viz., - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/30342/7/07_chapter 3.pdf · 83...
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BlOCKIUllCAL Cl.OCit Mf!CHAHlSMS
Rhythtd.c oscillation has bi.!en unequivocally re<;arded
as a funda.aantal propertt of life {:.icheving et al., 1978 J
3cheving, 1976). Hhythms of varying fl'-EF;u::.ncies ( circacilan
ar.Kl/or circannual ate:.) nave oeen d,eteet.ed in several
physiolo.Jical and met.a~'olic varid~Jles, viz., cholesterol,
glucose, triglycerides, phospholipids, free fatty acids,
corticost:Jrone, tostost,,rone, insulin, thyroxiM,
5-hydroxytryptaraina, _Sialic .Jcid, uric acid, urea, phosphate,
sodium, potassium, c.1lcium, ~iliriWin, protein, JlJUJ:U.n, globulin,
.Jlk.J!ine phos,>hatase, transaminases, lactic dehyJroJenasa,
80
GPT, ate.,. in plaSH1a and/or serum 1 glycogen, Got, GPT,. e~-Hal:lH,
acid pho&i)h-:ttasa.LDH, tyrosine trzmsamtnase, lJN,~ RN~ etc:., in
liver and rtmny other tissues (H~intxu:<J. 1974 ; Halberg and
i\hl.Jr(ln, 1980 ; ltalL;.;~r o at <d .•• 1911 ; Halb~!rg, 1974 1 Sf:b9v1ng
et al., 1978 ; iJ,aye:rsb>lch, 1976 J Halberg et at •• l900h
it()'mW,c;:t J il!Ost of the :H.udies are <:onfined to flla:mmls itu:luding
h\lman bein<;;J&. In notlllla!m;:~al..ian and other lower fl!rtebrates
re;.}orta on rhytl\J:5ic variations in the physiolo~:i.cal and
:~tabolic correlat·1S are r<H.atively limited (Sctw..as&t~Va.nn,. 1911,.
Urown. Jr., 1•:;73). However, rhytrodc l!lQtor activities in
fishes have been studied extensively (Thorp~t, 1918).. Rhytbml
(circadian and/or circannual etc.) in carhohydrate, lip~d.
erythropoiesis, oxidativ~, endocrinological, and reproductive
parawet.;rs have bQen I'<"Jll documented in f.ishes (Fernandez
cmd nan<•s, 198·J ; D'Jbhunty at al., 1980 .J .:iimpson, 1976 i
Ya;ldshit.a, 1969 ; Billard and Greton, 1978 • Jonas and
uilinski, 1965 ; Leach and Taylor, 1977 ; Valtonen, 1974 a Larsson and rrzmga, 1977). 3ut the mannC1r in which the
periodici tie a of these cycles have been de! tett:.ined is based
m.ostly on visuc.~l reauing of the macros~opic chronogrilms.
Furthermore, not a sin.Jlu piece of report is avail:lLl!! on
rllyth .. ls of cave fishes (Da.rr, 1976).
In the present chapter, ,lfl attempt has .:nan made to
Jn.:JerstJnd prope.cti(!S of S)me of the biochemic<ll clocks of
a cave fish. The author has Oiapl()yl.ld the lat,Jst .w.Jilable
81
mathenutical modal to validate citCf.idian rhythms fo:r glycogen
and lact<l ta eont·~nts in mU$Clc, lac:t.l te in livor, alkaline
phosph<l tase dnd acety lcholinestorase enzyme aeti vi ties 1n
gill, and acetylcholinesterase activity in brain tissue.
1wenty four fishas, approximately of same body weight
and len9th, were seh<:tGd from the stock. a'lU<&riUlll ( 40 l.i ~e
capacity} and wa·re transf<.'lrred to unother smaller aquarium.
They w.::re accli::;;ated to UJht-dark phot<;)pceriod,with 12 h~tur.s
o£ li']ht. alternating with 1. hf'urs of darkness (light awitclled
on at oa.oo and off at 20.00 h) fox twant·1 one dar·s. rhe
temperature of the experimental rOO>;;~ remained near constant
(26 ,t. 4°C). Four fishes w<&re sacrificed t>y docap:itation
at each of the following time points, viz., 09.00, l3.ou~
l.7.oo, Ll.OJ, 0l.,Xl, and 05.00 h. Muscla• liver, gill and.
brain :;ere recoverea froo autopsied fishes, froz<>n at -200c
in a deeP-frigidaire until utilis2d in biochemi~l assay.
~evels of ,;lyCOJOO and lactate in muscle, lactate in liver;
..1Cti vi ty of alkaline phosphatase & acetyl.ebolinestcrase in
'Jill 01nd acetylcholinBster;J.ae in brain were assaJed according
Glycogen
;lyco y'n content ,Jf tha ::tu!lclo t13:>ue \':JS d'~t !rmi,ned
!1y th~ mathxl of Jeiftar ?t ·ll. (1950) lft•H' hot 1l<:tl1
ax truction.
82
Principle
Concent.rat<;ld sulphuric .acid hydrolyses ()lycosidt.e
bond to give mon•:.~saccharides which are fu.:rthar dE~hydrnted
to f~furnl ~nd its derivatives. the furfural reacts with
antlu::.me (lO...kato, 9-10 dihytiroanthracene) to give a blue
<;~reen coruplex ..
30 a of pot...s<>ium {l<;.oH} pGll~ts dissolve>d in 100 ml.
of distilled water.
2 l Y. $odium sJlphate solution
l 9 of sodium sulph,Tte (Na2:>o4 ) dissolvad in 100 ml
of distillud water.
3 95 i; :c. tha nol
95 ml of absolut: et:1an.:>l ilkld& to 100 ml with
J.2 'J of ;J:lt::r,Jne disS:JlV·"'-' in lw ml. of 95 ~~ sul;)hUX'ic
acid {H2 ~:.}4 ). This ro.:~'}Unt \l.lS fresnly pre:'·'red.
~it.andard glucose solution
20 mg of glucose d1ssolvad in lvJ ml nf Jistilled
IYJter .:md diluti!d to 1 litre. ~ ml of thia solution
cont.1ins !.JJ ~J of •Jlucoso.
83
.ieighed samples of muscl&& we:n~ hlten in test tubes
containin9 3 ml of 3J ;?. Kc»i solution and ctiguted in boiling
water bath. After cooling the tubes, o.5 mJ. of l ~ Na-2 so4 and 3.5 ml of 95 ;< ethanol <tere added to eacll tube to
prc:ipi tate cylyeog m.
