Mitochondrial function and free fatty acid levels in rats with portacaval shunt

Mitochondrial Function and Free Fatty Acid Levels

Rats after Portacaval Shunt

4 Augu I 197Y

s

:'1 l: [) I l .~ L JU L R~ ~ I

.

In

I I

A. LOCHNER,

A.

J.

BE ADE,

J.

C.

.

KOTZE,

J.

E. RO SO

P. J.

KRI K.

D. L B D RIO,

SUMMARY

The effects of portacaval shunting on the oxidative phos-phorylation process of mitochondria isolated from rat liver and skeletal muscle were evaluated and correlated with mitochondrial free fatty acid (FFA) contents. ADP/O ratios, respiratory control index and Q02 values were significantly depressed in liver mitochondria from porta-caval-shunted rats; these changes were associated with decreased mitochondrial FFA contents. The mitochondrial function of skeletal muscle was unaltered.

5. A/r. med. J., 56, I I (1979).

Pona aval shuming has been reponed IQ ha e certain beneficial effects on two inherited disease, viz. glycogen sIQrage disease and familial hypercholesterolaemia."· However, the exact mechanism whereby this procedure achieves these effects has not yet been established. Recent work on animals has shown that the metabolic con-sequences of portal diversion are more complex as well as more profound than has been realized.""

Relatively little is known about ubcellular change occurring in liver tissue after portacaval shunting. In rats, the significant loss of liver glycogen content ha been shown to be related to the decreased food intake, whereas hepatic atrophy after portacaval shunting appear to be directly related to the shunting of portal blood away from the liver'" Thi phenomenon could be due

LO the lowered insulin supply, whi h ha been hO\ n to

be a major ponal factor responsible for maintaining cell size and number.'o ynthesis uf cholesterol and of total fatty acids was found to be imilar in portacaval-shunted and control rats.'

Ischaemia of the liver. produced by clamping either the portal venous blood flow or the hepatic artery blood upply, resulted in a significam depression of mitochondrial function."'" which was attributed to increased mito hon-drial free falty a id (FFA) content." ince shunting of the

Department of Medical Biochemistry and Metabolic UnH. Departm.ent of Medicine. University of S((~lIenbosch. and National Research Institute for Tutritional Diseases of the South African 1\1edical Research Council, Parowvallei. CP :\, LOCII~ER. D.Se.

:\. J.

S. BE:\fADE. D.se.

/. C.

N. KOTZE.B. C. HO:-;S

\1.

P.

J.

l.'RIEK,B.\·.SC .. :-1.:-rED \'ET (PATH.) I). LABADARIOS. PH.D

./. E. ROSSOUW, ~I.D,

DJle received: 12 December 1978.

Reprint requests to: Or A. Lochner, Dept 01 Medical BlochemlSl!Y.

niversily of Slellenbosch Medical School. PO Bo, 63, Tygerberg, 7)05

RSA.

portal blood away from the liver could possibly induce chronic i chaemic condition \ ithin the cells, this tudy wa undertaken to evaluale the effect of portacaval shunting on the mitochondrial oxidative pho phorylation proces a well a on the mitochondrial FFA content of liver and muscle. The role of dietary intake in portacaval-shunted rats was also evaluated by studying mitochondrial function in appropriate pair-fed and ad libilllm-fed con-trol animals.

MATERIALS AND METHODS

Animals

ale BD 9 ralS (230 - 2 0 g) were used. The animals

were housed individually in metaboli cage fitted with wide wire-mesh bottoms. The animals were allowed to adapt for a period of I week before experimentation. A reversed light-darkness cycle was adopted and the rats were sacrificed in the mid-dark pha e.

Four eries of rats were tudied: (i) portacaval-shunted rat (PCS); (ii) ham-operated rats, pair-fed to PC rat (S); (iii) normal control rats, pair-fed to PC rats (PFC); and (iv) normal control rat, fed ad libilUm ( LC).

The porta aval shum operation was performed a de cri bed by Lee el al." The sham operation was per-formed under imilar conditions and the ham-operated controls were individually mat hed in term of i chaemi time and weighl to PC animals. The mean ischaemi time wa 13,1 I 0.3 min.

All animals were observed for 42 day from the da of operalion, fed once per day, and the food intake and body weight recorded. On the 42nd day, 24 hour after their last feed. the rat were sacrificed.

