Skip to main content

Full text of "DTIC AD0030251: GLYCEROL PECTATE AS A BLOOD PLASMA REPLACEMENT"

See other formats


UNCLASSIFIED 




AD NUMBER 

AD030251 

NEW LIMITATION CHANGE 
TO 

Approved for public release, distribution 
unlimited 



FROM 

Distribution authorized to U.S. Gov't, 
agencies and their contractors; 
Administrative/Operational Use; JAN 1954. 
Other requests shall be referred to Office 
of Naval Research, Arlington, VA 22203. 



AUTHORITY 

13 Sep 1977, ST-A per ONR ltr 



THIS PAGE IS UNCLASSIFIED 














Because of our limited supply, you are requested to return this copy WHEN IT HAS SERVED 
YOUR PURPOSE so that it may be made available to other requesters. Your cooreration 
will be appreciated. w 




NOTICE: WHEN GOVERNMENT OR OTHER DRAWINGS, SPECIFICATIONS OR OTHER DATA 
ARE USED FOR ANY PURPOSE OTHER THAN IN CONNECTION WITH A DEFINITELY RELATED 
GOVERNMENT PROCUREMENT OPERATION, THE U. S. GOVERNMENT THEREBY INCURS 
NO RESPONSIBILITY, NOR ANY OBLIGATION WHATSOEVER; AND THE FACT THAT THE 
GOVERNMENT MAY HAVE FORMULATED, FURNISHED, OR IN ANY WAY SUPPLIED THE 
SAID DRAWINGS, SPECIFICATIONS, OR OTHER DATA IS NOT TO BE REGARDED BY 
IMPLICATION OR OTHERWISE AS IN ANY MANNER LICENSING THE HOLDER OR ANY OTHER 
PERSON OR CORPORATION, OR CONVEYING ANY RIGHTS OR PERMISSION TO MANUFACTURE, 
USE OR SELL ANY PATENTED INVENTION THAT 'MAY IN ANY WAY BE RELATED THERETO. 

Reproduced by 

DOCUMENT SERVICE CENTER 

KNOTT BUILDING, DAYTON, 2,-0 H 1 0 



A 





BURKE RESEARCH COMPANY 

11281 East Nine mile Road 
VAN DYKE, MICHIGAN 



ANNUAL PROGRESS REPORT 
Including Technical Report For January 195 2 * 

TO 



OFFICE OF NAVAL RESEARCH 
UNITED STATES DEPARTMENT OF THE NAVY 

BIOLOGICAL SCIENCES DIVISION 
CLINICAL BRANCH 

LT. COMMANDER WILLIAM J. PERRY, HEAD 



FROM 



BURKE RESEARCH COMPANY 



O.N.R. RESEARCH CONTRACT NONR - 86 0 ( 00 ) -NR -102-008 
''Glycerol Pectate as a Blood Plasma Replacement" 



REPORT NO. 21 

For Period: May 5, 1952 to January 31, 195^ 



URS F. NAGER 




Glycerol Pectate as a Blood Plasma Replacement 



Summary 

Among the naturally occurring hydrophilic colloids, those of cit- 
rus peel have been investigated for use as a blood plasma replacement. 
According to earlier biological studies, degraded citrus pectin, a 
partial methyl ester of polygalacturonic acid (60 to 70$ carboxyl groups 
methylated), has shown suitable oncotic properties and a low degree of 
toxicity. However, the so-called pectin sols have not gained the gen- 
eral acceptance of the medical profession because of certain short- 
comings which may have been due to the high percentage of free carboxyl 
groups responsible- for the precipitation of calcium pectinate. 

Attempts have thus been made to prepare suitable derivatives of 
pectic acid (product used having between 10 to 20$ of the carboxyl 
groups methylated) that would no longer precipitate with calcium ions. 
With the intention of blocking the free carboxyl groups of pectic acid, 
by esterification with a hydrophilic alcohol, propylene glycol pectate 
has been prepared. Biological studies on preparations esterified be- 
yond the point where calcium precipitation was possible have shown best 
results with propylene glycol pectate samples containing about 15 $ re- 
sidual unesterified carboxyl groups, but certain toxic symptoms attrib- 
utable to a propylene glycol ester were encountered in these studies. 

In comparative biological studies, glycerol pectate has proven to 
be an effective blood plasma replacement free of toxic side reactions. 

Its preparation has been accomplished by esterification of a commercial 
pectic acid with glycidol. In studies on artificially hemorrhaged dogs, 
effective restoration and maintenance of plasma volume and blood pressure 
have been obtained with 4$ solutions of glycerol pectate in saline at a 
relative viscosity of around 4 and at an esterification degree of 85$. 

In vitro studies have demonstrated that the oncotic properties of gly- 
cerol pectate are primarily due to the unesterified carboxyl groups. 
Hydration and/or charge effects on these carboxyl groups appear to con- 
tribute to the molecular configuration; the number average molecular 
weight seems to be considerably below that of plasma. 

Introduction 



Ever since the last war there has been a tremendous effort placed 
on the development of a satisfactory artificsl plasma replacement fluid 
for the treatment of hemorrhage, shock, burns and other conditions. Al- 
though adequate amounts of human blood plasma, have been available for 
normal needs it became obvious that these supplies would never be suf- 
ficient to meet the requirements of the armed forces and the civilian 
population in case of a major catastrophe. In spite of numerous attempts 
only a few synthetic preparations are available today which approach the 
requirements of a satisfactory plasma replacement fluid, the two of most 
importance being dextran and polyvinyl pyrrolidone (PVP). Neither human 
plasma nor the just mentioned most promising synthetic plasma, replacers 
have yet reached the stage where they could be used universally without 
any ill side effect. 




