolism of essentia1 fatty acids (SFA) and the effeas of essential fatty acid deti- ciency in various species. little is known of their exact function except 8s precursors of the prostaglandins (Nutrition Reuiews 27, 67 (19t i9) ) . Evidence for altered func- tion has been obtained with mitochondria from rats with an essential fatty acid defi- ciency, such as decreased ADP/O ratios and respiratory control (R. M. Johnson, J. Nutrition 81,411 ( I = ) ) . Changes such ati these have been the main basis for (UT-

surning that the decrease in EFA concen- tration in membrane lipids results in mi- tochondria) damage, since tight respiratory control is aseociated with an undamaged mitochondria1 membrane.

ADP/O ratio is the increase in oxygen uptake caused by the addition of ADP and is a measure of phosphorylation efficiency. Respiratory control is the ratio of oxygen consumption in the presence of added ADP to the oxygen consumption after ex- penditure of ADP. Intact, undamaged mi- tochondria are “tightly coupled,” i.e., they have high respiratory control indices, whereas a low respiratory control index is considered evidence of mitochondria1 dam- age.

It is well established that mitochondria from animals with a deficiency of essential fatty acids are less stable upon isolation, perhaps as a result of increased phospho-

lip- A activity. which could result in more rapid hydrolysis of membrane phoc pholipids (J. A. Smith and H. F. DeLuca. J. Celf. Biof. 21, 15 (IS%): M. Waite and L. M. Van Golde, Lipi& 3, 449 ( 1 S 8 ) ) . Recent work has shown, however, that when isolated in hypertonic sucmee or mannitol containing EDTA to chelate heavy metal ions, mitochondria from defi- cient rats give nonnal ADP/O and respin- tory control velum (T. It0 and Johnson. J. Biol. Chem. 239. 3201 ( 1 9 % ) ) . biation in isotonic sucroee, however, results in greater mitpchondrial damage (Smith and DeLuca. loc. cit.).

In view of the complex nature of EFA deflciency in animals, the report that l i m leate stimulated growth of hamster d b in culture (R. C. Ham. Science 140, 802 ( 1 S 3 ) ) raised the possibility that cultured mammalian cells might be a simplified test system in which to obtain mitochondria depleted of EFA. after culture of the cells in lipid-deficient media. Subsequently, L. E. Gemhenson. J. F. Mead, 1- Hamy. and D. F. Haggefly. Jr. (Biochirn. Bioph)~. Acta 131, 42 (1937) ) investigated the effect of linoleate and arachidonate on growth. lipid composition. and respiratiaa in cultured HeLa cells. HeLa cells are an established cultured strain of epithelial cells derived originally from a carcinoma of a human cenrix.

In the absence of lipid in the medium.

March 19701 NLTRITION REVIEWS 69

the cells failed to grow, and addition of serum albumin caused no improvement. Supplementation of the basal medium with fetal calf serum produced the great- est gmwth in the seven day test period. However, supplementation with linoleic acid had 39 per cent of the effect of serum. and arachidonate, at an even lower con- centration than linoleate. was 56 per cent as effective as serum. ‘

In the cells cultured on lipid-free me- dium, the proportions of oleate and pal- mitoleate increased. Those of linoleate and arachidonate decreased to almost unde- tectable levels, but without an increm in the pmpo~ion of eicoeatrienoate, which does increase in animals fed a diet lacking EFA. Supplementation with linoleate in- creased the proportion of linoleate and de- creaned that of oleate, but caused no in- crease in amchidonate. Supplementation with amchidonate inmaned the proportion of arachidonate. No figurea were given for fatty acids of chain length longer than arachidonate. With succinate, alpha-ketoglutarate, or

beta-hydmybutyrate an the oxidizable atbetrate, ADP/O ratio and respiratory control were reduced in “deficient” cells cultured on the lipid-free medium contain- ing albumin, in comparison with the cells supplemented with calf serum. The cells cultured with linoleate or arachidonate Bowed values more like that of the cells grown with calf serum. Th e absence of arachidonate in the cells grown on linole- ate results from the fact that the subline of HeLa cells useq’by these workers ( H e h Sd cannotcionvert linoleate to ar- achidonate (Haggerty. Cerschemn. Ha- rary. and Mead, Biochem. Biophys. Res. Commun. 21. 568 ( 1 s ) ) . This is most probably also the reamn for the absence of an increase in eicasatrienoate in the cells cultured in the lipid-free medium. However, the fact that linoleate. without being converted to arachidonate. stimu- lated growth indicated that linoleate and

arachidonate may be functionally inter- changeable in these HeLa cells.