Th~ cont<lnts in ec"iC:h tuno were stirrad ~tith a glass
rod and the rod WJS washed ir. tll:? tube itself with a small
quantity of <Jthan;>l. Th;: tubas ,·,rn:.] jently heated, cooled
and .1Umv d to "t:.1.1 r>V~rai·,:;ht for cmaplate precipitation of
•JlYCQ·Jon ;,tt room t >:.'lpa.r:.rttl.'t~. !hli! Bext ~Y th<i tu.bo$ l'i~u·e
c .ntl.'ifuvud dt 3·JV0 .rpm for lf> c:linuws., The suj)llrn~tilint was
disqa.r;.t ;d .11'ld th.a r"'siduu 'NilS dissolv<o:d in al.>out 3 ml of
distilled •~<~tar :md .r;:pr;;c;i.pitatod >dth 2.0. mJ. of 9;:J _.;
ethanol. It wc1s c•.:mtrifu..J\Od a.Jain ;.;oo supernatant we~& discarded,
The pr. c;;.~;i L:. \;.ls fin,:jJ.ly dissol vc•d in 5·J ul of distill~
,:ctt ·r. ·..;c;ti;:ntion of ~;lycor;en 1'/.::lS c<Jrri"'d out as follows a
o r.Jl of ;.Jli ,uot W•lS transfdrr;;td to a wide mouth boiling tuoo.
This served as a tost s.:~r:1ple. 5 ml of standard glucose solution,
cont:Jining lJ.J ~;J of glucose; vns t::~k;:n in another boiling tube.
This .-;.on·eJ c;s st.md~1.rd. 5 ml of .Jlass distilled ::~ater was
taken in a thi.:d t~st tube. This served i1S a bl;mt.
fhe t<ves containin,;; t .•3t 1nd stHld 1rd solutions and
olallk .. :re pL:~c.1J in ic·~ cold water. Jry ~nJ cla:Jn JlJss beads
.:.JrJ l13 :d t.> ,>J:>JV ~nt. i).JmpillJ f Jll uillg 3ddi tion Of ·'lllthron~
r9ag$nt. 10 ml of freshly prapa~ud an~1rone reagvnt (4) was
adJod to each tube with a iast flowing p.ip&tte. The contents
1rJere mix;:ed. by sl1<lkin;J. The tubes were then transferred t.o
:j tnilini;J wator !;!CI.th for <~x:C~ctly 30 minut•e and th~;~n cooled
to room tcmr:.>erature. The optical density (o.u.} of the teet
dnd sbndard solutions were read agoinst blank at 6::-:~; nm in
S:}G\tal.
'tho amount of the- tJJ.yco-;cn '""'s calcc~latad from
the ecpJu tion
1).9 of ')lyco-)en in 5 ml of .1liquot = ----loll X S
;.) = o.D. of standard solution
l.ll = ,;onverd:.m factor of •Jluco-~ , to Jlycogen.
Lactic acid
K.nown ·.vei,;hts of ~:~UScle tissue N<;re horno<Jeniz•~d in
lJ ;-~ trlch.loroac-)tic >cid (tc::,) <~nd the homo:r n.~t.es ,•,ere
C3ntrifUJ0d ;;.t 3JJJ r;>r.l for 1:':> r:linutes. The supern:1t~nts in
vach •.. ,:;e >Nre used for the estimation ~~ lactic acid by the
me thvd 'J f i3 J rk;l r ,1nd .j\.lltr,IO ra :>n ( 1941) •
Principle
->lucose .1nd other interfering llU':eriah of ;1rotein
fr~oJ ·xt"'·.c;t dre .r.amoved ;:;y Van .:>lyke-.ialkowsi !119thod of
treatment 11i th copper sulphate and calcium hydroxide. The
solution io than he;~ teu with cmlcQntra t'"d sulphuric acid.
Lactic ~:•ci.d is thus converted tv acetald\>lhtde, the lattez
is then <ktcrn.i:>i!d by relcti::>n with p.-hyd£oxydipllenyl in
1 20 I'· Co;:;;Jer ~\llPhate solution
~.
6
2011 g of Cu:Jo4 • oo2o ctisnolvtH.i in 150 ml of •.-nt'!l'r
witll gentle heal;in~n ttuil solution wm~ cooled .:<lt..l later
diluto;J to 1 Utre ·•itn distilled ;·.rater.
4 ;.-: C:>;J(:Ja:C sulph3toii solutl.un :
l vol'-.1•:1_,. of 2.; '' copper s..~l,)ilata solution (1) was
•.til ;bi t.:> ) volu;'la5['Jith d:i.wtil!~ l•nter nnd th~rouJhly
-, ·JyJr"~··.rJ'p''·'n •1 ~-- · , .... · '~ Vf-.1 ..L th.. • .lo\.::'<.:Jt.'Hioo 1
5 Y. NaOH s:>lution ; 2 ml di<Jtilled ~~;)t·3l!' ud.:bd >·lith
sliqht w.:1rming ·11'\d stirrin9; the solution then made
to l..J,J ml ,fith distillod \·Htar and ntorod in brown
bottle •
. ~.215 :;; oi 1m:::e: l~t!liULil 1-~ct t<.~ d;!.ssolved in l.OO ml
of :.li;:;'tillod .:;:. ter !>1 1 li u·c voll..tt:<otric flask J l m1
of concantl.\1ted suJ.phi.Xic .:LCid <.!OOGd ; tJw whole uade
t,.; O!W lit.i:>? .1i t!\ t.li.;.; t.ille!.\ l<<:~tol:' GlllU tl'lttl:OU\,lillV mixed.
i !.is .,,av>:;; l m;~ lnct.ic ~,:;iu in ? ml. of soluti;;~n and was
ut.:..i..<lo for :1 lon;;; tir,J~ ;vlwn k"pt in .lZ<!fl'iger;:;tor.
5 ml of s to eli: s t:m,br'l soL; tion (1) C:ilutoo to lO'.J m1
in t;L .. sc sto; .. ·pu.rcc,; vol.Jti•£:tr1c fLwk witi. distilled
;~atc•r .'lnd r.d.xod tho.~:ou;Jhly J tl)iS solution (}3Ve o.Jl mg
ldc tic .otc:id p-er r.tl and ,v-;s peer a red fre~h claily.