- 2 - 



Since relatively few compounds have so far been investigated as 
synthetic plasma replacers, very little is known with regard to the pre- 
ferred class of compounds that will yield the most satisfactory material . 
However, present knowledge indicates that a polysaccharide-type compound 
may be superior to a protein- type or strictly synthetic compound. It is 
only logical to assume that besides dextran a variety of other materials 
exist in nature useful as plasma replacers. The objective of this pro- 
ject was, therefore, to search in the group of naturally occurring hy- 
drophilic colloids for such a material. Of particular interest appeared 
to us the plant colloids among which notably those of citrus peel (pec- 
tin), cactus and plantago are available in unlimited supply. Due to 
time limitations placed on the extent of our research, we had to restrict 
our studies to one material. Pectin was selected since it already had 
shown distinct plasma replacement properties, was known to be non-toxic 
and did not accumulate in the body tissues. However, in the manner in 
which pectin had been heretofore prepared and used, pectin had never 
gained the general acceptance of the medical profession. 



Earlier Studies on Pectin Sols 

Pectin solutions introduced by Hartman et al 1 , subsequently also 
referred to as pectin sols^, have been prepared from commercial Pectin 
N.F. by thermal degradation of a 1.5$ solution to a relative viscosity 
of about 4. Such prepared pectins represented a partial methyl ester of 
polygalacturonic acid with about 50-40$ free carboxyl groups as sodium 
salts . 



The affinity of the unesterified carboxyl groups of pectin towards 
calcium, other alkaline earth and heavy metal ions is well known. In 
fact, pectin is made use of, for example, in the jelly manufacturing as 
a thickening agent or in medicine as a detoxifying agent, in heavy metal 
poisoning. 



The percentage of free carboxyl groups of these pectin sols was in 
the range where precipitation with calcium ions had to be expected. 
Furthermore , it would be anticipated that the great number of free acid 
groups present in such a polymer produces a distinct cation- exchange be- 
havior with selective affinity for cations according to their size and 
charge. For example, such a polymer may bind calcium more strongly than 
sodium. It appeared obvious that pectin sols when introduced as the 
sodium salt into the circulatory system would react, by double decomposi- 
tion, with other salts such as those of multivalent ions; such reaction* 
in turn may seriously affect the electrolyte balance. Recent animal 
studies made at the Henry Ford Hospital on salts of polygalacturonic 
acid, having about. 25$ of its carboxyl groups esterified with methanol, 
caused extensive sludging in the circulatory system followed by immediate 
death. 



^.W. Hartman et al., Ann. Surg. 114, 212 (1941); J.A.M.A. 118, 

? ll6l (1942), 121, 1157 (1945) 

G.H. Joseph, Pectin Sols for Parenteral Use. Review publ. by 
California Fruit Growers Exchange, Ontario, California. (1950) 






- 3 - 



The original concept in the present program, was to block off the 
free carboxyl groups of pectin to prevent precipitation of calcium 
pectate or pectinate and to eliminate the chances of deposition of such 
salts in the tissue. The plan was to investigate completely esterlfied 
derivatives of pectin. Essentially two groups of highly esterifled de- 
rivatives of polygalacturonic acid are known: (a) alkyl polygalactur- 
onates, e.g., methyl polygalacturonate obtained by reacting polygal- 
acturonic acid or its naturally occurring partial methyl ester (pectin) 
with diazomethane at low temperatures and (b) hydroxy alkyl pectates, 
e.g., hydroxyethyl polygalacturonate prepared by reacting polygalact- 
uronic acid with ethylene oxide in the presence of water. Of these two 
groups comprising derivatives of the carboxyl groups the hydroxy alkyl 
esters were preferred because of solubility considerations; the ultimate 
aim was the preparation and testing of glycerol pectate (glycerol poly- 
galacturonate ) . Glycerol pectate promised to be the derivative of choice 
because of the extremely low toxicity and the possibility of metabolism 
of glycerol if it should be liberated in the body by initial metabolism 
of the infused pectate ester. Since but little information was avail- 
able on the preparation of glycerol pectate, and since glycidol, the 
esterifying agent required for its preparation, was difficult to make, 
it was first attempted to prepare the propylene glycol pectate to de- 
velop a practical preparative method and to verify our working hypothesis. 



Studies on Propylene Glycol Pectate 

Although original attempts were made to prepare our own poly- 
galacturonic acid from citrus peel, for economical reasons resort was 
made to commercially available pectin preparations. Among these Pectic 
Acid No. 75^ and Pectin N.F.3 proved to be most satisfactory starting 
materials available in unlimited quantities. Pectic Acid No. 75 was 
found to have 80 rfc free carboxyl groups, the rest being esterifled by 
methyl groups. Following the general principle proposed by Deuel** for 
esterification with ethylene oxide, Pectic Acid No. 75 was esterlfied 
with propylene oxide in the presence of water. No matter what variations 
of the reaction conditions were made, a considerable excess of propylene 
oxide (at least 9 times the theoretical requirement) was needed as a 
substantial portion of propylene oxide was hydrolyzed to propylene gly- 
col due to the presence of water. Studies with regard to the reaction 
mechanism revealed that a small amount of alkali was a desirable cat- 
alyst for esterification. This was already present in Pectic Acid No. 75 
but had to be added in esterification studies with purified pectic acid. 