The effects of fatty acid supplementa- tion in beating rat heart cells were also studied (Cerschenson. Haraw, and Mead, Biochim. Biophys. Acta 131, 50 (1967)). Previous work b d shown that serum lipids were necessary to maintain-beating (Nutrition Reoiews 24, 180 (19fX); Har- ary, R. McCarl. and B. Farley. Biochim. Biophys. Acta 115, 15 (1966)) . Cells incu- bated in the basal medium without fetal calf serum stopped beating within two days. Addition of linoleate or arachidonate did not maintain beating. but beating was maintained, although at a slower rate, by a combination of linoleate and palmitate or by palmitate alone. With alpha-kete glutarate as the oxidizable substrate, ADP/O ratio and respiratory control were reduced in cells flown on the lipid-free medium plus albumin. These values were normal in the cells mpplemented with linoleate or arachidonate, but not with palmitate. Linoleate and arachidonate levels were high in the cells cultured with linoleate, since linoleate can be converted to arachidonate by rat heart c e b which are still capable of beating (Haggerty et al., loc. cit.)

One might ask whether the inability of the HeLa cells to convert linoleate to ara- chidonate was an adaptation to many gen- erations of cell culture or whether it was characteristic of a cancerous cell. since the beating heart cells could still convert l ins leate to arachidonate. That the lack of abilit)’ to form arachidonate could have arisen from dedifferentiation in culture was demonstrated by the fact that rat heart cells which have dedifierentiated lose the ability to form arachidonate from linoleate (Haran.. et al., Lipid Metabolism in Tissue Culture Cells. G. H. Rothbht m d a Krirchersky. Editors, p . 25. W s - tar Institute Symposium Monograph No. 6. J96i).

However. \V. StoKel and A. Scheid

[Vol. 28. No. 3 70 SCTRTTIOS RK3'IEWS

( H . - S . Z. f . physiol Chem. 338,205 (1%') subsequently reported that HeLa cells. under their conditions, converted linoleate to arachidonate. PoFsible reasom for the difference between the results of Haggerty and co-workers and those of Stotfel and Scheid are either the subline of HeLe cells used or the culture conditions when con- version of linoleate to arachidonate was studied. In the experiments of Haggerty and co-workers. the cells were grown on glass Petri dishes in a serum-free medium in which "C-linoleate was the only fatty acid added. The duration of growth was not given. but it was at least two days. since they stated that "the medium was changed every two da-w." In the work of Stotfel and Schpid. the cells were grown for 12 houm in roller culture in a medium containing 10 per cent calf serum, then "C-linoleate added, and the incubation continued for 20 hours before the cells were harvested. More recently. studies have been made

of the composition and function of mito- chondria isolated from mouse fibroblasts cultured on a lipid-free medium (Y. Ka- gawa. T. Takaoka. and H. Katsuta. J. Biochem. 65, 799 ( 1 5 6 9 1 ) . 'Ihe cell line used, LP.,. is a subline of the fibroblast line L929. which originated from C,H mouse fibroblasts. This subline had been mown for several veam in their laboraton. on a chemically defined, lipid-free media (DM-120) which contained 19 amino acids. all of the B vitamins required by the mouse and rat, plus ascorbic acid, and minerals. Fat-soluble vitamins were not added. The "macro" minerals were pro- vided as known salts. Values for iron. cop- per. and manganese were aLw given. but the form in which these were provided was not stated. A value for zinc was not stated. Antibiotics were not added.

Approximately 10" cells were incubated at 37" C. in 10 ml. medium in stational?; culture. with the medium renewed twice

weekly. Under these conditions. the cells showed a tenfold increase in seven da>s.

