Effect of n-propyl thiouracil on muscle

Effect of N-Propyl

Thiouracil

on Muscle

Itrgestioll of a 0.03’:; solut ioll of rb-propyl thiorlracil car~sed the development of :I hypothyroidic condition itr rabbits. Colrcrlrrelrt with this devrlopmellt. champs were observed itr the testlve a11t1 properties of skeletal mllscle. I.Xectrotl microscopy indicated a definite degenerative rhallge ill the myofibrillxr organixatioll of I hr back mr&e. The etleymat ic activity of the cotlt rart ilr protcilr, rn!-ositr B, isolated from the back mnscle of the rabbits was altered by the n-propyl thioLlraci1 treatmellt. The changes in enxymatir activity which occ,Ir were most readily see,, OII the addition of ati enzymatic activity activator sr~rh as et hylcllediamille tetraarct ate at high iollio strength or magnesitnn rhloride at, low ionic strel&h. Both act ivnt ors increased thta enzymatic activity of the myyosill B from the n-propyl thiorn-xi1 treated animals to :I greater estellt thatl the mgosill B from the court rol atlimals. The maximum vclority of the ellzymat ic reactioll derived from Alirhaelis Alenton kilxtics for the myosilt B from the rabbits treated with the goitrogell was greater thall the valises obtained with t hc control animals. The absolllt e valrtrs of the adetlositle triphosphatase :rctivitJ- of t hex myosilt B decreased with age hlrt t hc relative differences bctweell the myosin B from 1hc relit ro1 and n-propyl thiollracil t rcated mlimals remailled rollstan

The goitrogenic action of the thioumcil derivatives has been recognized for many years (1). The biosynthesis of the thyroid hormones is altered by wpropyl t’hiouracil (2). The major effect of the 1’TU’ on the t,hyroid is to inhibit the formation of diiodotvrosine from monoiodotyrosine. This inhibit,ion results in decreased concentrations of both triiodothyroninc and thyroxine. Both the tot,:rl iodine upt~ake by the thyroid and the uptakc of org:micall~ bound lodint: h\ the thyroid are dccreused on administr:~tion of I’TL. In addit,ion to changes in th\-roid hormone levels, t8hcrc is :L charxteriatic change in t’hc thyroid tissue -\\-hich is wadily qxwnt on histological cx:rmiwt,ion (3). Fl~pcrplasia is itltluccd :md both the Lvctight oi the thyroid and the h~~pcrplastic changes incrcwe during the course of the PTU trcntment. kkuninatiou of t,he thyroid tissue sho\vs increase in cell height, formation of papillac~, disappwr:Lnce of stainable colloid and distension of

blood vessels. Changes in the weight of the heart parallel weight changes of t,he t,hyroid. Large increases in the weight of t#hc heart (relative to cotkrol anim:ds) have been reported. In many cases myoardial changes ure :dso observed. In these C:LSCSfoci of cnlcificntion xith necrotic muscle fibers in thcsc areas have been show1 in the mgocardium. The structural changes in the myocardium have bee11 at8tribu~td to h\rpothyroidism. Changes in b&h heart and sltelet:d muscle :a: common second:q\- effects of chronic hypothyroidism (1). I II gcnclal muscle mtl ~w~l;ness occur. The firm tl\-stro~,Il~ normal tcst~urc of skeletal muscle is lost and tllc tissues appear spotqq~ or “mudh\‘.” Sccrosis of the ni\wcle libw in loc~:~liaetl ;wc;w has been obserwtl. Tlw niyolibrill:tr org;miz:lt~ion is intcrruptc4 ill t’hcse areas (-7). (‘ross AriaLion is dccrc~wc!l and fragment~:Aion of t,hc niyotibrils \vith ni:~rlif:~l ac~iitn~~l:~tioii of glycogen has :rlso lwcn Ob-