’Sunkist Growers, Ontario, California 

r H. Deuel. Helv, Chim. Acta., ^0, 1523 (19**7) 



\ 



\ 




- 4 - 



It was then discovered that esterification may be accomplished more 
conveniently and economically in a heterogenous system, where Pectic 
Acid No. 75 was suspended either in an excess of propylene oxide or in 
an appropriate solvent thereby reducing the amount of water to a minimum. 
In this manner, complete esterification was achieved with as little as 
twice the stoichiometric requirement of propylene oxide; e.g., with a 
charge consisting of 10 parts Pectic Acid No. 75, 7*5 parts propylene 
oxide, 40 parts pentane, 1 part water complete esterification resulted 
with a reaction time of 20 hours at 35°C . At the end of the esterif- 
ication, indicated by neutral reaction of the solids, the ester was is- 
olated simply by collecting the solids by filtration, rinsing with ace- 
tone and drying. The crude propylene glycol pectate was then dissolved 
in water, bleached with chlorine dioxide and clarified. The filtrate 
diluted to proper concentration was then treated with two volumes of 
acetone to precipitate the ester. The precipitate was isolated by de- 
canting the superatant. liquid, slurrying the solids several times with 
fresh acetone and finally drying in vacuo. 

Several batches of propylene glycol pectate so prepared were sub- 
mitted to Dr. F. W. Hartman of Henry Ford Hospital Laboratories for 
evaluation on animals# In loading experiments on mice and rabbits no 
signs of acute toxicity were found. When administered to dogs subjected 
to severe experimental hemorrhage (up to 85 $ blood withdrawn in 3 steps) 
the preparations positively were shown to be capable of restoring and 
maintaining plasma volume and blood pressure. The levels employed were 
in the range of 3*5 to 4.0$ in saline adjusted to pH 7.3* However, when 
given to dogs in large doses, occasionally serious side reactions were 
observed such as edema of the lungs and the heart. The intensity of 
these toxic reactions were variable and appeared to be associated with 
particular batches of propylene glycol pectate. In vitro, differences 
in oncotic pressure could be demonstrated among the various products. 

A careful examination of all the samples submitted for testing revealed 
a variation in the degree of esterification. A high incident of toxic 
reactions and low oncotic pressure could be correlated with a high degree 
of esterification. A series of tests on dogs with preparations adjusted 
to various degrees of esterification revealed that highly ester if led 
preparations were unable to support plasma volume and blood pressure, 
and that low esterified material, in turn, was excreted too rapidly. 
Furthermore, these studies clearly indicated an optimal degree of ester- 
ification of around 85$. Consequently, a. certain amount of free carboxyl 
groups was essential to attain satisfactory oncotic pressure in vitro 
and adequate support of blood pressure in vivo. Nevertheless, propylene 
glycol pectate was not further pursued because of the toxic side re- 
actions encountered which were very much alike those reported on poly- 
propylene glycols and esters thereof. 



*A11 references herein to such animal evaluation studies are based 
on oral reports only. 



Studies on Glycerol Pectate5 



- 5 - 



Efforts were then devoted to the preparation of glycerol pectate 
from Pectic Acid No. 75 and glycidol. After considerable initial dif- 
ficulties glycidol was finally obtained in 60-70$ yield by reacting 
glycerol alpha -monochlorohydrin with caustic soda. Due to scarcity of 
glycidol only a limited number of esterification studies could be made 
and attempts to esterify in a. heterogenous system have been unsuccessful 
so far. However, these studies have shown that glycerol pectate can be 
prepared in a homogenous system using a charge of 1 part of Pectic Acid 
No. 75, 2 parts of glycidol and 4.5 parts of water and a reaction time of 
24 hours at 40°C. Glycerol pectate was then isolated according to the 
procedure outlined in Table I. 



TABLE I 



Preparation of Glycerol Pectate 



Pectic Acid #75 

21$-C00CH3 

68$-COOH 

ll$-C00Na 



(2.4 Kg) / Glycidol (4.8 Kg) / H20(l0.9 Kg) 



Agitated 24 hours at 40° 



Glycerol Pectate Reaction Mixture 



a) 



b 




bleached with 4.5 liters C10 - 2 
solution (approx. 0.3$) 
diluted with 5-5 liters H 2 O 
filtered on K-7 clarifying pad 
precoated with Dicalite SS 



Filtrate 



a) precipitated with equal volume of 
acetone (about 22 liters) 

b) isolated by decanting supernatant 
liquid, reslurrying precipitate with 
acetone, and drying 



Crude Glycerol Pectate 



a) 

bj 

c) 






redissolved in 24 liters H 2 O 
added 180 ml saturated NaCl solution 
precipitated with 24 liters acetone 
and isolated as above 



Purified Glycerol Pectate (2.1 Kg) 

Degree of esterification: 88$ 

^ rel. "-'-'7 (c r 4$) 

^u.S. Patents being applied for. 