When the composition of the cells grown in DXI-lXI was compared with that of cells grown in a combination of 90 per cent DM-120 and 10 per cent calf serum by vol- ume, the Dh.1-120 cells contained more lipid esters (microequivalents per milli- gram protein). but the lipid phosphate and cholesterol contents were similar to the celis from the medium containing serum. The relative proportion of saturated fatty acids ranged from 35 to ,38 per cent. the proportion of monounsaturated fatty acids from 59 to 65 per cent, while tho proportion of polyunsaturated fatty' acidti was less than 1 per cent. The mrrenponding valuen for the cells grown on the medium con- toining wrum were 42 to 57 per cent for saturated fatty acids, 23 to 5:l per cent for monounsaturated. and 6 to 14 per cent for polyuneaturnted fatty acida. No explana- t ion was given for the I elat ively wide varia- tion in the proportion of monounsaturated fatty acids in the cells supplemented with serum. The calf serum unnaturated fatty acids were 29 per cent C 18:2, octadeca- dienoic acid (presumably linoleate). 2.2 per cent C '20: 3 eicoaetrienoic acid and 2.4 per cent C 20: 4 e imte t r aeno ic acid (pre- sumably arachidonate).

Celb were aLw grown in DM-120 with radinactive inorganic phosphate ("Pi) added to label the phospholipids. No dif- ferences were observed in the distribution of "P in phospholipid classes in mitochon- dria from the cells grown on DM-1% or on DM-1'20 plus linoleic acid.

As expected, the addition of linoleic acid produced large changes in fatty acid com- position of the phaspholipids. In the ceph- alin fraction. the proportion of monounsat- urated fatty acids decreased from 58 to 25 per cent, and the proportion of linoleate increawd to 3 i per cent. In the lecithin fraction. the proportion of monounsatu- n t e d fatty acid ( C 18 : l ) decreased from

March 19701 SLTRITIOS REVIEWS 51

46 to 24 per cent. and linoleate increased from 0.9 per cent to 24 per cent. No values were given for arachidonate. Presumably, these cells are unable to convert linoleate to arachidonate (R. P. Ceyer. A. Bennett. and A. Rohr. J. Lipid Rex 3, 80 (19621). Almost no polyunseturated fatty acids were detected in the cells grown on DM- 120.

The DM-120 medium lacked inositol, although it had been reported that most cell lines in culture require inositol (L. Levintow and H. Eagle. Ann. Reu. Bio- chcm. 30, 608 ( 1 9 6 1 ) ) . Alkaline hydrolysis of the lipids, together with paper and col- umn chromatography of the hydrolysis pmducts. indicated the presence of inmi- to1 in !he phmpholipids. T h e synthesis of Cog from methyl."C-methionine was a h demonstrated.

Mitochondrial fatty acids from the cells grown in the lipid-free medium aLw nhowed almorrt no polyunsaturated fatty acids. A small amount of C 2O:O was de- tected, but unsaturated fatty acids con- taining more than 2p carbon atoms were not identificd. C 18: 1 was the major fatty acid in the beta position (about 60 per cent) of the glycerides in both total cell lipids and mitochondrial total lipids. Anal-' .wis of the lecithin and cephalin fractions of the mitochondria showed approximately 56 per cent C 18:l in the lecithin fraction and 75 per cent in the cephalin fraction. The values for C 16:l were 15 per cent and 8 per cent. respectively. in the leci- thin and cephalin fractions. i'accenic acid (the ? m ~ - i s o m e r of oleic acid) was approx- imately per cent of the C 18: 1 acid.. in the total cell lipid and 24 per cent in the cells p u n on DM-120 with 5 per cent calf serum added.

Addition of linoleate reduced incorpora- tion of "C-acetate into "C-fatty acids. as well as decreasing the amount of "C-mono- unsaturated fatty acids and conversion of

ues were given. the authofi commented that they detected also small amounts of di- and tri-unsaturated fatty acids labeled with "C. The q m h r o m e spectrum of the cells from the lipid-free medium was simi- lar to that of the cells flown with linole- ate.

ATPase activity, in the presence or absence of oligomycin. was unaltered in whole ceUs grown on the lipid-free me- dium or in mitochondria isolated from these cells. Olipmycin inhibits oxidative phosphorylation as well as ATPase activ- ity in mitochondria or submitochondrial particles. This mitochondrial ATPase ac- tivity is required for oxidative phosphory lation. Oligomycin sensitivity of ATPase activity, in turn. depends upon another mitochondrial fraction requiring phospho- lipid for its activity.