Mitochondrial Studies

itochondria ere prepared from liver and skeletal muscle (quadri ep) li ue for the tudy of oxidative phosphorylation. Two mitochondrial isolation media were used: (t) sucrose (O,25M). Iris-HC! (I m 1), EDTA (I

mM) (pH 7,4) was u ed for liver lissue; (ii) KCI (O,18M), EDTA (10 mM) (pH adjusted to 7,4 with tri~ base) wa u ed for muscle tis ue. All available liver ti sue and the quadriceps mu cle from both leg were placed direclly into ice-cold i olation medium and minced finely with sci sor . After wa hing the tis ue 3·4 time 10 remove

all traces of blood, fre h i olation medium was added before homogenization (>12 volumes per g ti sue). The liver was homogenized at maximum peed with a Polytron PT 10 homogenizer (I 3 seconds), whereas the muscle tissue was homogenized for longer period (4 X 5

I 2

MEDIE E TYD KRIF 4 Augu tu 1979

Morphometry

• Results expressed as mean ± SEM.

Pvalues indicate significance of difference from ALe. Numbers in parentheses indicate number of animals.

Five blo ks from each animal were taken at random and electron micrographs taken at an initial magnification of 6 40 and printed at a magnification of 17100. The urface area of the mitochondrial outer membrane a well a the urfa e area of the mitochondrial cri tal' wa deter-mined by the u e of quare latti es.'"

72,63 - 2,46 72,92 -+- 4,64 NS 87,10 - 2,45 <0,005 48,18 ± 6,92 <0,01 71,49 - 8,63 93,24 ±13,47 77,96 -+- 6,75 141,06 -+- 3,90 147,13 ±13,04 NS 178,32 ± 5,44 <0,001 114,78 -+-10,87 <0,05 6,29 ± 0,41 5,89 ± 0,54 NS 5,25 -+- 0,36 NS 3,45 -+- 0,62 <0,005 6,53 ± 0,40 7,05 ± 0,52 NS 5,93 -+- 0,45 NS 3,99 ± 0,61 <0,Q1 2,79 -+- 0,08 2,42 -+- 0,08 <0,01 2,41 -+- 0,06 <0,005 2,07 -+- 0,19 <0,005 3,41 - 0,13 9,65 ± 1,31 3,31 - 0,07 10,68 ± 1,63 3,36 - 0.15 8,70 0,99 1,87 - 0,06 1,62 - 0,06 <0,02 1,68 - 0,05 <0,05 1,43 -+- 0,11 <0,005 ALC (6) PFC (8) P S (8) P PCS (9) P Skeletal muscle Substrate: qlutamate PFC (6) S (8) PCS (9)

RESULTS

Mitochondrial Oxidative Phosphorylation (Table

f)

Liver: Mitochondria, isolated from tbe livers of ALe rats, using sucrose-tris-EDTA as isolation medium, with glutamate and succinate as substrates, yielded ADP/0

ratios. RCI and Qo, value similar to tho e reponed by other workers."'"

Mitochondria isolated from Jivers of PCS animals exhibited significantly depressed ADP/0 ratios as well as RCI and Qo, values, compared with the other 3 groups. This ignificant depression was evident with both glutamate and succinate a substrate. Compared with ALe mitochondria, the percentage depression in ADP/O, RCI and Qo! of PCS mitochondria averaged 26°0, 45°(, and 34"{, respectively, with glutamate as ubstrate. Figs 1

and 2 show oxygraph tracings obtained from liver mito-chondria of PFC and PCS rats. which clearly illustrate the marked differences observed in the parameters of mitochondrial function.

TABLE I. LIVER AND SKELETAL MUSCLE MITOCHONDRIAL

OXIDATlVE PHOSPHORYLATION OF CONTROL AND

PORTACAVAL-SHUNTED RATS'

Liver ADP/O RCI Q02

Substrate: 9'utamate ALC (6) PFC(8) P S (8) P PCS (9) P Substrate: succinate ecolld ) or until no whole panicle of tissue \ ere een.

Liver mitochondria \ ere prepared a described by ordahl

eEal."The final pellet wa upended in ucrose-tri -EDTA

medium at a concentration of 30 - 40 mg mitochondrial protein per ml. keletal muscle mitochondria were iso-lated according to the method of 10le er al.'· and finally upended in K I-EDTA. yielding a su pension medium containing 10 - 1- mg mito hondrial protein per ml.