32s

JACOBPOE

AN11 STAPLETON

served. Nuclear and mitochondrial changes accompanied the myofibrillar disruption. Changes in the properties of muscle on administration of PTU are to be expected since such changes are common in chronic hypothyroidism. However the effects of 1’TU treatment on skeletal muscle have not been examined. Such induced effects will be similar to those observed with chronic hypothyroidism only if the changes are due soleI! to the disturbance of the thyroid function. If any additional changes in metabolism are induced by PTU, or if there is any direct effect of the I’TU on the muscle itself, the changes in muscle caused by PTU administration may not directly parallel the changes produced in cases of chronic hypothyroidism. In this work n-propyl thiouracil has been administered to rabbits and the skeletal muscle tissue has been examined to determine t’he changes caused by this treatment. The enzymatic activity of the major contractile protein myosin B extracted from normal muscle and from muscle tissue of animals treated with PTU has been compared in order to determine if there is an) difference in the behavior of the isolated contractile protein unit. X4TERIALS

ANI)

METHODS

A solution of 0.03’;‘,, n-propyl thiouracil was used as the drinking water of New Zealand white rabbits for various time periods ranging from 7 weeks to 5 months. Control animals of the same age were paired with 1he n-propyl thiortracil treated animals and maintained under identical condit)ious with ordinary t)ap water. Both a control and a PTU-treated animal were killed at, the same t,ime, and the back mttscles were excised immediately. Samples for electron microscopy were immediat,ely fixed with glutaraldehyde and postfixed with osmiltm t,etraoxide. Sections of silver or gold color were stained with lead citrate and uranyl acetate. Parallel preparations of mgosin B from control and PTU treated animals were made immediately aft,er sacrifice. The myosin B was prepared by the technique previously reported (F). In the final precipitation the muscle protein was washed with water as suggested by Perry (7) to remove soluble proteins. In t,he centrifngation of the myosin B solutions, ottly the central portion was retained. The myosin B was st,ored in 0.5 M KC1 at 2’ with the pH adjusted in the range of 0.8-7.0. The COW centration of t.he stock solution WRY kept, between

2-3 mg/ml proteitl. The protein concentration was measnred by the micro Kjehdahl technique and the fact,or 6.25 was used to convert the nitrogen concettt,ratiott to protein concentration. The thyroid was removed from the rabbits within 20 minutes of sacrifice. The t,issue was placed in Ringers solution and later fixed for histological examittatiott. A ttertt,ral formal-saline solution was used for fixing. The fixed t.isstte was stored in 7O’,Y;ethanol. The adenositte t.riphosphatase (ATPase) atativity of the myositt B was measured by a method which was adapted from a technique suggested by Lecocq attd Tnesi (8). The method employed here was identical with the exception that trichloroacet,ic acid rather than perchloric acid was used as the prot,eitt precipitant. This change elimittat’ed precipitation of potassittm perchlorate and ettabled the met,hod to be used wit,h solutions over a range of potassittm chloride concetttrat)iotts. This mel.hod was standardized by measuremettt of standard phosphat,e samples. The myositt B ATPase2 act,ivity obtained by this method was compared to results obtained ott the same protein soltttions by the Fiske-SubbaRow method (9). Both methods yielded identical resttlt,s. A Beckman l>BG spertrophotometer was ttsed in both methods. All ATPase activity measttrements were at 25” in 0.02 M Tris buffer at pH 7.1. The activity was measured within 1 week of preparation. The ATPase activities of a sample from the control and a sample from the PTU treated animal were measttred always in the same experimental sequence ttnder identical conditions to eliminate as far as possible aging effects in the myosin B. Comparisons have been made only from measurements in the same experimental sequence. Dottblc distilled water was used throughout. All salts were of reagent grade quality. Sigma Chemical Co. crysfallitte grade disodium ATP from eqrtine muscle attd Ntttritiottal Biochemical n-propyl thiortracil were used. RESULTS

An example of the histological changes observed in the thyroid is given in Fig. 1. The upper part of this figure shows the thyroid tissue from the control at 10 (la) and 50 (lb) times magnification, while the lower part of this figure shows the tissue from the PTU-treated animal at similar magnification. The thyroid tissue from the PTU-treated animal shows the characteristic degenerated changes expected from PTU treatment. A characteristic hpper2 ATP

= Adenosine

t,riphosphate.

pl2lsia is apparent lvith increase in cell height, dis appearance of st:tin:Lhlc colloid, formation and dist’ension of blood vessels. Of papillae Th e thyroid tissue shown here is from an nni ma1 treated with L’TU for 120 days. In all

wscs examined varying from 52 to 233 d: LVS of 1’TU trextmcnt~ :I degenerate change in the t,hyroid IV:IS apparent. The degree of degeneracy incrwsed with t,hc length of I:hc I’TIT treat mcnt