\ 



1 



The purified glycerol pectate prepared as outlined above, dis- 
solved much more rapidly and showed a greater water solubility than 
propylene glycol pectate. It gave practically colorless and non-foam- 
ing solutions. For reasons of comparison with the propylene glycol 
ester, glycerol pectate was tested on animals at a concentration level 
of 4$ in the relative viscosity range of 4 to 5 and with a degree of 
esterification of 85$. Solutions suitable for intravenous adminis- 
tration conforming to the above specifications were prepared in the fol- 
lowing manner. The purified glycerol pectate was dissolved in the proper- 
amount of 0.9$ saline to give a 4$ solution and then clarified by fil- 
tration in the presence of a small amount of Celite, analytical grade. 

The filtered solution was thermally degraded at 100°C to lower the re- 
lative viscosity to around 4, which usually required about two hours. 

The degraded solution was checked for degree of esterification by tit- 
rating the free acidity of a deionized aliquot. This determination per- 
mitted the calculation of the amount of alkali required to adjust the 
solution, by partial saponification, to 85 % esterification. For stabil- 
ity reasons, it was preferred to keep the degraded solution at the 
natural pH (of around 4) and add the alkali just prior to administration. 

In studies at the Ford Hospital, glycerol pectate proved to be a 
very promising plasma, replacer. According to Dr. Hartman’s findings, 
glycerol pectate did not give the toxic side reactions observed with the 
propylene glycol derivative. Plasma volume of hemorrhaged dogs was 
quickly restored to normal and maintained, and no evidence of deposition 
in the tissues could be demonstrated. 

In view of these results. Dr. W. J. Perry of the Office of Naval 
Research, arranged a more extensive evaluation of glycerol pectate at 
the Naval Medical Field Research Laboratories of Camp Lejeune. The 
original testing program proposed by Capt. C. 3. Galloway and Dr. S. W. 
Handford, was to study glycerol pectate at concentration levels of 3, 

4, 5 and 6$ at a relative viscosity of around 4. Also, it was agreed 
to study the 4 fo level at a relative viscosity of around 3* 

Solutions in saline containing 3,4,5 and glycerol pectate were 
prepared as described above and then thermally degraded at 100°C. to 
a relative viscosity of around 4, requiring 1,4,7 and 11 hours, re- 
spectively. Due to the prolonged heat treatment, the 6 % solution showed 
considerable darkening and was therefore considered unsatisfactory for 
use in animals. Oncotic pressure determinations on the 3, 4, and 5^ 
solutions of glycerol pectate neutralized and adjusted to 85 $ ester- 
ification are shown in Graph I. The 4 % solution gave a final oncotic 
pressure comparable to human plasma and to the 6 $ dextran solution used, 
as reference material. With respect to plasma the 3$ level was hy- 
potonic and the 5 % level accordingly hypertonic. 



■■■■■■■■■■■■■■■■■■I 



■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■I 
-■■■•■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■I 
'■inn ■■■■■ 



■ ■■■■■■■■■ aaaaB aBSBaaaaBB aaaaa BBBBflBaBBaaflBIMMBBHMBflHMBH^H^HiHHBHBBHBflaaaBaiOTaBBaaaaBI 

■ ■«■» 

■■■■■■ ■ ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■ )■■■■■■■■■■■■■■■■■■■■ 

■■■■■■■■■■■■in ■■■■■■■■■■■■■■■■■■■■ ■■■■■■■■■■ MM«ililSSSlSSS5iSS5ii5ii5iii5iiii5ii5iiiiiil 

■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■a ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■! 

■■■■■ ■■■■■■■■■■■■■■■■■■■■■■■■■ ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■BBaaaKBaal 
■ BBBBBBBBBBBBBBBr"PII''rPBB-«>Pl a «l' , P'P'a>»PBBBr;Br:in»PP'i"lxCflB7FPPBI«r*BB~P*-»pn|rBB aa PBBBBBBBBBBBBBBB 
BBBBBBBBBBBBBBBB'.'n'i.'Jl'P IHIMJ-J-I 1 ("U'M VMII.-l'l 1 ! - I'l IB » L« l 1 • t IIIIBIHIBBBBII 

IIBIBIIIIIIIIIIlH«MHM"**l |lllaa * Ha| i* ,IHBaHaMIIIIII *9 Bllal>a ■■■■■■■■■■■■■MaMKMHMJIIIMBIlIBBIIIB 

bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb 

BBBBBBBBBBBBBBBBBBBBBBBB»T« a PP*»P»PBTBrPBB"r>P>9 , 9nF«'l«"*r»B«lfBB a 9 a <tP*^ a >BBBBBBBBBBBBBBBBBBBBBBBBB 

■BBBiaMiiBii»»BHmii j -.-iJ"A:i l . l ar"ii •<jti-. > u^9':r^i-.Ma ji!L? a ! . aaaaaaaaaaaaaaaaaaaaaaaaa 

aaaaaaaaaaaaaaaaaaaaa aaaaaa 

iiaaBaaaaaaaaaaaaaaaaaaaaaaaaaaaaiaaaaaaaaaaiaaaaaaaaaaaaiaaaaaaaaaaaaaaaaaaaBBaaaaaaBBsaaH 
Sbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb5bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb«bbbbbbbbbbbbbbbbbbb 
BBBBBBBBBBBBBBBBBBBBBBBaBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBflBBBBBBBBBBBBBBBBBBBaBBBBB 
BflBBBBBBBBBBBBBBBBBBBBflBaBBB73PHPPnrrBIBrFafl*Brnfla'«Bfl"BBI a l'='raBBBBBBBBBBBBBBBBBBBBBBflBBBBB 
aaflaaaaaaaaaaBBBBBaaaaaBaaaBM iv ;«'"<pi>: i T9> ill aaaaa 