The oxidative phosphorylation proces of liver and mu cle milOchondria was tudied polarographically a described by ordahl er al., j The incub:.Ilion medium for liver mitochondria contained KCI ( - m ), tris-HCI (50 mM), K,HPO. (12.5 mM), MgCL (5 mM) and EDTA

(I mM). Glutamate (5 mM, tris salt, pH 7,4) and succinate (5 mM, tris salt, pH 7,4) were used as substrates. The incubation medium for mu cle mitochondria consisted of sucrose (O.25M), tri -HCI (10 mM, pH 7,4) and K,HPO. ( ,- mM). u ing glutamate (5 mM) as substrate. To produce state 3 respiration. a O,I-ml aliquot of DP (containing 400 - 450 nmol ADP) was added. The exact amount of ADP added (which i equal to the amount of ATP formed) wa determined spectrophotometrically. u ing a millimolar extin tion coefficient of 15,4." The incubation temperature for these studies was 25°C. Mitochondrial protein oment was mea ured by the method of Lowry et al."

The following indice of mitochondrial function were measured: ADP/O ratios (nmol ATP produced per nalOm oxygen onsumed); mitochondrial oxygen uptake

(00,) (state 3: natoms oxygen uptake in the presence

of ADP mg protein min; state 4: natom oxyp.en uptake after pho phorylation of ADP mg protein/min); respi· ratory control index (ReI) (ratio of oxygen onsllmed in the presence of ADP to that after phosphorylation of ADP).

FFA Determination

On the 42nd day blood was drawn under ether anaes-the ia from anaes-the abdominal aorta in all rats and collected in EDTA. FFA were extracted from plasma and mito-chondria according to the method of Dole and Meinertz" and analysed by a Beckman model GC4 gas chromato· graph as des ribed.'· The total FFA content of each ample wa al ulated a the um of the individual FFA mea ured.

Preparation of Specimens for Electron Microscopy

Block obtained from the liver immediately after the rats had been killed were diced in precooled (4° ) 3°" glutaraldehyde in 0.1 M phosphate buffer (pH 7,2) into I-mm' blocks. These were fixed in 3Un gllllaraldehyde for

24 hour and po tfixed in I().n 0 O. in Millonig's buffer (pH 7.3)'· at .f°C for 2 hours. The block were then de-hydrated in a graded series of ethanol solutions and in propylene oxide, embedded in Epon 812. sectioned at 100 nm. and stained \ i[h uranyl acetate" and lead citrate." The ec[ions were examined with a Philip EM 300 electron micro cope at 60 k .

4

Augu t

1979

SA

MEDICAL JOURNAL _Iln

ADP/O:I.12

RCI :2.94

Q02: 106.8 nA O/mg prot/min. PAIR-FED COYfROL RAT

Sub'>lrate : Glutamate

The total FFA content of mitochondria i olated from PFC, Sand PCS rat liver was significantly lower than that of ALC rat livers (P<O,OI, P<O,OO I and P 0,005

respectively). Analysis of the individual FFA fractions showed a tendency of all FFA (except C14: I) to be decrea ed in mitochondria from PFC, S and PC rat livers. ]n these 3 groups the mitochondrial Cl 6:0, 18:0 and C20:4 content were significantly lowered compared with those of LC rat.

Liver and Skeletal Muscle Mitochondrial FFA

Contents (Table

IT)

AOP/O: 2.74 AOP/O: 1.83

RCI : 6.43 RCI : 7.63

Q02:63.6 nA O/mg prot/min. Q02 :1~5.snA O/mg prot/min. Fig. 1. Oxygraph tracings of liver mitochondria from PFC rats. (Substrates: g:utamate (5 mM); succinate (5 mM); amount of ADP added: 438 nmol.)

PORTACAVAL SH NTED RATS

Fig. 2. Oxygraph tracings of liver mitochondria from PCS rats. (Substrates: glutamate (5 mM); succinate (5 mM); amount of ADP added: 446 nmol.)

The reduced food intake in Sand PFC rats affected mitochondrial function: mitochondria i olated from PFC rat livers had a significantly lower ADP /

°

ratio (P<O,OI) than mitochondria from ALC rats, while the RCI and Qo, values were unchanged. Mitochondria isolated from livers of S rats had significantly lower ADP/O ratios com-pared with Jiver mitochondria from ALC rats, wherea the Qo, values were increased. These phenomena were ob-served with both glutamate and succinate as substrates.

Skeletal muscle: Mitochondria isolated from the quadri-ceps muscle of S, PFC as well as PCS rats exhibited no changes with regard to ADP/a, Qo, and RCI alues.

Subs/rate: Glutamate ADP/a:1.34 RCI :1.68 Q~: 39.1 nA O/mg prot/min. '" ;; o -0 C .;

'"

E

.