330

JACOBSON

Gramsmuscle No.

days

PTU

52 65 98 120 195 233

big recovered g muscle

~~~ Control

147 156 111 293 121 296

PTU

113 110 130 2H 132 201

Control

-4.2 7.5 1.8 3.4 2.0 3.7

ANI)

per Difference in recovery (controlPTU) mg recov~$&er g PTU

3 .5 0.0 3.7 3 :1 0 .9 2.7

0 .7 1.5 1.1 0.1 1.1 1.0

The duration of the 1’TU treatment, the n-eight of the muscle and the recovery of the mgosin B are given in Table I. The texture of the back muscle as it n-as excised from the animals was visibly different after YS days of PTU treatment. Before this time the texture of the whole muscle appeared normal to a visual examination even though definite degenerated changes were observed in the thyroid tissue. In all samples excised after 9S days of the PTU administration the muscle was spongy and the tissue mushy. In no case XE it difficult to excise the back muscle of the control cleanly in one piece. HoLvever in the animals which had prolonged treatment with PTU the tissue tended to pull apart and it was much more difficult to completely excise the whole muscle. As indicated in Table I, the total weight of the back muscle from the control \Iras greater than from the back muscle of the animals treated with PTU with the exception of the animal pair in which the I’TU treatment was for 193 days. However there does not appear t’o be any definite trend in this data and there is no significant increase in difference as the 1’TU treatment was contimled for longer periods. This difference may be ascribed to individual variation in the animals. The percentage of recovery of the myosin B from the cont,rol was in all cases greater than from the animals treat’ed with I’TU. If the values were averaged lvithin each series t,he mean vnlues (4.3 for the control :md 3.3 for the I’TU t’rcated) would not bc significantI\ diff’erent since the stand;wd deviation of the means are 13 and 1.6. Ho\\-ever :IS seen in the

STAPLISTON

last column of Table I, if only the individual pair are considered in which all experimental conditions of preparat’ion (such as reprecipitxtion t’ime, stirring time to dissolve precipitate, etc.) were identical the yield from the control is higher than from the PTU treated. The mean value for this difference is 0.9 + 0.5 standard deviation. This latter observation can only be used as an indication of a trend since no attempt was made to maximize yields of myosin B. Such an attempt would have required a sacrifice in the purity of the protein preparation. Electron microscopy indicated that most of the ultrastructure of the muscle tissue from the PTU-treated animal was normal and identical to the control sample. However there were definite areas in the muscle tissue from the I’TU-treated animal which \vere abnormal. In these areas the myofibrillar structure is disorganized. An example taken from such a11 area (21,000 magnification) is shown in Fig. 2. The distance between myofibrils increases, cross striation is lost and the regular arrangement usually seen in muscle tissue is not observed. -\Iost of the mitochondria in the sections from the PTU-treated animal examined appeared normal. There were some cases in \\-hi& the mitochondria appeared to have some dense inclusions. However, no rearrangement’ of the mitochondrial cristae into urlusual configurations was observed in the sections studied. In a few areas central migration of the nuclei \vas observed, but the nucleus in these areas appeared normal. ?\lost of the nuclei contained prominent nucleoli. T,ipofuscin granules and lipid droplets were observed. In Fig. 3 the =\Tl’asc activity of myosin B in 0.5 x potassium chloride solution on addition of 1.O rnhr ATI’ is shown as :I function of protein concentration. No measurable difference in ATl’asc activity is observed between the t\vo myosin E samples when the protein concentration is less than 0.2 mg/ml. At higher protein concentrations the ATI’ase activity of the control myosin B is about 0.5 pmoles Pi3/g/‘sec greater than the ~ZTl’:wc activity of the myosin B extracted from the PTU-treated animal.

332

JACOBSON

AND

STAPLETON