aaBBaBaaBBaaaaBBaflaaBBflaBBaaaaaaaaBaBaBaBaaaBflBBaBkBaaBBBBaaaBflBiflflflBBBBaBaBBBBaaBBaaflBBBB 
aaaaa aaaaa aaaflaaaaaa aaaaa aaaBfl aaaaa aaaaa aBBaBflBBaBBBBBBaaaBBaBflBB aaaaa BBBBaBBBaaaaBflaBflBBa 
BaBBBaaaBaaaaaaaBaaBBBaaaaBaaBBaaaaBaaBaaaaaaaaaaaaaaaaaBaaaaaaaaaaaBaBBflaaaaBaaBBBBaBBBBa 
aaaaa B aaBBBaBBBBaBBBBBBflBBaaBBaaBBflBBBBBBBBBBBBBaBBflflaBflBBBBBBBBaBBBBB aaaaa BaBBaBBBBBBflaBB 
Baaaa a BaaBBaaBBaaBaB aaaaa aaaaa BflBaa aaaaa BBBaaBBaBBaflflaaflaaBBBBBflBaBBBB aaaaa aaaaa BBBBaaBBBB 
aaBBBBaaaaBaaBaBaaaaaaaflBaBaBBBaifliiBflaBaaaaaaiaaaaaaaaaiaaaaaBaaaaBaaBBBaBBBaaaaaaaBBaBBB 
flflflflflflflBflflflBflflflBBflBflflflBIflflBBBfllfllBI BBBBI ■BBBgBBBBBBBBBflflflflBflflflflBflBBfllBBBflBBflflflflBBflflBBflBBBfl 
BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBflBBBBiBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBIBBBBBBBBl 
BBflflfllBIflfl BBBBI IflBIBIBBIIIflBfllflBBIIBflflBIBIflfllllBBBIIIflllfllBBIIBIIIflBBBIBBBBflflflBBIBBflBBflflBB 
aaaBB aaaaa aaaBBBBBaa aaaaa aaaaa BBaBBBBaaBBaBaBflBBBBaflflaBaBaBBaaBBBBBBBBBBBflaBBBBaaBaflaaBaaB 
flBBBflflBBBBflBaBBBaBBBBBBBBBBBBaBBBBBBBBBBBaBBBBBBBBBBBBBBBflBBBBBBaBBflflBBBBflBBBBBBBBBBBBBBflB 
BfllBBflflBBIflflBBBflfllll BIBB 11 IBBI BIBB IllIBBIIIBIBBIBBIflIBI BBBBI IflflBIlIBBBIfllflflllBBflflflflBBflflflflB 
aaaaB aaaaBaaaaB aaaBB ■ a bbb b a BBBBBflBBBBBBBBaBBBBBBB a bbbbbbbbbb aaaaa aaaBB flBBBBaBaaa aaaBB aaaBB 
flBflBBBBIIfllllllBIBBBIflflll BBBBI BflBBBIBIBBIIIflllllBB BBBBI BBBBB BIB BflBBIflBIBBIBIBBBBIBflBB IB Bflfl 
aaBBaaaaaaaBBaaBBBBBflBaBaaaaBaBBBBBBBiaBflBaaaBBBBBBBBBBaaaBBaBaBaaBBBaflaaaaBBBaaBaaBBBBaBa 
BaaBaaaaflBBBBflBBaaaaaaaBBaBaBBaaBBiaaaaaaaaaBBBaaBBBBaBflaBBaaBBBflaBaaBBBaaBBaBBaBBBBBBaaBB 



aaaaaBiLri:iBBaaBBBaiaBaBBBBaaaBBaBBBBBaiBBiiaaBBBaBBBBaBaBBBaflaBP^UBaaakM.'aiutj.ri'jiiaaaBBB 
flBBIBBBflfll BBBBB BBBBB BBBBB BBflBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BFiflfl BBBBB fll BBBBI IBB BBflflB BBBBB 
BflBBBflflflfll BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBPiflBBBBBBBBBBBBBBBBBBBBBBB BBBBB 
BBBBBBBBBIBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBPiaBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB 
Baaaa aaaaB BBaaaaaaBaaaBaBaaaflaBaBBaBaBBBBBflaBBBflBBflBaBBBnaaBBaBaaaBBBaaBBBaaaaaaBBBBaBBBaB 
aaaaaaaaBB bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbf aiBflaa aaaaa BBBBaaaaaBaBBBaBBBaaflaBBB 
BBBHBaaflBB bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb aaa i, i*fl bbbbb abbaa bbbbb bbbbb bbbbb bbbbb bbbbb 
5a|aBBBBaBaaaaBaBBBBflBBBaBBaBBBBaBflBBBBBaaaflBaflBaaBr^aBBaBBaBBBaaBBBaaBBBBaaBBBaBBBBaaBaaB 
■aiBBBBBflB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB '-BBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB 

■aaaar 1 -naaaaaaaaaaaan — i 

iBaaaB?<£i. , 'j bbbbb bbbbbbbbbb bbbbb bbbbb bbbbb bbbbb bbf',ab bbbbb bbbbb bbbbb bbbbbbbbbb bbbbb bbbbbbbbbb 
E agSiaBBI bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bp abb bbbbb bbbbbbbbbbbbbbbbbbbb bbbbb bbbbbbbbbb 