,..

o .0 E

"

c c

'"

c~

"''''

"'.0 EE

-"

_z c o§U 0--' ~« -E " ' 0 :g.= c", 0 0 -Bc

0::

.:: 0 E::O -0

"'-Eo "-0 0 E~ cm_o tn:-= "'c

'"

"0'U; '" ~~ 0 ' " ~o ~:o o.~

+1

'I

C/) Z M CO l"i 01 <0

_....

o

0

+1

tl

01 M M 'I" cri N o

...

_ 0 In N "lif N r- <.0_ ,..:<ti L!') CX) N U')

+I+I~ I~+I~

~gVgv~

ON 0 l"i - a J r- 0)

'I +1

~

0'1" MO!. N U ' ) M '1"- CO <0 O N 0 0 N~ N l"i

I 'I

~

tl

~

+1

~

CO'l" <0 CO COO ,.... Lt)

<D.n

l"i

as

:g~

b1

~ O~ 0 0

1

+1

~

'I

~

I

~

.... '1" CO N <0'1" M M

.n .n

~-; ~ U') N

o

+1

U') 01

o

+1

'I

0 .... co M "t'1". M N. 0 0 0

I+I~ I~+I~

010 <0 N CO CO M

l"iN l"i l"i

a> U en ::>

LnCC

CO

E

LnaJ

CO

--Q_Q-a..---Q.._Q-a..

uu

e

C/) .lSUU

e

C/) '--lu. U .s!-lu. U ~<Cc.. C/) c.. ~<cc.. C/) c.. ::i C/) o 00 N 00 00 tu

o

I-....

o N <0 o <0 W - l U C/)

~

- l

«

I-w - l w ::.::: C/)

o

z

«

a: w

>

:i ~ C/) I-Z W I-Z

o

u

«

_u. u. - l ~ a:

o

Z

o

J: U

o

I-~

184 A MEDIE E TYDSKRIF _{4 Augustu 1979} The total FFA content of skeletal muscle mitochondria

of PFC and S rats wa significantly lowered (P<O,02 and

P<O,OOS re pectively). However, the total FFA content of

PCS rat skeletal muscle mitochondria was unchanged. Skeletal muscle mitochondrial C 16: 0 content was signifi-cantly lowered in all 3 experimental groups.

Electron Microscopic Findings

Itrastructural morphometric analysis of the hepato-cytes of the 4 different groups showed that the only rele-vant change present wa the significant reduction (300~) in the urface area of the mitochondrial cristae membranes in PCS rats compared with those of ALC rats. 0 statis-tically significant differences were observed in the number or size of the mitochondria of liver tissue of

pes

and ALe rats (Figs 3 - 6).

DISCUSSIO

This paper is the first to describe marked alterations in mitochondrial structure and function occurring in rat liver tissue after portacaval shunting. Whether these mitochon-drial hange are due to events occurring in the liver immediately after portacaval shunting, or to events occur-ring duoccur-ring the 42-day period after the operation, has not yet been established.

Portacaval shunting induced lasting liver mitochondrial functional changes which persisted when the mitochondria were incubated in a suitable medium under favourable

Fig. 3. Electron micrograph of ALC rat liver (x 10500).

Fig. 4. Electron micrograph of

pes

rat liver (x 10500).

4 August 1979 SA MEDICAL JOURNAL

185

Fig. 6. Electron micrograph of

pes

rat liver (X26 000). conditions. Oxidative phosphorylation of mitochondria isolated from the liver of PCS rats was significantly de-pressed with both substrates studied: mitochondrial ADP/0 ratios as well as RCI and

0o,

values were sig-nificantly lowered (Figs I and 2, Table 1). The percentage depression was greatest for the respiratory control index (glutamate: 45

%;

succinate: 39%) which is considered to be the most sensitive index of mitochondrial function.'~ This was due to a decrease in the rate of state 3 respi-ration, as well as to an increase in state 4 respiration rate. It is also of interest that glutamate oxidation is more sensitive to injury than succinate oxidation. The depression in RCI and 00, averaged 45% and 34°~ respectively with glutamate as substrate, compared with 39% and 19% with succinate. These findings suggest that oxidation of AD-linked substrates may be more susceptible to injury induced by portacaval shunting.

These significant changes in the oxidative phospho-rylation capacity of mitochondria isolated from livers of PCS rats were substantiated by the finding of a reduced surface area of the mitochondrial cristae membranes which are intimately associated with the processes of electron transport and oxidative phosphorylation.'"'