Procedure
2 nl of prot::<in fr~::c s:..~pcrnat.Jnt, 5 ml of fr,:s•1ly
;:.r . .,:· •. ;r<~<1 li thiu:n Ltct .• te standard solution .:md a :re:~,ymt
cl.lnl< (5 rul of ni~till:!'<J ·.J::tor) .mre taken in three separate
;:;.;,;; t..- H >.~ 1 1i t . .L .JS ; .L ol of 2U 1• Cr':>;)er sulph.• tc solution
,, .~ J J~a·.< to .J .. l ;:11 ·"lJ ttun r~'le to lJ tal ··1ith dis tilled •~ater •
.Jne 'J of po;'Jdere<.i ~ll( Jd) 2 cus 1 .. J ·<.i to dc.~Cll tu·.,o ·.;i ~h 'l
spatula. They v.~era stoppered :tnd shaken vigorously until
uniformly dispersad. The tubes \'lore kept for an hour with
frequent shakiwJ and latar e:ertrifuged at 3000 rpm for
15 minutes. 1 mt of supernatant vla& taken from eacb of
aoov& tubes in separuw tube& with an internal diameter
of 18 to 20 •• <l.05 ml of 4 ;1. CuSo4 (2) was added to
a.:~cb tube. .;}ubsaquently 6 ml of <:billed analax' f'2S04 was
added• initially drop i.>y drop, with uniform mbin9 of the
contents, to each tube. t~l the tubes ~re kept in a
boiling water botn for 5 minute& and cooled to 2;)0r; ~
below in running cold vtrlte:r:. ;J.l m1 of P-bydroxydi.phenyl
na>Jent was the-n added drop by drop \o eaeh tooe.
Precipitating reagent was dispersed again throughout the
solution quickly and uniformly by shaking. All the tubes
were incubated at 30° :1:. 1°c for 90 seeonds and were allowed
to cool to room ~mpera tare. The opti<:al density was
measured against blank at 570 nm in Spekol. The wlues
are exprees;:d as i.19 lactate per 100 mg fresh weight o~ muscle.
Non-specific pbosphosaoDOe$'teraaee (i\J.kaline phospatase il.C.
No. 3.1.3.1 and Acid phosphatase ~.c. No. 3.1.3.2)
Homo Jenates of fresh tissues were prepared in u.25 l'1
ice cold sucrose solution to approximately 5 i~ w/v 1
eertritu,Jed 3 t 300J rpm at 4°C for 1'.) rlinutes ;-~nd supernatant
w~s used for enzyme assay.
Pd.MJ.J)l•
Enzyme extract.. on 1ncubatic.n w1 th buffered aodiua
p-glycero:phoaphate, :t:llleaa~a iru>rganic pnoaph.at<J by
ena·~t1c hodrolyds., The inor.Janic phosphate reacts Vlith
an acid nolybdate solution to form phosphomolybdic acid.
The phosph:lmol)'bd.f.c acid is X''ilduc&d 'oy addition of 1. 2. 4
amino...noptholsulp!lonic acid reagent to produce blue colour
(Fiske and Subba aao. 1925). The intensity of the colour
developed is directly propotional to the amount of i~ganic
phosphate. which in tw.'n indicates the enzyl!liltiC activity
of phosphatase.
H.aa;enta
1 AlkaUne phosphatase aubetrate (pH lO.o); o.5 g of
sodium p-Jlyc~rophosphata and 0.424 g sodium diethyl
barbituri.lte dissolwd in 10J ml of distilled \Vater
and stored under a layer of petroleum ether in a
refrigerator.
2 Acid 11hosphatase subs tra t:: (pH 5.0) :
o.5 g of sodium P-glycerophosphate. 0.242 g sodium
dietnyl barl.liturate and 5 Ill of 1 N aceUc acid
dissolved in 100 all of distilled water 3lld stored
undar:"l.:.1er of petroleum ether in a refri']erator.
69
3 30 1- T:ric:hloroac:&Uc add (TCA) :
30 g .of !CA dissolved in 100 1111 of distilled water.
4 5 ;.~ !richloroac:otic acid ;
5 9 of TCA dissolved in 100 ml of distilled ~t.er.
5 10 N Sulphuric acid
4"50 ml of con;;:,}"ltr:.toaaulphuric ~ctd added to 1300 ml
of distill~ v1ater.
6 ;;>tock standard phosphate st:~lution :
o.35l g of pure dry monopotassium phosphate dissolved
in tiistille<i wa te:t in a l Ut:te volumetric: flask J
10 ral of 10 N ~so4 added and tho whole diluted to
l litre .vith distilled water. Thb solution contains
J.4 m,; of phosphate in 5.0 ml.
7 ,Jorking st..ltlddrd phosphate solution·.
6.25 ml of stock phosph:~te solution (6) tak.un in a
100 ml volumetric flask J 16.7 ml of 3C 1~ r..;.\ a<.ided
and the whole diluted ~vith distilled wat•Jr to 100 ml.
This solution contains 0.04 ~J of phosphate in 8 ml
8 ,\::T.JOnium oolyW<.~ tc :>olution ·.
!>.a g of a;:unonium molyndate dis3olv.'CI in 40 ml of
distilled 1vat·!r ; 60 ml of lu u ~~04 add :d to 1t
and the vlhole diluted to 200 ml with distillfrd water.
9 15 ;-:. &oJium bleulphite so1ut1o11:
15 g of sodium bisulpili~ dissolved in 100 ml of
distilhd w.:.~t·n:·.
10 2u Y. Sodium su1phi te solutbn :
20 g of sodium sulphite dissolved in 100 ml of
distilled water.
11 .\!llinonaphtholsulphonic acid re:1gent :
97.5 ml of 15 ;~ socii wn bi-!Zulphi te solution taken
in a ';Jlass st.:>ppered me::~su:ein;;; cylinder ' 0.25 g of
1.2.4 aminonaphtolsulphonlc ac~d and 2.5 ml of 20 Y.
sodium sulphite added and shalutn until the powder
dissolved. The solution was then transferred to a
brown 9l1ss iJottle and stored in cold.
Test·.
9.00 ml of buff~red sut,str.:te of r.:-:!Jir.~d pH and
1.0 ml of axtrac:t \ll·:.!re shak::n in a t,st tuu.c. The
ra:~ction r.tiJCturG w..1s incu:;.ltad for \>J r:U.nutes at
J~C. Th•:.! reaction \V<lS stoj)j)Od , 1 addin J .: 1nl of
3; t' TCA.
91
Conuol:
Control s3t11ple was prepared with 9.00 ml of buffered
.substrate. 2.0 llll of 30 :;{ TCA and 1.0 ml of ext.ract
l3oth tas t and control sarnples wel'e centrifuged
at 3000 rpm for 15 minute$. a.o ntl each of t'o?st sample,
control !lample, v~!d.ng at.Jttdard phosphate solution (7),
and 5 it. TI:A as blank Vtrere tak.,n in fou;,. t1!lst tubes ~aduated
to 10 ml ; 1.0 ml of anuoniu~lybdate added to •Jadl tube
and mi~ed tnornu\ihlY ; o.4 ml of a."!l!ncmaphthol~ulphonic
acid :te.Hj .~nt them added to e:'leh tube 100 mixc:d thoroughly.
tut:es '.F~rcJ left for ::; minutvs and optical density was read
ilc}aine t '1 H\k i:.l t (>(,;) nm in 3pekol. The values are expressed
Protein content of the oxtr.:1ct t•tas dot~~rmined
by the method of Lowry t?t a1. (1951) as described 1n Chapter 1\1
Acetylcholinesterase (a.c. 3.1.1.7):
0.25 m sucroGo iiulution to a,J[JJ:Oximdt.:1!( :.> ~~ w/v; centrifugoo
at 31XJO rpr:~ at 4<t for lj minutes 5 supe.rnutant \lc>a used for
'?nZ:YtU•.1 a!h.l:f ·l :.hu colo.~:itOOtric floJ\.hod of .. lotc<~lf (1951),
as :<lodifiod ; 1 .• urali .J-i:;hnati:.lss {1967).