^i!i^i^^iiiii::::S:E£issE::EEEEEE:::ss:r^:=s5S5Siiilss:iis£E:EE=:E:E::EEs:iiii:iisE3^ 

laaaa^naBBaaflaBBaaBaBflaaBaaBBaBBBBBBBBaaBBBFaBaBBaBBBBgggBBBaflBBaBBflBaBflBBBaBBPcwaaBaaaaaaB 

l^MMfaaaaaBaaaaaaaBBBaaaaaaBBBaaaaBBaaaaa-jBBBaBBBaiBBBaBBaaaaaaaaaaBaaBaarhBaaaaaaaaaBaaaB 



BBBa^B3’:r'1BaBBBBBBBBBBBBBBBBBBBBBaBBBBB^-BBBBBBBBaBBBr'r4BP:FF- ra ^Fa a <BBBBaCaBBBaF---aBBBBBBBBBl 
■Baa.a*.^l’ , JBBBaflBBaBBBBBBBaaaBBBBaaaBBar.BflBBBaBBBBBBfli:/^BL:V^^L!C. t L:JlBBBFMaBBPr:xaBBaaBBBBaBaBB| 
BB— -aaaaa aflBBBBBBflflaBBBBBBBBgBBBBaBBF JflBBBBB BBBBBBBBBB BBBBBBBBBB BB’9aaa B t^aBflBBaaBBBBB aaaaB 
aa aa a aa aaa BflflBflBIBflfllflBflBBflBBBBBflflBflB'ABflflflflflBlIBBBBBIBBBIBBBBBflflBBBflP^ BBBBB BflflflBBflBflBflBBBfl 
BBBB. aaaaa aaaaB BBBBBBaBBB aaaBfl bbabb a^BaBBBBaaBBBBB aaaBB BBBBBBaBBFSFiiaaBiT'iar/^FairBB '■iiaaBBaa 
aaaaa aaaaa aaBBaBBaaBaBaaBBaaBBBBBBBrflaaBBBBaBaBBBBBBBaBBBaBBBBBE''^aflBBaB.y^at\L/aa.ri. > aMBBBBBB 
■BHBBflHBaa aaflaa aaaBB BBBBBBBBBBBBBBajBBBB aaaaa bbbbbbbbbb BBBBBBBr.^a bbb BBaBaaBanaaa aaaaa aaaBB 
^■Kaaaai bbbbbbbbbb bbbbb bbbbb BBBB r i bbbbb bbbbb bbbbb bbbbb bbbbb pabbb bbbbb bbbbb bbbbb bbbbb bbbbb I 

^■a^flBBBa BBBBBBBBBBBBBIBBBflBBBBBrBBBBBBBBBBBBBBBBBBBBBBBBP:,.4BBBBBBBBBBBBBBBBBBBBBBBBBBBBBl 

^■SjiBaaBIBaflBBBaaBBBaaflaflBaaflBB»4BBBBaBBflflBflBaBaaBBBaaBar:>caBBBBBBBBBaBBBBaiBBBBBBBBaBBBBB| 
^^KsBmr.HBBBBBBBBBBBBBBBBBBBBBB'jBBBBBBBBBBBBBBBBBBBBBBrAaEaBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBl 



I IZ BBBBI I 

l^aaaaai 



■ aa iBBi as laBBaaflara bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb aaaat I 

is ■■ P.BBBI i waaBaflBBBi is laaaaaa aaai ia t* ■bbbbI 

ibbi ibbi iA! in bbbbb bbbbb bbbbb bbbbf abpbb bbbbb bbbbb bbbbbbbbbbbbbbbbbbbbbbbbb 

I HBKBaaBiaBBaBaBaBaaBflBBaflBaNBBBflBBflBaBflaBBaafla^BaEaBBBBaBflaaaBaaaBBaaaaaaapr^aflBBBBaaBBl 
flaBavBaBBaflaBBBBBaflaBBBaBBaaraaaBaaflaBBaaBBBBBFaBa’iaBBaBBaBBaBBflaaaaaaBBBr^oaBaaBaBBBaaaaal 
BBBt.IBBBBIBBBBBBBBBBBBBBBBBB'iBBBBBBBBBBBBBBBB'SABBraBBBBBBBBBBBBBBBBBBBBr-ABBBBBBBBBBBBBBBBBi 

aaaBgaaBagaaaaaaaagaaaagaaagagBaBBBBBBBBBBBF^BBB^B aaaaB aaa aa aaaaB BBF^iiBBBaBaBaaaBBBaaaaBBal 



BBflflTBBflfll BflBBaaBaaBBBaflBBB iBBBBBBBBBBBBBB'-.ABBBABBBBBBBBBBBBBBBBPr-BBBBk- T. 4Bn>F^r Bfc IklBBaaBB 

■Baapapj^^'j bbbbb bbbbb bbbbbpabbb bbbbb bbbbf abbbIi bbbbb bbbbb bbbbb fabbbbbbbb bbbbb biibbbbbbbbbbbbb 