The exact mechanism by which portacaval shunting affects mitochondrial function of the liver is not yet known. However, it appears to be a specific effect on liver tissue, since mitochondria isolated from skeletal muscle of PCS rats functioned normally (Table l). The reduction in food intake by PCS rats could play a role

in the above ob ervations. since mitochondria isolated from the livers of both PFC and S rat had lower AOPtO ratios when compared with ALC rats. However. portacaval shunting also aused an additional reduction in

RCI and

0o,

value.

A similar depression in mitochondrial function has al 0

been observed in ischaemia of the liver. Production of ischaemia by either va cular occlusion of the median or left lobe" or by incubation of excised liver tissue in a moist chamber at 3

rc"·"

resulted in reduction of mito-chondrial ADP/a. Rei and 00, values. Although the hepatic blood upply following portacaval hunting in rat has not yet been determined. the hunting away of porta-caval blood could induce an ischaemic situation in the liver and thereby affect mitochondrial function.

Elevated mitochondrial FFA levels have been suggested as the cause for the reduction in mitochondrial oxidative phosphorylation observed in hepatic ischaemia." A 6 - 7-fold increase in mitochondrial FFA levels was found after 2 hours of ischaemia at 38°C." However, the result obtained in the present study are not in line with the above theory, since the FFA levels were significantly lowered in liver mitochondria from all 3 experimental groups. The effects of lowered mitochondrial FFA level on mitochondrial function have not yet been established. Since these levels were observed in both liver and skeletal muscle of all 3 pair-fed groups, thi could possibly be related to reduced food intake.

The depressed mitochondrial oxidative phosphorylation process in myocardial ischaemia has been shown to be associated with increased levels of tissue FFA, while mito-chondrial FFA levels remained unaltered .... The possibility therefore exist that mitochondrial function in PCS animal may be associated with increased tissue levels of FFA. However, this remains to be determined.Ithas been shown that the FFA-synthesizing capacity of the liver is not affected by portacaval shunting, since the incorporation of IYC-acetate into FFA is similar in all groups'

Another factor to be considered is the depression in plasma glucose and insulin levels observed in PCS rats.' Addition of glucose and insulin to the perfusate of hypoxi perfused hearts caused a significant imorovement in mito-chondrial oxidative phosphorylation.'" However, it is clear that further work is necessary to elucidate the exact mechanism whereby portacaval shunting affects ubcellular changes in liver tissue.

In summary, the results obtained clearly showed a sig-nificant depression in mitochondrial function of liver tissue occurring after end-to-side portacaval shunting, which is an indication of the significance of maintained portal circulation in hepatic function.

This reduction in mitochondrial oxidative pho pho-rylation capacity could contribute to the impairment of liver function often associated with this procedure.'" The reduction in food uptake observed in the e animals also appears to be of significance in the interpretation of the results.

The authors wi h to thank the South African Medical Research Council and the niversity of telleobo ch for financial upport.

186

SA

MEDIESE TYDSKRIF 4 Augusru 1979

REFERE 'CE.

I. Helmann. R. E. and Mercer. R. D. (1969): Surgery, 65, 499.

2. Boley. S. J .• Cohen. M. I. and Gliedman, M. L. (1970): Pediatrics.

46, 929.

3. Starzl. T. E .. Brown, B. L. Blanchard. H. et al. (1969): Surgery.

65. 504.

4. Starzl. T. E .. Chase. H. P.. Putnam. C. W. el al. (1973): Lance'.

5.

~ie?n40'E.

A .. Mieny. C.. pitl. L. el al. (1975): Ibid., I. 32.

6. Cywe. S.. Davies, M. R. Q.. Louw. J. H. e, al. (1976): . Afr.

med. J., 50, 239.

7. Rossouw. J. E.. Lahadarios. D.. Vinik. A. I. el al. (197):

Metaholism. 27. 1067.

Rossouw. J. E.. Labadarios. D. and De Villier. A. S. (1978):

S. Afr. med. J.. 53, 1024.

9. Magide. A. A .• Press. C. M .. Myant. B. el al. (1976): Biochim.

hiophys. Acta. 441, 302.

10. Starzl, T. E .. Porter. K. A. and Putnam. C. w. (1976): Metabolism.

25, 1429.

11. Daniel, A. M. and Beaudoin. J. G. (1974): J. surg. Res .. 17. 19.

12. Boirne, L, Smith. E. E. and Hunter. F. E. (196): Arch. Biochem ..

128. 704.