function of ATP concentration. The data for two different rabbit pairs is shown in this figure and the experimental values for the second pair is indicated by the primes (‘). In 0.4 M potassium chloride solution there is no difference between the control myosin B and the myosin B from the PTU treated rabbits. In the presence of 1 ml\r EDTA the ATPase 2.5

2.0

Y

o.ol/ 0

: 0.1

0.2 mg / ml

03 MYOSIN

0.4

EDTA

1.5

0.5

I3

FIG. 3. Activit,y as a funct,ion of myositl B concentration. 0.50 M KCl, 1.0 rn~ ATP, pH 7.1, 25”, 0, rontrol; +, PTU-treated allimal.

In Fig. 4 the ATI’ase activity of myosin B is shown as a function of potassium chloride concentration. In these experiments the protein concentration was kept constant and the ATP concentration fixed at 0.5 rnl,r. Since the protein concentration \vas slightly less than 0.2 mg/ml, the ATPase activity measured should be less than the maximum value obtainable. There is very little difference between the ATPase activity of the two myosin B samples over the whole range of potassium chloride concentrations (0.060.50 M). The difference in enzymatic activity between the two myosin B samples is less than 0.1 pmoles Pi/g/set which is within the limits of experimental error of the determinations. The normal activation effect of EDTA(10)4 is seen in Fig. 4 for the control myosyl B. However in the presence of 1 rnM EDT& the activation effect on the myosin B isolated from the PTU-treated animals is considerably greater than the control. The ATPase activitv of the myosin B from the PTU-keated animals is 1.1, O.S, 0.4, and 0.4 pmoles Pi/g/set greater than the control values in 0.24, 0.30, 0.40, and 0.50 M potassium chloride solutions, respectively. In Fig. 5 the ATPase activity of myosin B with fixed protein concentration in 0.4 M potassium chloride solution is shown as a 4 k;l)TA

=

ethylene

diamine

tetraacetate.

0.0

I

o-

I 0.2

0.1

I 04

I 0.3

1 0.5

E’IG. 4. Activit,y as a function of potassirml chloride concentration. 0.5 mM ATP, 0.19 mg/ml control, 0.18 mg/ml PTU-treated animal, pH 7.4, 25”, 0, control; +, PTU; 0, control with 1 rnM EIITA; X, PTU with 1 mM ElIT.4.

x /’

PTU E DTA

I

0.0

0

I 0.2

I 0.4

I 0.6 mM

I 0.6

I 1.0

I 1.2

i 1.4

ATP

FIG. 5. Activity as a function of ATP conceIltration. 0.40 M KCl, pH 7.4, 25”, 0.24 mg/ml control, 0.21 mg/ml PTU treated, 0, control; +, PTU treated; 0, control with 1 mEvl ETITA; X, PTU treated with 1 mnx EDTA. The primes indicate data from a second rabbit Dair.

EFFECT

OF S-PROPTL

TIIIOUI:ACIT,

activity of the myosin B from the I’TUtreated animals is greater by about 0.5 pmoles P;/‘g/sec than the ATPase activity of the control myosin B. The AT&se activity of myosin B decreases on storage of the myosin B even under optimal conditions. It is well lmonn that there is an initial large decrease followed by a slo\\-cr decrease in the activity. Hence for comparison of samples of myosin B it is necessary to compare samples of approximately the same age. In Table II, the ATPase :tct,ivity of myosin B from various pairs of animals with increasing duration of treatment of PTU is shown for samples measured in 0.4 M KC1 and 0.5 m&l ATP. All measurements were taken 2 days after the final protein centrifugation. The data is an uvernge of t\vo points with deviation less than +.05 in the absence of EDTA and a deviation less than f.11 for measurements in t’he presence of EDTA. There is no significant’ difference bet’neen the myosin B from the control and the PTU treated animals in the absence of EDTA. In the presence of 1 rnM EDT.4 there is an increase of 0.32 pmoles Pilg/sec in the mean of ATl’ase activity of the I’TU treated compared to the control. This increase is greater than ca11 be accounted for by the standard deviation. In O.OG I\I potassium chloride solution (l:ig. 1) there is IW significant difference betwzen t,he ATl’ane activity of the t\\-o myosin B samples after addit,ion of 0.5 mnf ATP n-hen t,he protein concentration is in range of

Control

I

65 i 98 120 195 233

.2-k .2-I .20 l!) .18 .2“

,\Iean

Activity

52

~