BBBe^BBBBIBBBBBBBBBBBBBBBriBflBBBBBBBBBBrABBBiiBBBBBBBBBBBBBFTIABBBBBBBBBBBflBBBBflBBBBBBBBBBBBB 
BBBBpBBBBI BBBBBBBBBBBBBBFABBBBBBBBBBBMBBBUBBBBBBBBBBBBPtiABBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB 
■aaBYBBaai bbbbb bbbbb BBBB r i bbbbb bbbbb p:jbbbbabbbbbbbbbbbp^ bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb 
BBBB 2, BBBBI BBBBBBBBBBBBBFBBBBBBBBBBP ABBBBflBBBBBBBBBBFiaBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB 
BBfli:BaBBiaBaBaaaaBaaaa’iaBBBaaaaBP4aaBMaaaaBaaBa’' a BBBBaaBBaBaaBaaaaBaaBBaaBBaBBBBaaiaaaaa 
BBBlo BBBBI BBBBBBBBBBBBFaBBBBBBBBFABBBliBBBBBBBBBFABBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB 
aaaaBaaaaiaaaBaBaaaBBajBBaaaaaar.aBBPuiaaaBaaardaaBaBaaaBBaaaaaBaaaaBBBBaaBBBBaBBaaaaBBBaBB 
aaaagBBBBI IBBBBBBaaBBr.aBiaBBia^aBflBMaiaaBBPFABBBBBBBBBBBBIBBBBBBaBBBaaBBBaBBBflBIBaBflfllBflaB 
anm bbbbb bbbbb b’jbbbbbbb^ bbbbb bbbbb bfabb bbbbb bbbbb bbbbb bbbbb bbbbbbbbbbbbbbbbbbbb bbbbb 
aaaaaaaaaaaaaaaaaa 

8S!nSiSHSwiiSSS!*m!uiir'ij3HBHHHHBBiHMHHHHHHi 

BflBiBaaBBBBap!iiBB»pSaBS*aflBr%aBai 

■aaBiBBBBBBaa'/BaBaPiaaKiflBP^BBaBfla^^^^^^^^^^^^^^^^^^^^^^^^^^H 

BBBBI BBBBB BBFBBBBFABBflflBPiBBBBBBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB 

■BBBgaBBBBBB'iBBBa^BBfcaa^a bbbbbbbbbb bbbbb BBBBBBBBBaBBaaaBBBBB Baaaa aaaaa aaaaa Baaaa aaaaa 

EHBEflflBBBP-BBBPABBIiaBF ABB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB 

BaaaiiaaiBB'jBBB'iBKaF^iaBBBBflBBB bbbbi BBBBBBBBaflBBBBB aaiaa Baaaa bbbbb aaaaa aaflaa aaBaa aaaaa 

IBlI'tTnl BflflflflPjBBB'ABFB’^AflBflBBflflBfllBflflBflflflBflflflBflBBflflBBBflBflflflflBBBflBBBBBBflBBBBBflBBBBBflBflflBBB 
WW 1 .I.M BBBBB BBB r AB3PAflBflflflflfl BBBBB BBBBggflBBggiBBflBBIBggBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB 
li ww aaaai BBBBMB',iPBr;flBBBBBBBBBaBBBaBBBBiBBBBBBBBBBBBBgEBB BBBBBBBBBBBBBBBBBBBBBBK.3*»-;ir3ii-ii.l8g 

RiiB 8 bbbbi ■BBpaBr.pF^BaBBBBBBBaaBBBBBBBaBggBB|gBggiBBBBBiBBaaaaaaaaaaaaaaaaiaHMaaaMaaaHa| 
IBIBI BBBBI Bflfl'iBFF^ABSBI . • ■BBBBBflBaBBBaaBilBBilaiiBBBIBlSaBi 
aaaaa aaBaiaBFarr^aaaaBaaBaaBBaBBBaBBaaaBBaaaaaiiaiBaaaBBaai 

I BBBBB BBBBI Ba'IF/' aaaBB BaBBBBBBBBBBBBBBBBBB Bgagg BgBBBgBBBBBBBB ■ 

BaaaBBaBaiBrF.'.aBBBBBBBBBBBBBBBaBBBBgBggBgilligggBBIBBBaaBaiBBBBBBBiBaBBBBBBBaaBBaaaaflHI 

I :',-'.' aaa ■ - j aaari.i.'aaaaaaaiiva 

iaBBBaBBBBBBnBBaaL3^BBBBBaaBj£W 

ESsSsssSIaisssssSSiSSaSSaSHI 



^^^^^^^^^^^^■aaiaaaaBiBaail 
_ ■HiiiaaaBaBaBBaaaBBaaaBaaaBaiBiaaaaaBil 
«a ia i a a» waaBi * sr^ »j air « I 



^^MWbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb bbbbb bbbbb bbbbb bbbbb bbbbb I 
aaaBaaaBaiBBBaaBBflBaaaBBBBaflaBflaBflaBaBBaBaaBBBaBBBBaaaaaaaaaaaBBaBaaBaaaaaaaaaaaaBBaiBial 



■IggflBBBBBflBflflflflflflflBflflflBgflBBgiBfl BBBBB BBBBB Bfll 
■iEiBIBiaaBaBBBBaBaBBBflBiiiBiBBBBailBBBIBBBBI 
■flBflflflflBflBflBBBBBflflBflBI BBBBI BBBBfl BBBBB BBBBB Bfll 