13. Rhodes. R. S.. De Palma. R. G. and Druet. R. L. (1977): Surg.

Gynec. Obstet.. 145. 719.

14. Lee. Amct. R. .. Engelbreeht. G. C. H. el cl. (1973): S. Afr.

med. J .. 47. 1596.

15. Sordahl, L. A .. Johnson. C .. Blailock. Z. R. el al. (1971): Methods

of Pharmacology. I, 247.

16. Mole. P. A.. Oscae. L. 13. and HolloSlY. J. 0. (1971): J. clin.

InvesL, 50. 2323.

17. Lowry. 0. H .. Rosenbrough. N. J .. Farr. A. L. el al. (1951): J.

bioI. Chem.. 193, 265.

I Dole, V. P. cnd Meinertl. H. (1960): /b d .• 235. 2-9-.

19. Lochner. A .. Kotze. J. C. '.. Ben.de. A. J. el al. (197): J.

molee. cell. Cardiol.. 10, 57.

20. Millonig. G. (1961): J. appl. Physiol.. 32, 1637.

21. Wat on. M. L. (195 ): J. biophys. hiochem. CyIO!.. 4. 475.

22. Reynold. E. S. (1963): J. cell. BioI.. 17. 208.

:23. \Veibel. E. R .. Kistler. G. S. and Scherle, W. F. (966): Ibid ..

30, 23.

24. Chance. B. and Williams. J. R. (1956): Advanc. Enzymo!.. 17. 65.

25. Lebninger, A. L. (1975): Biochemistry, chapL 19. p. 509. 'ew York:

Worth Publishers.

26. Boime. I.. Smith. E. E. and Hunter. F. E. (1970): Arch. Biochem.

Biophys.. 139, 425.

27. Lochnel. A.. Kotze. J. C. N. and Gevers. W. (1976): J. molec. cell.

Cardiol.. 8. 575.

2 . Lautcrburg. B. H .. Sautter. V.. Preisig. R. et aJ. (1976): Gastro·

enterology. 7]. 221.

Pain

H. W. SNYMAN

On the letterhead for this conference appears an apt

quo-tation from Keats:

'Pleasure is oft a visitant but pain clings cruelly to us'. Allow me to say:

'From different times and mental climes came Milton and Keats but sensitive each to the deep barb of pain,'

In order to quote the earlier Milton: 'A sense of pleasure

we may well spare of life perhaps and not repine

but live with content which is the calmest life. But pain is perfect misery the worst of evils, and excessive

overturns all patience.'

The medical profession has always been under pressure to supply public explanations of the diseases with which it deals. On the other hand, it is an old characteristic of the profession to devise comprehensive and unifying theories on all sorts of medical problems. Both these state-ments apply to pain - one of the most important and clinically striking phenomena and expre sions of man since his origin in the mists of time.

Family of Medicine, Universit)' of Pretoria

H. \V. SNY 'JAN. :\l.B. B.CH .. M.D .. Professor alld Dea/l

I,allgur.J1 add res at the Pain Conference. Cape Town. 23 February 1979.

Need I emphasize the obvious that pain as such cannot be directly observed; what we do observe are persons claiming that, and/or behaving as though. they are in pain. What then is this pain? The definition 1 submit follows that of Engel ' and that of Fabrega and Tyma:' Pain is an unpleasant perception which the individual explicitly refers to his body and which can represent a form of suffering. The emphasis is thus on perception, unpleasantness, and the link with the body or physical apparatus in order to distinguish pain from other unpleasant perceptions such as guilt, sadness, 'mental pain' and even nausea. Further-more, the affective properties of pain in this instance fea-ture more than the purely sensory properties.

Within the great diversity of human types, the way in which people will react, what they will say and how they will behave when experiencing this unpleasant sensation called pain will vary considerably. What they say and how they behave are observable as the external accom-paniments of the presumed internal state of pain; these are grouped and referred to as pain behaviour. There may be movement, involuntary and voluntary, and changes in demeanour and in facial expression. More important and helpful to us is what they say in attempting to describe and to qualify the pain experience. This is the linguistic dimension of pain, the pain language within the langua3 usage of that person and the people to whom he belongs.

The response to and avoidance of noxious stimulaticn is an elemental factor in the adaptation of all living Sy3-terns and thus also of man. We see pain as a warning and thus as a protective mechanism. Man's capacity for sym-bolization, however, introduces a different dimension t'J the problem. it is not only a central nervous system but