PJ:i.nclple
CH.., - 0 - C\l;l. CH20H ...
I
Clia c~
I
u:;1 .W ·(CH3)3 + H:;tl iCl .. N-(Cf~s)3 + HCQI.ii
I
OH UH corresponding acid
Acetylcholine chloride Choline
;l.emoval of acetylcholine p~r unit time is measured
by comp.'lrison of its initial concentration in a ref~rence
tube .. i th the finul conce-ntra t!.on in exp<:rimental tube •
.. ~cetylcholine reacts vtith hydroxylamine to form correspc;nding
acylhydroxc•nic acid, ':lh:i.ch forms ~ stroo<Jlj'C. coloured farric
hydr:>xar!u te ,,1 th ferric salts.
Reagents
1 ;.i/15 phosphate ouf:hr(nH 7.4)
16.72 g of disodil.lll hy<Jro 1 ·n ph0sph.?te (i~a.Ll!P04 ) and
93
2.12 g of l'ilOnopotasai\lm dihyd:cog<m phosphate (~a4)
dissolved tn 100 ml of double distilled water.
2 J.004 rt ,\c;•tylcholine solution,
::itock solution (0.04 Ml was prc:pared by dissolVing
o.7466 g of ac«atyleboline chloride in 100 ml of HCl
of pH 4.o. ->toc:k solution >liaS diluted vdth 9 volU!Il9s
of phosphata buf'0.r (l) before uaut.
13.9 g of hydroxylamine hydrochloride dissolved in
lChJ ml of distilled ~vater.
4 3. 5 N sodium hydroxide :
of J.istiliad wator.
5 Alkalin0 hydl:oxybmine solution;
•iqual volumes of hydrolCyl:Jmine hydrochl.Jr~de SJlution(3)
c~nd 3.5 ~~ H.aOII solution (4) were milC~d to.pther before
use.
Hydrochloric acid (ltl):
'l:>lU:.10 :Jf cii.;tlll J 1\tlt.r.
7 0.37 ;,t Ferric: c:hl;.>rida solution
10 g of forric chlor:tde dissolved in 10\J ml of
O.l H HCl.
a 0.2~ M sucros~ solution
l ml of extr"ct vr\$ udded to 1 ml of buffered substrab
solution ; incubated :fo;.: 3i) :;Uay·;aa ;;. t; 37°t,l°C ; reactlon
was sto,Jp\ld by tiN ciddi tion of 2 ml of ulk.--:1lina hydroxylamine
b~'drochl.;ri.:l.?. Tho t:h<Js wJra sba~;.en thorou]hl.y .:md l m1 of
HCl (l:l l1Gl:ll2o) w..;s ~Jd,?d with further shaking lnd the whole
>'1·1S cantl:ifugod. 2. 5 ml of suptlrnatant was hken in •'l test
tube an~ 0.5 ml of ferric chloride solution was added. The
contents •wre a')ain thorou;;hly lllixed. Zero tilu0 controls
vnre m<>.intained ';::y adding 2 ml of alkaline hydroxylamine
hydroclll-.~ride prior to the oddition of the en~ylllO extrac:t.
Colour of the hydroY.ami.c acid fornod .os read at 54~) nm
a::pinst the bl:mk. The bl:mk cont1in·1d 1 >ll of \)uff :r
insta:1d of ~;:..~£fer subs tr.>ta :aixtur '•
The activity of tho enzyme 1'/,js d :t·.:rminod u1 the amount
of unreacted acetylcholine ct\loride left. aft~r 3.; ::dnutes of
95
incubation. 'fhe intensity of the coloUJ." at the end of the
incui:>;;,Uon ,Jives the nmount acotylcholine left unhydrolysed.
concentration and subst:c~te concentr .s tion left unhydrolysed,
the act\Hl l~v<)l of the '30ZJll'lO activity >m& G:;;tiwat'd and
e:<;rr-i.Hl<)'~d 5.n :" ~:101es of acet1•lcbolina chloride hydrolysl#d
p>:J: mg l)rotein per il:Jur. Protein content of th.:~ extract was
dot0rrainect~LO'>JI'Y ct ;:,l. ( 1951).
Statistical methods
uata (m<::an :J:. 1 St:) wc~re pbtted by the convent!.onal
way :.,Jainst clock tulur. i.liHter .. n>t fe..ttures of tinto-:iependent
v,,;;L.iJJ..,,s ,,,ere 2<;;$,;sseti. ..;t..:;U.,\.ical analysis :for comparison
of J:Coups of uuta •l~t:Ci.:: ct~rriod out by one W<lY analysis of
anll :::.intz, h/77). vill,Jla co;;inor was employt.>d to dctortaned
rhythm parar:Je ~;,_,;rs, viz.., HK!Sor, :;::Jpli twJe • ,md acroph.lse
(Halberg :t ,;1., um, 1972 ; !lelson ct al., 1979).
Results
Mu•cle glycogen
tioa sc1lc. ;; 't' ,~,>r;J 3U :j 'Ct<)d to 1!1-llJsis of v •ri ,mea.
fable lJ).
The muscle glycogen content was found to be highest
at ul • ..;u h. rne muscle had lowost .%lount. of glyco;Jen
stor.lS -1t. 13 • ..); h (Tal,le 17). Joth the time points are
exactly l~ hours dp.u·t :fr.Jm ~;ell other. dhen both h:l.ql'utat
.md lowvst mt#ans Wc!t:J coii<p<.u::'.!d~ u statistically s1•;:;nif1cant
•i.lif:. :ronco . .~~,s witnessed (l.i:.mc .. m• s .Jul tiple-range test,
'fabla l7). 1:110 pce.tc.:•r,t ci·:~m-Je fl:'u'll low,)st to hi:Jhest. mean
,iasul ts of tho cosinor <::Ml_isis of d~ta :t<;v;wl detection
of statistically si.;rrificant circ.'ldhn rhythra in the lmlSClet
glycog. n content of cave fishes (Table 19 1 fiql..l.t'~ J.8). Tbe
zero-a1:1pli tude hypothesis W-lS rc}Jct.:d at 2.9 r~ .l.ovcl. The
acro;;hccse w.J.s lo..:at d <lt ... 16° {01.07). with 9':> Y. con~id,.;.nc.e
intorVcllS between -339° {22.6 h) and -53° (03.;)3 h)(Tavle !9).
