Acta Physiol Scand 1981, 113: 33-36

Early changes in ATP and cyclic AMP levels

in experimental critical skin flaps

GORAN JURELL’ and BERTIL B. FREDHOLM2*

*Department of Pharmacology, Karolinska Institutet and the ’Department of Plastic Surgery,
Karolinska sjukhuset, Stockholm, Sweden

JURELL, G. & FREDHOLM, B. B.: Early changes in ATP and cyclic AMP levels in
experimental critical skin flaps. Acta Physiol Scand 1981, 113: 33-36. Received 5 Dec.
1980. ISSN 0001-6772. Department of Pharmacology, Karolinska lnstitutet and Depart-
ment of Plastic Surgery, Karolinska sjukhuset, Stockholm, Sweden.
A cranially based pedicle skin flap, 2 cm wide and 7 cm long, was prepared in t h e midline of
the rat dorsum. The contents of ATP and cyclic AMP were determined in the proximal,
middle and distal parts of the flap. The basal level of ATP was 1.3iO. 1 nmol/g dry weight.
Following the operation the level of ATP fell rapidly. The fall was more marked in those
parts of the flap that eventually do not survive. During the first 12 h the level ofcyclic AMP
fell at least as rapidly. However, thereafter the cyclic AMP level remained constant or even
increased while the ATP showed a further decrease. It is suggested that the rapid fall in
ATP may be used to assess the eventual survival of slun flap. The results are also
compatible with the opinion that a period of metabolic stimulation in the skin flap starts
12-18 h after operation, i.e. at the time when the adrenergic nerves in the flap degenerate.
K e y word.7: Skin, skin flap. ATP, cyclic AMP

The skin flap procedure is an established method irreversible damage (e.g. L e Page 1964, Chaudry et
for covering tissue defects. However, sometimes al. 1974, Fredholm & Fronek 1974). These changes
the flap becomes critical. i.e. the distal parts of the in A T P appear to be due t o a reduction in blood
flap may not survive the operation and if necrosis flow and to a simultaneous metabolic stimulation
occurs the entire procedure is a failure causing con- (cf. Fredholm & Fronek 1974, Fredholm, Belfrage
siderable pain and discomfort to the patient. The & Blaschke 1977). In several tissues including sub-
prevention of flap necrosis is therefore of vital in- cutaneous fat the degree of metabolic stimulation
terest to the plastic surgeon and considerable effort may b e estimated by determining the level of cyclic
has been made to understand the underlying adenosine 3’,5’-monophosphate, cyclic AMP (Fain
mechanisms (e.g. Reinisch 1974). 1973, Fredholm et al. 1977).
The eventual survival of a skin flap is probably In the present study we have therefore deter-
determined already during the first 24 h postopera- mined the levels of ATP, cyclic AMP and lactate in
tively (Jurell & Kaijser 1973). GIinz & Clodius experimental, critical skin flaps during the fifst 48 h
(1972) have shown that tissue pH values may be following operation. The results suggest that the
used as a parameter for predicting viability. Recent- survival of experimental skin flaps may be asseksed
ly, Hoopes & Im (1978) report that in flap areas, by following the early changes in tissue ATP.
later becoming necrotic, the glucose levels fall
rapidly and that the lactate content is elevated
throughout the flap between the 6th and 24th hour MATERIAL A N D METHODS
after operation. These authors suggested that meta- Female Sprague-Dawley rats (Anticimex strain) weighing
200-300 g were used. They were given free access to food
bolic changes in the flap may play a significant
and water. Under light ether anesthesia the dorsum was
role in skin flap necrosis.
In several tissues a fall in ATP-levels is related to * To whom reprint requests should be addressed.