.21 .21 .20 .18 .22 19

-

:ontro1 PTU

+lms EDT.4

0.7i

0.83 0 .03 0.50 0.60 0.76 0.90

0.18 0.40 0.80 0.88

0.70 3x.15 81)

0.65 *. 10 s 1)

1.2i 1.13 1.01 1.10 1.25 1.15

0.50

-

f

1.25 17 SD

:3x3

ON JlUSCLI;

0.19-0.18 mg/ml. As sho1v-n in E’ig. 6, with similar conditions to those given above except for the presence of 5.0 m&r magnesium chloride, the activity of the myosin B from PTG treated animals is greater than from control animals. The data reported are from two different rabbit pairs with 52 days of PTU treatment and n-ith 120 days of I’TU treatment. The differences observed in the presence of magnesium ions at low ionic strength is further illustrated in Fig. 7. This data is from one rabbit pair in 0.03 M potassium chloride solution lvith 0.5 rnlr magnesium chloride added. The protein concentration \vas kept constant at 0.06 mg/ml. The top curve marked I’TU 1 (-- .--) is the ATl’ase activitv of myosin B from a PTU treated rabbit -in \\hich the activity was measured immediately aft’er the preparation (O-l da! storage). The next curve marked Cl (- -) is the activity measured for the myosin B from the control animal in the same experimental sequence as the PTU treated (O-l day storage). The third curve marked PTU 2 (- -, with data indicated by +) is the ATPase activity of the same myosin B from the I’TUtreated animal measured after -1-S days of storage at’ 2” in 0.5 31 pot8assium chloride solution (pH 6.S). The bottom curve marked , with data indicated by 0) it; the C2(activity of the same control mvosin B after 4-5 tllys of storage. I:or both fresh :md

PTU +1mv EDT.4

1.73 1.57 1.63 1.88 1.92 1.89

oo-

0

01

02 mg/ml

03

04 MYOSIN

05 B

FIG. 6. Activity as a frmction of myosin cetltratioll. 0.06 M KCl, 5.0 IllM MgCls, 25”, Rabbit 1, 52 days of PTU treatment, 2, 120 days of PTU treatment. 0, control pair 1; +, PTU treated rabbit pair 1; 0, r&hit pair 2; X, PTU-treated rabbit,

B conpH 7.4, Rabbit rabbit control pair 2.

334

JACOBSON

ANI>

stored myosin B at low ionic strength the ATPase activity of the myosin B from the I’TU-treated animal is greater than from the control animal in the presence of 0.5 rnA#r magnesium chloride. The usual decrease in ATPase activity of the myosin H occurs on storage but the relative difference betlveen the two myosin B samples remains. In Fig. S the ATl’ase activity of the myosin B samples for a different rabbit pair (296 days PTU) is shown as :L function of ATP concentration. The measurements were made with similar conditions as reported in Fig. 7 (0.03 nr KCl, O.fiO rnM l\IgCl,), however the protein concentration has been increased to 0.197 mg/ml for the control and 0.122 mg/ml for the PTU sample. The ATPase activity of the myosin B from the PTUtreated animals is higher than the ATI’ase activity of the myosin B from the control sample. Since the ATPase activity of myosin B is concentration dependent, I\Iichaelis lIenten kinetics are not wholly applicable. The maximum velocity of the ATI’ase activity of the myosin B (vlnnx) has been calculated in 0.03 11 potassium chloride solution in presence of

_.-.

6.0

-‘-

PTU (1)

/’

0

I 0.2

I

I

I

0.4

0.6

0.6

mM

mM

ATP

FIG. 8. Activity as a fllnction of ATP concentratioll. 0.03 M KCl, 0.5 my 1LlgC12, pH 7.4, 25”, 0, 0.197 mg/ml control; f, 0.122 mg/ml PTU treated.

0.5 ml1 magnesium chloride with a fixed protein concentration (data from Figs. 7 and 8) from a modified Michaelis :\lenten plot (S/V vs. 8). The value of vlllnx calculated varied with the age of the myosin B and with the protein concentration. However the vlrraxvalues for the control myosin B are in the range of (i-10 pmoles Pi/g/set while the values for the myosin B which had been extracted from the PTU-treated animals are in the range 11-16 pmoles Pi/g/set. DISCUSSION

/’

0.0

STAPLETON

I 1.0

ATP

FIG. 7. Bctivity as a function of STP concentration. 0.03 M KCl, 0.5 rn~ MgCls, 0.06 mg/ml myosill B. ~ --, cotltrol measrwed O-1 day after preparation of the myosin B; -. -, PTU-treated measllred O-1 day aft)er preparation of the myosin B; 0, -control measured after i-5 days storage at 2”; +, - - - PTU-treated measured 4-5 days after storage at 2”.