The m;,sor vns of 0.31 (mr.J/100 mg), with <lO amplitude o.04
(lll<J/hU mg).
Muscle lactate
rlesults of analysis of variJnce revaal st~tistically
signific:.nt time .~ff?ct (P < o.o!'>) on the content of lactate
in t.ha ouscle ti~sue (Tabla 18).
The max1.mum conc<1ntration of lacta tu in the r.l'Jscle
WdS ca:ord<:d 1t l3.uv h and ;;linimum ;:~t lv.uO h (Table 17).
Both the m-~.:.10 v.J!ues diff,)red st;tisticJ.lly significantly
97
...t stJ tis tic<Hly llliynifici:lnt ciJ.'..;.:~dian rhythm in Inuscle
lact<:~te cc;me<~ntrations Wd& ct~tl1!cted (Taole 19 ; r'i'JI.U:e la).
i!u prol:J.Jbili ty t:n t. tita .~;a?li '"v..l.<;~ -..£ tnis t:hftltJ\ is not
'ihiildJ. t.o zero ~~"'a :~;;.1 '' (Jaal~ 19). The ilcroph~\$0 •Nas
Liver lact.lte
,;;e.1n ;nlue at J5 •. JJ h :nd that of J.o,J;::st .1t 11 • .;: h (;~i·;ure 15).
uoth the tin:.? ;)olnt::; •·J.!:CO .1 0 ) •• rt from e,1cll -.~t.l<Jl: '.Jy 13:.;;".
·' :> \.·J tis ticJ.lly SiJnificant cir;; dian rnythm in the
l.•ct.1t.• conc·nt.r1tions of livor ti~suo .'/JS not v.,licJ.,ted
with the help of coSinor rhytlll!lom(:try. lbwover, the
acx·o!Jh;;se 1'1<.13 :litn>?ssed at -48° (03.2 h) with a meso:.:
18.2 (Jlg/lW mg), iJnd an amplitude 2.56 (ilg/l•JO mg)
('raLle 19).
Gill alkaline phosph.::~tase
iJcJta .wura')as <t. l .:;,~) n:.ve i;c~en ;'lotbd as o fut11::tion
of ti;;1e (Pi Jt.l:t<l l~). ·.,hul ts of atMlysis of v.u:l.;n<;o; did
hi·]hes t rocordad r.1van w~•s 42 ;.; •
th0 Jill (l:::.,le 22). 'io.vev:?t', t:·~~ .lC.i.';ipi·us.; :1.1S '.i ·:ectad
:~t -167° (11.13 h), ,;ith •l :J so.r of 2.~~4 (y_i )i/:nJ/h), alld
Fl . •'">)li tud~ J. 56 (.t] •Ji/n:J/h).
Gill dcetylcholinestarase
.... -> .'i"f ..~ .... . d on -.:, h tt,.J. nld
99
Maxi.11Uill enzyme activity was noticed at 17.00 h and
1~1inimum .1t u9.·)) h. ihc lo·ln>t ra.:an value signiiic.:Jntly
diffar:•cl fron that of tha ni jhc)St (P ( 0.01 J fable 20).
'l'he p·!l:'Cc·mt <:iunqa from tho lowest to highest .r:)corded
1\. .,t 1 t.iot.ic<<llY oi•Jnificant circadion rhythm w ... s ;~lso
" Lin·.'d ··1lt.io tb1 J;1lp .CJf til 1 cosincr anill·rds of the data
te;t ·:ns 1'c1j:·c~J:d .. t 1.7 ;.• lovul. The ci.rcadi.m acr'.>i>llase
vns rL t·~ : t: · .. t. -·''"'·p ( 1-:P •. J7 hj, with 9~ 1:. confidence
Bruin ~catylchblinesteras&
(Tacle 21).
to .31 . : •
100
i\ statistically significant circadian rhythm was
valid,.'lted for the~ acetylcholinesterase activity in the
b:nin tissue with the help of codnor a""'llysis of the
data (I<tble 22 ; Figure 19). The assumption of &ero
amplitude hypothesis was r~jected at 3.8 t. level. The
eirc,ldian acrophase was d(~te>cted at -271° (16.07 h). with
95 ;t. confidonce intorvals between ..:J30° and •312° (T.tnle 22).
The muor Willi of 2.9 (jlll!Ol ;:.t;h/UifJ protein/h) with an ati'IPlitude
o.312 {~l ;i;h/rJKJ protein/b).
Dlscuss1oa
Present results clearly demonstrate the presence
of circadian rhythm in the conwnts of muscle :;~lyco;en,
muscle lactate, and in the acetylcholineaterasa enzyme
activities of ;ill and train tissues of cave fish.
Nemas;h•4U!Q gwz.ardi.when subjected to LD 12tl2 photoperiod.
Since liter3ture on biological clocks of cave fishes is
completely lacking, it is probably the first information
of such a kind. However, a statistically si:mificant circadiaJ1
rhythm -!1 ther in the content of liver lact::~ te or in the
alkaline phosphatJse activity of gill tissue was not detected.
This clearly indicates th1t oven though fishes live inside
the CJ vc!rn under eompl•.:> te darknoss their biochJ:nic 11 clocks
could be synchronis-.!d ll:r .JrtHieial photoperiods. ,\t presont
101
1t b not possible to explain why rhythms 1n livJr lactate
and alkaline phosphatase activity in gill tissue did not
appe'lr following exposure of the fishes to UJ 12c 12
photopedod for 3 week1:r. Could 1t be that still longer
period of accliraat.i<m is required for the expression of
rhythm in the above two biochamical paramet&rll 1 furthel'mOre,
invrssti\;l;at.iona ;)f rhythm in all tne above parameters of cave
fishes under natural condi ti()ns would be of great physiolQ<Jte.al
importance.
l'be aerophase of tha circadian muscle glycogen rhythla
appeared at -16° (;)J..o7 h), •vitll 9!> ;4 confidence inteJ:val
ran;;ing between -339° and -53°. Qv.lar the single transverse
circ:,dian time scale there Wi1S a 38 ;4 varianility from
lowest to hi·Jh<.!st recorded means. Although glycogen is
found in almost all tissues, muscle and liver tissues are
met.) -olic,,lly more imPOrtant in this re ;ard. Usually
glycogen func ti:ms as a res"rva of glucose and, dGp•~nding
upon the requir:a.Jnts, it is readily broken down into glucose
to meet energy demands of th~ ~ntire organism{Newsholme and
.:;tart, 1973). Present results, ther.Jfore, St.IJ':)est thilt
probably thare is less ;lucose utiliz:.~tion in the cave fishes
during mid-night 1Vhich in turn allows maximum accumul'l tion
in the form of musclo glyco-Jen during this time of the <:ircadl:u.
tlrae scala. .>ince we do not have J.ny d,J td/informa tion on
circadian rhythm'> -11ther in Jlyco;Jen syntn.t.ase or in glyco<;Jen
102
phosphoryhse aetiviti<?S of this cave fish, or .any other
fish, it is difficult to make any suqljlostion at present.