T h e requirement for phospholipid has been shown by the fact that activity was abolished by treatment with phoepholi- pase A, which removes fatty acids from the beta position of phoepholipids. Since es- sential fatty acids occur chiefly in the beta position, this mna evidence that EFA were necessary for activity (Kagawa and E. Racker. J. B i d . Chem. 211, 2461

However. respiratory contml and ADP/O ratio. with succinate as substrate. were lower in mitochondria from cells grown on the lipid-free medium (DM-120) al- though oligomycin inhibition of endog- enous respiration of whole cells was not altered. It was stated that omission of lipid from the medium did not aHect oxidation of NADH by mitochondria1 fragments. The authors also stated that no apparent ditferences in mitochondrial morpholop were indicated by phaw contrast micras- copy of the cells grown on the lipid-free medium. It is unfortunate that no electron microxope evidence was presented. espe- cially since the reduced ADP/O and respir-

(rw)),

C-stearate to "C-oleate. Although no V a l - a t o p contml may reflect damage upon I 8

72 SlTRmOS RESlEWS [Vol. 28. No. 3

isolation. Comparison of isolated mitochon- dria with those in siru would give some answer to these quest ions.

From these experiments. it is clear that vnrious t y m of cultured mammalian cells may differ widely in their requirement for unmturated fatty acids since HeLa celLs mav require linoleate or arachidonate. and certain lines of L cells do not. However, Kagawa and co-workers indicated that they were able to cultivate a subline of HeLa cells on a protein-free. lipid-free medium containing inmitd and, even in the L cells, the reduced ADP/O ratio and respiratory control in the mitghondria from the cells grown on the lipid-free me- dium are evidence for mitochondria1 alter- ation. either in nitu or from wlation.

Admittedly, cultured cells may repre- sent dedifierentiated qstems removed from the condition of cells in intact ani- mals, and the metabolic properties of a given cell type may have been altered as a result of the conditions of culture used for propagation of the cells. Nevertheless. they present a system for obtaining mam- malian cells devoid of EFA or high in EFA. depending upon the p w t h medium, a condition which cannot be obtained in the intact animal. Therefore, the use of these cells presents a potentially valuable s p tem for studying how functions in animal cells may be altered by wide changes in the fatty acid cornpodtion of variouo lipidn which appear to be required for thew function8 in the intact animal.

RENAL DEFECT IN MAGNESIUM DEFICIENCY

Magnesium i R known to be intimately related to calcium and phosphorus metab ol im. and is a h required aa a cofactor for a great many enzymes. including thase concerned with oxidative phmphorylation bee Nutrition Reviwn 26, 65 (1958) ) . Magnesium deficient animals are known to develop metastatic calcification, and administration of magnesium in the diet has been shown to enhance urinary cal- cium excretion and to reduce formation of calcium oxalate stones in rats deficient in vitamin B, (Nutrition Recierrs 25, Xlf ( 1 9 3 ) . Guinea pigs are even more sus- ceptible than rats to deposition of calcium and phosphoms in the wft tissues. dental abnormalities, and powth inhibition with magnesium deprivation.

D. L. >lever and R. A¶. Forbes (Roc. SOC. G p . Rial. M e d . 128, 157 ( 1 s ) ) were unable to demonstrate that mineral ac- cumulation in the kidnehs. like the usual h>percalcemia of magnesium deficienq.

was absent in parathymidectomized rats even when magnesium deficient. Other recent evidence has led to the suggtstion that ex- magnesium m y actually in- duce a h w r p q a t h p i d state in the rat (H. J. CitelmaA. S. Kujoli, and L. C. Welt, Am. J . Phyniol. 215, 483 (JW)). E. R. Morris and €3. L. O’Dell (Roc.

SOC. Exp. Biol. Med. 13% 105 (1969)) pur- sued an earlier observation by O’Dell, Morris and W. 0. Regan ( J . Nurrition 70, IW (1960)) in guinea pigs made deficient in magnesium. Although the level of blood phosphorus became elevated in these animals, who are unusually ceptible to magnesium deprivation. blood calcium remained normal. This suggested a renal disability rather than h-yperpara- th>-roidism, and renal “impairment” has been previously obsewed in rats or cats on diets low in magnsiurn. Thus, these authors explored the changes in renal function of the guinea pig with magne-