The histological changes shown in Fig. 1 are in agreement wit’h reports of previous workers (3) which indicate t,hst administw t,ion of ,I-propyl thiouracil in drinking water causes characteristic thyroid changes similar t.o naturally occurring hypothyroidism. After prolonged treatment n-&h PTU the texture of the back muscle is similar t’o that previously report’ed in naturally occurring hypothyroidism. There appears to bc no question that the administration of wpropyl thiourncil in the drinking water of rabbits induces thyroid changes similar to naturally occurring hypothyroidism. The changes in ultrastructure of the back muscle induced by. the PTU t’rentment are similar to those mduced by chronic hypothyroidism (?i). In both cases most of the muscle appears normal and thcrc exist scattered arcas in which the myofibrillar organization is disrupted. In both cases

glycogen nuclear migration, granules, ionic strength increase the ATl’ase activity lipofuscin bodies and lipid droplets are of the myosin B from the PTU-treated rabbit readily observable in the muscle tissue. In to a greater extent than the control myosin our sections of muscle from the PTU-treated B. The differences in enzymatic activity ma? animal \VC did not) observe the radical be due to changes in t’hc individual muscle changes in mit,ochondri:t noted in the sec- proteins I\-hich form myosin B (myosin A t’ions from t#he hypothyroid human biopsy. and actin) or t’o a change in the mvosin This difl’crence may reflect a fundamental L&actitt junction. In the latter case “it is dif?crence bet,\vecn I’TU treatment and possible that there mum\’be no me~surablc chronic lt>.pothyroidism but may be due to changes in the properties of the isolated ittdifference in degree of hypothyroidism and dividunl muscle proteins. Such an cfYect length of I’TIJ trcntment (1 months) com- could be c:~useclby a differettce in t’he nutnbet I)nred to duration of hypothyroidism in the of tnyosin -4 units bound to actin to form lnwious report (2 years). myostn 73. This t’ype of diffcrcnce has been The ch:mges induced in the muscle tissue suggested to occur in myosin H isolated from and the myosin H by prolonged treatment dy&rophic animals (13). This type of difiercnce could also be used t’o account, for the \vith I’TU may be due to t’he induced hypothyroidism. It is possible that other difference in relative recovery From \vholc metabolic ch:anges occur on admittistrntiott of mwclc and from isolated myofihrils of 1’TC which in turn also :ifYect the mtwlc, or vitamin Ii:-dcficicnt :tnim:ds. there may be ;t direct effect of the I’TU on t’he muscle. It is impossible at, this time t,o di&ngnish bet~wcn t,hese possibilities. However the myofibrillar ch:mgcs in muscle induced b\, I’TU :tdministr:ttion appear to p:wallel the cliwtges occurring in hyqwthyroidism in humans and animals. The rccover\~ of m\-osin l-3 from XVhole muscle tissue from r&t&s treated I\-ith 1’TU 1. :rppwrs to be less than from the control :tnimals. It is possible that this difference is 2. due t,o the \v:Ltery tinture of the muscle tissue ;tf’tcr 1’V.T :~tlminist~r:~tioti. This effect is .3. similar to results reportotl for I\-hole muscle from vitamin l~;Sdcficic~tttrabbits (11). HOD4. cvcr thk result c:umot, be applied to 5. determine the m\win 13 content’ of isolatcil tnyofibrils. In the case of vitamin 11;deficient 6. r:tbbit,s t,lw twovcry of n~yositi I3 from isol:~tctl myofibrils is greater than normal 7. (12) \vhile the rccovcr~ from \\-hole muscle is IM tBll:ul IloYnxrl (11 1. 8. IMinitc tliffercnccs in ettz)mat’ic :tctivit\ have been shown bct’\veett IlcJrXKll mywin H !I. and niyositi l< ert>r:tct,ed from rabbits which have been niaint:~ined for a prolonged period IO. \vith I’T1’ :~dtled to their drinl;ing water. 11. 11tiy cl~igcs obsorvcd appear to matketlly del~ntl on the experimental conditions of t’hc 12. tne:tsuretii(~nt . L1dditioti of an ctizym:tt,ic activity nctiwtor such as I+XWA at high ionic st8rength and magnesium chloride at low