3 -8 15879 Arta Physiol Srund I13

 G. Jurell a n d B . B . Fredholm

cence was recorded with a Packard 2008 liquid scintilla-

tion spectrometer set out of coincidence using repeated 6 s
readings. The sample readings were compared with a
standard curve prepared at each assay, 0.5-100 ng
sodium-ATP (Sigma, St. Louis).
Determination o,f cyclic A M P . Adenosine 3',S'-cyclic
monophosphate (cyclic AMP) was assayed essentially as
described by Brown et al. (1972) directly on neutralized
perchloric acid extract. Binding protein was prepared
from bovine adrenal cortex. 8-3H-cyclic adenosine 3',5'-
monophosphate (27.5 Cifmmol) was obtained from the
Radiochemical Centre (Amersham) and authentic unlabel-
led cyclic AMP from Boehringer, Mannheim. Recovery of
added cyclic AMP of the homogenization step was essen-
12 24 36 48 tially complete. Purification of the cyclic nucleotide on
TIME AFTER OPERATION [HOURS] short Dowex I-X8 columns did not alter the results and
hence this was generally omitted.

mal; (V- . -V) the medial and

the flap (see inset).
.---.
Fig. I . Changes of lactate with time in different parts of an
experimental skin flap in the rat. Each point represents the
mean of 3 determinations. ( 0 3 )represents the proxi-
the distal part of
Determination of lactate. Lactate was determined
enzymatically (Hohorst 1965) directly on the perchloric
acid extracts using reagents obtained from Boehringer.
Mannheim.
Stafisrics. Results are usually expressed as means 2
standard errors of the mean (SE). Statistical hypotheses
were tested by Student's t-test for paired or unpaired
shaved and a cranially based pedicle flap, 2 cm wide and 7 vari2tes.
cm long, with the base at the shoulder gridle was created.
The entire flap, including the panniculus carnous was
raised from the fascia covering the backmuscles and fas- RESULTS
tened back with 4 interrupted sutures at each side.
At different times after the operation (0, 2, 4, 6, 12, 18, The basal ATP level of the skin flap was 1.2720.1 I
24, 36 and 48 h) the animals were killed. Three defined pmol/g dry weight and the basal cyclic AMP about
portions of the flap were rapidly cut out and immediately
600 times lower, 2.0220.11 nmol/g dry weight.
frozen in liquid nitrogen. The proximal piece of the critical
flap almost always survives, the middle part shows partial There were no significant differences between the
survival and the most distal part would go into necrosis in proximal, middle and distal portion of the flap with
its entirety in all animals. The frozen tissue was weighed regard to either metabolite. This suggests that there
and crushed between metal blocks and homogenized in I S are no major difference in tissue composition be-
ml 0.3 M perchloric acid containing 1 mM reduced
glutathione using an ultra-Turax homogenizer. After cen- tween the different parts of the flap. Similarly, there
trifugation with 10000 X g for IS min at 4°C the superna- were no major differences in the lactate content in
tant was kept frozen at -20°C until assay. different parts of the skin flap (Fig. I).
Determination of dry weight
The changes in ATP and cyclic AMP content of
Following the operative procedure there is extravasation
the flap are illustrated in Fig. 2 and 3 respectively.
of fluid and edema develops. In order to correct for this The corresponding lactate levels are shown in Fig.
metabolically inert water, all metabolite levels were ex- 1. From these data it is evident that there were
pressed per unit dry weight. After the wet weight had been relatively small changes in the proximal part of the
obtained the flap pieces were dried to constant weight
flap-the part that always survives following opera-
at +70"C. Usually 24 h were sufficient. The ratio dry
weight/wet weight was calculated for proximal, middle tion. There was a slight, but statistically significant,
and distal parts of the skin flaps at each time point. The fall in the ATP content from 1.36 to 1.07 pmollg
values was 0.36 at 0-time and was reduced to approximate- during the first 48 h following operation. Cyclic
ly 0.3 after 4 h (data not shown), suggesting rapid de- AMP showed a biphasic response pattern in the
velopment of edema. In the most distal part of the flap the
ratio dry weight/wet weight was further reduced to 0.25
proximal part of the flap: a significant fall that oc-
after 18-24 h. curred during the first hours with a minimum at I2 h
Determination of ATP. The ATP concentration was postoperative followed by a significant increase
determined by the luciferine-luciferase method essentially with a maximum occurring at 24 h after operation
as described by Stanley & Williams (1969). Dessicated
(Fig. 3). Lactate levels were almost unchanged in
firefly tails and lanterns (Sigma, St Louis) were
homogenized in 0.1 M sodium arsenate, pH 7.4 and kept the proximal part of the skin flap (Fig. 1).
cold for about 12 h to reduce blankreadings. The lumines- In the middle part of the flap, which only partly