Possibly, a similar machanism(s), as in I!II!lnwah. might
be operlting l0Z~ding to exhibition of circ<~dian rhythm
in the contont of ;rruscl,~ ~~lycogan,
~xistence of circadian rhythm in ~he content of
;nusch lac to< te has bll<~t!l r'Jpor tc1d in snak0s ( Gra t& and
autchison. l'J17). How-~ver, too above repOrt eo:nsist5of
obaervutions a"t two tima points only over a ci.rcadian
scale. t\dde this, tlla authors h<tVe not utiltzod inferential
st,>tistics to validate circadian rhythm for u1U#Hile lactate
concentrations. rn tile pr-:~sent study• however, lactate
C.)ntent of rausclu has been determined at six time points
and a rhythm was detect·Jd by cosinor analysis of the data.
rhe hi;;llest lactate accu.nulation in the muscle 0cf cave fishes
was found to be at -162° (lO.a h). Conversely, the circadian
bathyph:Jse wi tl'l respect to lactate accumulation in the muscle
w<.~s <.iGtected at -342° (22.8 h), with 95 y. confidence intervals
ran,JinJ .l:!t•~-><:ln -313° (22.87 h) and -11° {0.73 h).
surprisin _;ly, there is a higher de ;re:e of ov.crla;>ping bet~ve~n
the acrophase timings of muscle Jl yco,;en :Uld ba thyphase
timings of muscle lact.l te. This SUJ'JuSts that th"re is an
invc~rse r ;1,• tionship between the ,1ccu -,ul.l tion of Jl yeo jen
.11\d lact.>te in the :-:1uscle tissue, i.e., ··men ooscle JlYCoJen
(;ontent of the cuve fish is lll.:lximum, lact.1to concentr,•tion
103
1s lowest. At tho .nonent H is difficult to correlate
the rallil of livJr lactJt·J in th& ,JICCltrtlul .. 3tion of muscle
glyco;,•m since dJta on liver Jl)"co;;~<!Jn have not been
collectl.td.
Circ&Jlan rhtthrn tn the concentr.:•tion of beta te
in the li\l'lU~ t:Lssue of a nu;:Jber of v&rto>.>rctt;:~s has boen
known (.>Llyers;J:lch., 1976 ~ .Jratz and Hutchison.. 1971}.
Ine absence of rhythm in tim livvr bctata in the
present case is surprising. HoW9v~z. thlll acropi'h:uu.t ;MS
cl~teet~d iilt -4~0 (oo .. ·.; h). i.~ •• appl'oxl.llhltely dJBing the
mid-scotophase •
. \lkdline ph:Jsp!utaso is an enzy!A$ which hydrolyses
v ;::bus )hcJSphate t?sters and rela tod comrrJunds (Long. 1971).
\l tluu ;h, in ·n:J:-::::~-lls, seru . .tu allalin<? phosphatase hns be'ln
kno:~:m to under ;o hi ih "lmpli tude circadian rhyth.'3 ( ;>cheving
-:Jt 11., 1')78). in the p:r;;;s:·,nt st.1d·;, a stati:>tically
siJaific·mt circ.Jdian ri<,tho in gill alkaline phosph:lta
CJpious urina. osmotic r.? ; !l-< ti)n is ef: · ct·~-1 throagh
u)t.1ke of ions t.:r Y.!Jh Jills. d1Jhar .. 1ctivi t1 of 'll~oline
phos;>lnt.Jsil, to promot~ actlv-· .;}take of ions from 1mbient
:aediu:n, is the rut-lral corollary.
,\el.J 1:.1 Vc>ly tlUCh hi:Jher activit; of .:tl.-<~line
phos.Jhat.1ae in the pres.~nt C>VO fiahas, ilS comp.lr~ from
104
thaiJ:" ep.io<)e<.m relative::~, (Chapter IV). l~ncts testimony
to incrH·3~,.o m><.1Jtil;: stress in c;.~v" environment and is
suncs·~ive uf c. tooch hi;bar r,:tte of active uptake of ions
to malnta.ln lllfi>.arosnotic ra;ul;d;.i,.n. :'ibsence of a cU:eadian
rhythm in the .1ctivit)' of alkalino phosphabse is again
>~<ltWi.ll in that l:.h<.t f~<;ht~~s are almost ~ontinuously ~nder
osribtic etr~ss in 1;iw cave Gnvirofl!llet\t.
Tho il.1J)Ol:i:.::.ne~ of z;cfitt.ylchoUne-a.cetylchclinestorase
in nouro tl:w•smiosion iS too well known. Circadian rhythlll
in th(• ac,)tylcholinest ·rase a{;:tivity in the brain tissue of
a nur.Jl.Jer of .:m.'i..t:k1ls, viz •• inVJrte.:·.t'at.0s (Pavan KU\llaJZ
et at •• 19'79). liz,>:t'ds (;~ubra(ili)!ny~'ll vt al •• l\#01 J l?aV/lfl
t(umu~ et al •• 1979) ma,::;nals (:,tuy.Jrs -~ch, 1976 ; ;;ichJ.ebaler
and Mayerst:~ach• 1974) has been well documont·ad. In the
pr<J•Jent stuJy also. a:;sumption of zero-at:lplltude hypothesis
for brain ac~tylcholinesterase of cave fishes was r~tjected,
sugjestive of occur:r,mc;, of .:acadian rhvthm.
Ch:>linc5tcr .. \'H) is an t.tnzym· . .: tha:t hydroly,~s ac~tyl
choline to for:.t ~&-~tic "cid .:~nd cnoline (,;Ultinson, 1')7 6).
ln otil()J.; .mi·1 ls ii:. h.~:> b~i~<:n SIJJ..,e; ;;ad that tho ,lmplitud.J
of wGetilCholl.n .:::,ytru is greater in CiJ.::~ th:m in other
ti::;suas (..:u' .:.·· :~c:.ny:n.., e? ;..tl •• l9Ul). Ho;revcr, in t;Jw prasent
study, tho Npli ~Jdo of th~ rhytho in ;Jill is found to btt
greu ter th:1n \..!:: t of tiL· a~.v11 t:J.Jo in the bl:ain. This
definitely su;,•J.:;sts the gr~Z~t.or im.>ortanc• of
acettlCi1olinente:r:d>t in active uptake tiHln in neuro
trans;nission s{sta:.l in this c«ve <iwoJ..U.ng apecies of n.an. /H.:rot)h:~ses .:~£ cir:adbn rhythm in Jill and brain
tissues !.Ui'pt'is:l.rt;ly overl..:l;>pect ;,u\1:1 wll*re .:let.ected dur1ng
tile lc~tc- li ;!'It i:c1ction of the W l2U.2 pboto~dod.