Actu Physiol Scund 113

survives, there were similar changes in ATP and
cyclic AMP but they were more extensive. The fall
in ATP level occurred more rapidly and was highly
significant already after 6 h (P<O.oOl). A further
progressive fall was found in ATP content of the
middle part of the flap. After 48 h only some 4 0 % of
the original ATP content remained (Fig. 2). The
cyclic AMP levels in the middle part of the flap
showed a clearcut biphasic response with a n initial
reduction followed by a stimulation (Fig. 3). Lac-
tate contents were significantly increased after 18 h
and onwards.
In the distal part of the flap, which without excep-
tion underwent necrosis, the fall in ATP was very I , - , , .
rapid and quite extensive (Fig. 2). Thus, already 2 h 6 12 18 24 36 48
after operation only 50 96 of the A T P levels occur- TIME AFTER OPERATION [HOURS]
red after 6 h and at 48 h postoperatively only 7 % of Fig. 3. Changes of cyclic 3 ’ , 5’-adenosine monophosphate
the original content of A T P remained in the tissue. (cyclic AMP) in different parts of an experimental skin
Also the cyclic AMP levels showed a very rapid fall flap in the rat. Each point represents ? S.E. 6 determina-
in the distal part of the flap (Fig. 3). After 2 h only tions except for that at 2 and 4 h which represents 4
determinations. (For explanation see legend to Fig. I . )
some 35% of the control valued was found. The
level of cyclic AMP found after 6 h were fairly
constant during the remaining experimental period. substrate, i.e. cyclic AMP/ATP. The ratio in-
In the distal part of the flap the lactate content was creased 2-3 fold in all parts of the flap between 6
increased already 6 h postoperatively and remained and 18 h after operation, possibly indicating an en-
elevated (Fig. I). hanced adenylate cyclase during this time.
Since cyclic AMP is formed from A T P some part
of the fall in cyclic AMP could well be due to a fall
in the substrate level. To get a better estimate of the DISCUSSION
degree of activity of the adenylate cyclase reaction The major finding of the present study is that there
we have therefore calculated the ratio of product to were substantial changes in the contents of cyclic
AMP and A T P of the skin flap that occurred well
before there were any clear morphological signs of
tissue damage. The extent of the fall of ATP-levels
in different parts of the flap seem to well reflect the
eventual survival of the part in question. Thus, in
the distal part, which never survives the fall w a s
larger than in the proximal part, which always sur-
vives. Extensive reductions of the cellular A T P
content are known to compromise viability and lead
to eventual cell death. Measurements of the A T P
level in a skin flap may give an early relevant prog-
nostic of the eventual survival of the flap.
I The large fall in tissue ATP content after the skin
6 12 18 24 36 48 flap operation may partly be due to the marked
TIME AFTER OPERATION [HOURS]
decrease in blood flow that occurs following the
Fig. 2 . Changes of adenosine triphosphate (ATP) with operation (Palmer et al. 1972). The remaining blood
time in different parts of an experimental skin flap in the flow may in addition be shunted through large non-
rat. Each point represent mean k S.E. of 6 determina- nutritive blood vessels (Reinisch 1974) leading to
tions, except of that at 2 and 4 h which represents 4
determinations. (For explanation of symbols see legend to local anoxia. This could also explain the rise in
Fig. 1.) lactate. The measurement of cyclic AMP levels