Similar li;nt-im:l.uced incrc..~sa in the acetylcholine ctsterase
enzyr,1e acti •li ty l'k<s baan rap;:>rted for the other <u'limnls
incl•Jding inV'artebrates as well as ~ls (Mayer&bllcn.l976J
~u.bramanyam et al •• 19Sl). Could it be t1·1at the light ind~
response of this enzytne is ~ tllroughout the animal
kingd001 1 Even tissut~f> of !>ighly specialized caw adupted
fishes are not sparad. Pur·thel!' studies on this a$pe<:t
would be rewarding.
Concluding, it is su;t;i<~~sl;ed that cave adapted fishes
retain tne ability of synehroniZd tion by the photoperiod,
whicn is SU0)c;O<Wd to lJe the strongest • • Zei t.Jei>e:t •' of the
environraent. ilovreV·Jr• this is true only for this
troglophilic fish. c~xtension of tnis study in a specialized
troglobite ~vould be revealing in clearing several existing
controversies in the field of chronob1o1ogy.
Ta
ble
17 1
Circad
ian v
aria
tion
s in th
e g
lyco
gen
and lactat~
con
tent o
f tissues o
f cave
fish
, !:!• evezard~
Clock h
ou
r T
iaa
ue
Variab
le (u
nit)
-------"--
-----------
Muscle
09
.00
* a
Glycogen
o.2S±O.O
J (m
g/1
00
mg)
Lactate
88
.JQ±
8.5
0
( ..,g/l.JO m
g)
l3.W
1
7.0
0
2l.u
u
Ol.~J
a-0-~----------
· -·--------------
o.2~v.o3 o.3Q
t0.04 u.32~o.o1
o.J~u.oe
95
. OO
;t;.17. oo 7
8. O
O;t.J.4. oo
66
. 50;t;,5. ·ro e,d
51.00:1:.4.50
o~.oo
o.J4
;t;,0.0
2 c,d
5
3.2
5;,!;.5
.00
Liv
er
Lactate
l8.5
Qt3
.oo
l7
t3.4
l (..,g/100 m
g) l6.0J~2.16
17.5Qt3.77
19.~1.70
20
.50
,tl.26
------------------------·--------------------------------------------
* M
ean ;t;. l
SE
(m
ean valu
e of
fou
r ob
servatio
ns)
a,b
,c,d
Differ from
mean v
alue o
btain
ed a
t 01
.00
, ~.oo,
13
.00
, and 09.00 re
spectiv
ely
: P <
o.o
5 in
each case (based on D
uncan•o mu
ltiple-ran
ge te
st)
Ta
blo
1a
~ ~
""~l"·~i'
r.~ V
' ,•L ·•tC
•' "U
''l'l .·~,{ u
•-.i y:,;,
~
.,..A.
'·•.J..
~4• \01
-.:;J '"
,, ~•
fo:r
stu<.iy
of
t:ioe
i.'oo:;t$ o
n
tiu:;.;ua JlY"~.
and
la
ctJ
to
con
tcJ:J.t o
f c.lV
G
fish
, J.
a'\/'.};!\:U:\li
""'" ·~-"'""'""'''''~
-•·----.,.,. _
__
,.,.._
.. -~ .. -
-·-.., ............. ,
._...,_.,.,,...,--.------...,-"""'---'-~-"---·-"'-'•'"...,.....,..__ _
_ ~,.,._..-,.,.-..,_
I"'''"
f is~t.JO
: ;ri :·')1
&
.:.·y~:rce _;j1jj~)
{) f
, ·~--lun ..
t-" ...
(un
it) o
f n
,u·u~oG
df
;l(jiUJ:e
v
.1ri ·1 ti•.;!n
--------~---· ___ ..... ....._, .... "
-·--·~"""' ---......----·-·--·"'-'· .... ~
"""-·-·-··"""""--""""'"-·'--' Y#~---.. ~ .... •-t~.~<~,.,.,,_ .....
V ...... ~ ~-
... ------·
\ 1!/CO
JOn
T-;>
t:ll (J.~:ti4
2.3 ....
--
\ r.lJ/ .i. j..J[;JJ) .1 th.i:l
'J.l:'OU
fl'\ .~ ~ ;4:;;. lC
:~.· ,1},1.
.. -
!' itne
U.d~~3
5 J.:..; .>6
:J.:}J
<
;. )':) .,u~cle
-· ,_,. ____ ,,,, ........ "'
·---->~<-*$• .,
-~-
·-"·---..,.,.~,-
T ;ta
l 14l·J4.9~~·
23
-
-l..i.l\,; t.n
.a .;i th
in g
rou
p,
15
2:). 1
4
10
4
16
.32
:'
'· . / t . JlllJ) T
i 657~.2:1
5 J.3
1.5
.A
., .... m
e -
-;'3.1.t4
<
' ';','• ,_ .... """
-----
f'()t,il-· ,~,.,...,._.__""'""'-''"'58.3::f3"""".....,
2~ • ...... ,, . .,.
W _
__
_ ,....~--,._...,. _
__
._
_:._
,.
~--"""'""'*"'
w••
Lt.(t..:t.Jt..e
"i th
in gro1..1: "~.o
~;;~~-l." J iS
'''9
"'::! ..
-;..l.v<Jr
",r;. •
..;.,.._
( -J/l <'..J!Jl:J)
Tim
e 5:'•.33
:> ll.u
7
~'·3'17 }
,).,;_.:6
-------------~ ... -
.,,._, "
·-~------
__ ........_... .......... ....,. ,~,.~---"'-'""''"~-
.... ,-... ,, _____________ _
* '
' \..
• •..I ~' ••
Wd.S
p•.:.Cfo.~:uuJ '.m
tiat;l :i .u.u;;t:.rT
t:•d in
Tab
la l7
Ta
ble
19
1
ahy
thm
om
etric su:nmary
basad
on least-squ.:~re fittin
g o
f 24 h cnsin
e fun
ction
(2
4 h •
36
0°) to d:Jta illustr~ted
in T
able 1
7
Tissu
e
Muscle
I.1vo
r
Variab
le (u
nit)
No.
.Vat a lim
it o
ba.lo
w
hig
h
Plla
Glycogen
24
0
.19
o
.39
2
9
(mg/lJU
!lg)
d p
b M
<:t, SE
tt.•
in d
egrees
(9:> Y. \...L
.) 9
0.0
29
0
.3lt.O
o0
l 0
.04
•16(-339.~53)
Lacta
te
24
4
1.0
0
14
0.0
0 43
0.0
03
7J..9t¢'4.00
22
.40
•1
62
(-13
3.-1
91
} ( j.1 <J/ J. .. .:.om
g) ---·-
Lacta
te
24
8
.00
2
6.0
0
13
( llg
/l'JOm
g)
Legend aa in
Table 7
o.21s 1a.2~.oo
2.56 ~a
¢If in h
ou
rs
01
.07
10
.80
03
.20
Tab.la
20
1
Circ
ad
hn
V-JrJ.,:ltio
ns o
f th
o enzym
e activ
ities in
thsu
ee o
f cav
e fisht
l:!• ey
nard
i
Tissu
e
Gill
Brain
Clo
ck n
ou
r v
ariable
(un
it) ------------------------------------
Alk
aline
ph
osp
hatase
(~gpi/mg/hl
09
.00
1
3.0
J
* 2.Q
Q,to.a
2.1~0.2
17
o'J'J
1. 90;t.O
. 7
21
.00
U
lo-!0
os.vo
2.3
0;t_
0.4
2
.2o
.t_J. 7
2.3
0;t.0
.2
----------------------·------,---------------------------------------.-'\cetyl c
ho
line-
a,b
ater3
se l.4
7z0
.2l
( 1-' m
ol
ilCh
/mg
/h)
Acety
lch
olin
e-
a l.01:t;P
.2l
stera
se
2.7~0.12
3.0~0.36
(;.! mol
fC.h/m
g/h)
2.5
t,0.3
2
3.2
;a.0.3
l
2.15:t;.0.09 a
l.~U;!:.0.20 a
l.S'>
.I:;,J.lO
2.~0.04 2.86j;,O
.OS
2
.45
j;0.0
6a
--------------------------------------------------------------------------------* M
ean ;t. l 3E
(m
ean ValiA&
of
fou
r obae.rvationa)
a,b
D
iffer fro
m me~n v
alue o
utain
ed a
t 17
.00
h t
P < o.05.
and P
< 0
.01
resp
ectiv
ely
(b
aa
ed
o
n U
un
can
•a m
ultip
le-rang
e tE
Hi>t)
~
Ta
b!&
2
1
: :\n
aly
sis o
f y,lrian•::<
a sum.:~a.t:"/
for
stud
/ o
f tim
<f
ofi:',Jcts
on
on;&fillll!
activ
ity
in tissu
es o
f cav
e fish,
r.J. ovaz:1rdi
--
--""~" "-·---"~""-''""--~-""
------T
is3u
e
Varia
ule
Gill
dra
in
(un
it)
Alk
ali no p
ho
sp
ha
tase
( tl9
p1
/mg
/h)
Acety
lcho
line-
ate rase
( 1-l
mo
l N
:;.h/mg/h)
:\cety
lch
olin
e
ster3
se
( j.Unol
i'Ch
/mg
/h)
~ource o
f Sum
of
varia
tion
sq
uares
-
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te! 25.31
Ji th
in g
rou
p2
3. 62
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e 1
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df
23 18 ~
--
To tell
5.4
66
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gro
up
s3.o
73
T
ime
2.393
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8
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group>-2.919
Tim
e lo
l89
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18 ~
23
1
8
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uare
... 1
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2
0.3
38
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9
-0.1
65
0.27\3
* ,\JlJV
A
was
perfo
rmed
on
data
il.lu
atra
ted
in T
able 2
0
F
--0.2
58
--2.S
Ol
--l.€::85
p
--> o.os
-.. <
eJ.05
--> o.05c
Tlb
le
22
s
''hl'th::.or;;(.!tJ":ic :,;u., ... j,~J:y
'h:IS
fJci on
.Leaat.sq
uare fittin
(] of 2
4 h
¢o
sioo
fun
etio
n
(24
h =
J(>,P
) to
1-iat<l. illu
stra
ted
in T
able 2
0
TL
HlU
9
Vnria!:>
l.:t !~o.
Data
limit
of
ob
s. (w
olt)
low
hig
h --
-----·------,·~------"----------~---~---··--~--"-
--·------~----·-----~--
--·
--·---·-··--
?r\a
p
b
:_~C.._ ·-
d "'~*-
~';;. ,.e. '"'
tl in
df!g
U<
:Ht
in hours
',~~
' -. ' ,·9
!)-;;;;;. /'~-
._ ........
-~
---·~·~·-........._ __ , .. .,
'-~·-·----~--------------
will
•lkalin
e
O)ho::o;>hutase
(j.~.gpi/mg/h)
24 ,;.1
1
4.1
2
< 1
o.9
84
2.2~J.22
o.a~6
-16
7
11
.13
--···.
u .. '
.... ~~--~--------·------------------------------,..-------~·
.\cety
lch
o.lin
-
aste
raaa
24
l.l.~
3.4~
32 o.o17
l.934J:.0.09 o.3
v2
-2
86
(-25
1.-3
21
) l9
.u7
( j.U
OO
l ,:.,::,hfm
g/h)
--~-----------------------------·· ·---------------..
....,, .. _______________ _ i\cety
lcho
11
n-
Urain
este
rase
24
2.3
0
4.0
8
27 u.03a
2.90J.t::>,.oo o.3
l2
-21
l(-23
0,-3
l2)
1S.o7
(tUD
Ol
.>C
h/m
g/h
)
...... .-------------------------------------------~~~
~M ........... ._
........ ~ .. ~~------~--~------
.. ----------------------~--
.. --------------------
~egendsas
in T
able 7
20 Of-----0 MUSCLE LACTATE
I I LIVER LACTATE
o------o MUSCLE GLYCOGEN
so
~0
• • • • • 0
LIGHT DARK
LD 12:121 ~I 0900 1300 1700 2100 0100 0500 0900
CLOCK HOUR
0 !"'l
1- X: :I: 0:: <!) < ....I 0
D~
tn 0 ..... 0
( 4/U!a~oJd 5wtpasoa)eJ !d 6rr')
AllflllJV 35\flVHdSOHd 3Nil\f)!W
0 0 ll'l 0
0 0 ..... 0
0 0 ..... N
0 0 c--.....
0 t---10
(f) .....
0 0 Ol
N 0 ..... N ,....
0 .J
0:: :::l 0 :I: X: u 0 ...J u
.-.. .s:. -c: 4 0 0 BRAIN Ql .... I I GILL 0 ... a. 01 E -"0 Ill Ill >- 3 -0 ..
"0 >-.s:.
.s:. ~ -0 E :t. ..... 2 > 1-
> i= ~ w .s:. D ~DARK u LIGHT < 0
LD 12:12 ~~I 0900 1300 1700 2100 0100 0500 0900
CLOCK HOUR
51 NGLE COS I NOR
-
SINGLE COSINOR