Arta Physiol Scond l l 3

36 G . Jurell and B . B . Fredholm

provides evidence that there occurs a metabolic CHAUDRY, I. H., SAYEED, M. M. & BAUE, A. E.
stimulation a few hours after the operation. Fur- 1974. Effects of hemorrhagic shock on tissue adenine
thermore, there was a tendency of increased pro- nucleotides in conscious rats. Can J Physiol Pharmacol
52: 131-137.
duction of lactate a t 24 h also indicating an enhanced FAIN, J. N. 1973. Biochemical aspects of drug and hor-
metabolism, a s also reported by Hoopes & Im mone action on adipose tissue. Pharmacol Rev 25: 67-
(1978). It is well known that catecholamines, by 118.
causing a P-receptor mediated increase in cyclic FREDHOLM, B. B. & FRONEK, A. 1974. High energy
phosphate compounds in adipose tissue. The effect of
AMP content, can cause metabolic stimulation in
hemorrhage. Acta Physiol Scand 91: 165-171.
the skin (see Voorhees et al. 1976). A combination FREDHOLM, B. B., BELFRAGE, E. & BLASCHKE,
of decreased blood flow and increased metabolic E. 1977. Changes in ATP and cyclic nucleotide levels
demands lead t o a particularly great reduction in during sympathetic nerve stimulation in canine sub-
tissue ATP levels (see Fredholm & Fronek 1974, cutaneous adipose tissue in situ. Acta Physiol Scand
99: 313-322.
Fredholm et al. 1977). The metabolic stimulation GLINZ, W. & CLODIUS, L. 1972. Measurements of
may be caused by noradrenaline released from de- tissue pH for predicting viability in pedicle flaps: Ex-
generating nerve endings in the flap, as described in perimental studies in pigs. Br J Plast Surg 25: 111-1 15.
an accompanying paper (Jurell & Hjemdahl 1981). HOHORST, H.4. 1965. L-(+)-Lactate. Determination
with lactic dehydrogenase and DPN. In: Methods of
Noradrenaline may also contribute to vasoconstric-
enzymatic analysis (ed. H.-U. Bergmeyer), pp. 266-
tion via an action or a-adrenoceptors. 270. Verlag Chemie, Academic Press, New York.
It is thus possible that the combination of a di- HOOPES, S. E. & IM, M. J. 1978. Skin flap necrosis in
minished blood flow and an enhanced metabolic guinea pigs. Plast Reconstr Surg 61: 748-752.
stimulation leads t o irreversible damage of the skin JONSSON, C.-E., JURELL, G., NYLEN, B. & PAN-
DEYA, N. 1975. Effect of phentolamine and prop-
flap. Therefore skin flap survival may be enhanced ranolol on the survival of experimental skin flaps.
by drugs that either increase blood flow or decrease Scand J Plast Reconstr Surg 9: 98-100.
the degree of metabolic stimulation, or both. We JURELL, G. & HJEMDAHL, P. 1981. Degeneration re-
have obtained results compatible with this hypo- lease of noradrenaline in critical skin flaps in rats. Acta
Physiol Scand 113: 285-289.
thesis (Jurell & Jonsson 1976, Jurell, Fredholm and
JURELL, G. & JONSSON, C.-E. 1976. Increased survi-
Hjemdahl, unpublished). val of experimental skin flaps in rats following treat-
ment with antiadrenergic drugs. Scand J Plast Re-
These studies were supported by Karolinska Institutet, by constr Surg 10: 1969-1972.
the Swedish Association for Medical Research and by the JURELL, G. & KAIJSER, L. 1973. The influence of
Swedish Medical Research Council (04X-2553). The tech- varying pressure and duration of treatment with
nical assistance of Mrs Louise Vernet is greatfully hyperbaric oxygen on the survival of skin flaps. Scand
acknowledged. J Plast Reconstr Surg 7: 25-28.
LE PAGE, G. A. 1946. The effect of hemorrhage on tissue
metabolites. Am J Physiol 147: 446454.
PALMER, B., JURELL, G. & NORBERG, K.-E. 1972.
The blood flow in experimental skin flaps in rats
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Acta Physiol Scand 113