- 7 - 



Further thermal degradation of the 4 $ solution to a relative vis- 
cosity of 3*3 effected a slight increase in oncotic pressure, as ex- 
pected. It was thus of interest to find out whether solutions at levels 
slightly less than 4$ may be prepared which upon extended thermal de- 
gradation may yield oncotic pressures equal to the 4 $ level. As shown 
in Graph II the oncotic pressure of 3 $ and 3 |$ solutions of glycerol 
pectate thermally degraded for 2 and 5 hours, respectively, were obvious- 
ly much lower than that of plasma or 6$ dextran solutions. Even at the 
3w° level a very much longer degradation time would have been necessary 
to raise the pressure to that of 6$ dextran solutions. This could have 
only been accomplished at a great sacrifice of molecular weight. It 
was therefore generally agreed to limit exploratory evaluation studies 
in animals to the 4 $ level in the relative viscosity ranges of 3 to 3?- 
and 4 to 4 |. Depending upon the results obtained under these conditions, 
the effect of varying the degree of esterification was to be investi- 
gated later on. 

Approximately 125 liters of 4$ solution of glycerol pectate were 
prepared for testing at Camp Lejeune, and for drying and stability 
studies. One-third of these preparations were in the lower relative 
viscosity range of around 3*3 (Lot #12-3A, 7 fl2-3B, 13-3A and 13-3B), 
whereas the major part was at a relative viscosity of 4. Table II shows 
the individual batches made and illustrates the degree of uniformity 
obtainable by proper degradation and partial saponification. 



TABLE II 






4$ Glycerol 
Pectate 
Lot # 


Relative 

Viscosity 


Unadjusted 
Free C00H 
meq/g 


Ester 

$ 


Adjusted^- 
Free C00H 
meq/g 


Ester 


Oncot. 

Pressu 

f 


11 




4.4 


0.433 


89.2 


0.590 


§5*2 


95 


12 -3 (A) 


3.3 




-- 




0.573 


85.6 


103 


12-3 B 


3.2 




0.496 


87.6 


0.598 


85.O 


-- 


12-4 (A) 




4.4 


0.457 


88.6 


0.580 


85.5 


90 


12-4 B 




4.3 


0.457 


88.6 


0.587 


85.3 


93 


12-4 C 




4.3 


— 




-- 






12-4 D 




4.1 


— 




— 




80 


13-5 A 


3.1 




0.488 


87.8 


0.601 


85.0 


— 


13-3 B 


3.2 








-- 




— - 


13-4 A 




4.2 


0.438 


89.I 


0.610 


84.7 


92 


13-4 B 




4.0 


— 




— — 






14-4 A 




4.4 


0.468 


88.3 


0.595 


85.1 


91 


14-4 B 




4.5 


— 










14-4 C 




4.4 


— 




0.604 


84.9 


79 



^Neutralized and partially saponified to 85$ esterification with 
sodium hydroxide except for Lot Nos. 12-4D and 14-4C which were 
neutralized and adjusted with sodium bicarbonate. 

2 In comparison with 6$ dextrsn 



* 



V 




SSBBf 

liim 

■ini 

Esisas 

Hi!!!! 






ittiiii3iiii»i Ki Sii illi»aHlilH 'H!>!li!!liiliilililiiBiSjj!jj 



111! 

in* 



iMi ■■■■■■ 

iiiiiiiS 



BBbbbbbbbbbbbbbbbbbI 

■■■■■■■■■■■■■■■■■■■■■■I 

■■■■■■■■■■■■■■■■■■■■■■■I 

Ilia 8 bHbb 

iHWWHiiiuBggg| 

■piiiB 

< BBHBBBBBHB ■■■■■! 



■■HMMBBBBBBBBBBBBBBBflBBBBl 

»Bi^ EaawrBaaa iiiiBggBgj«iiig| 
Mfijgi ■ * » II » • «(») • U iHHMjjMMMl 



'■■■I 

■Ban 

■an 



[laaaa j ggg ; 

■BgggggglMMHl 

iaaaaaaaaaaaan' 1 -ii'.' 

BaagaaaaaaaaJI 
aaaaaaaaaaaa 
bbbbbbbbEbbb 



MpWJIi 



MP Vtf-WWf 



IS 

is: 



aaBBBBBBaaaBl 

laaaa aaa aaai 
aaiMH| 
■MmM 

ilaia aa a aa 



■ga 

IBB 



S3 



aaiaaB 



KiSKH 

ir-naaaai ■BBaaaaBaaaaaaaBBa aaaaaa 

::::: a 



:!!:! 



u: 



■>!!! 

aaa 



aaa 

aaa 



■aaaaar aaaaaaa aaaaa 
■HaBraaaaBBBB wa 
iaraBBBBBBaa 



' iH’.aari 

■aaagaaaaaaaaagaaaag aar.# 5 ■W 



BBBBB BBBBBBBBBB BBBBB! 

B iBaaaaBaaBgBaaaaaBBai 

laBBaaBBaBEaBaaaBBar 



H MBBaBBBPBBBaBBBBia 1 

■ I BBB V'^aaBflB BBBBB 



BBBBI 

algal 



888 

aaa 

■ ■■ 

SI! 



■BaBBBBBBBBBaBBB^a 
■a gaaaBaaaaagar.al 
» aaaaiBBBaaaraaB 
aufliiaaflariBBSI 
■aaaBaaa^Baaaai 



Bin 

aaaaaa 
■■■■aa 
■ggggggggggggiBBU 

BBBaBBaaaBBBaBBaaa 

aaBaaaaBBaBBaagBaa 

BBBBBBaaiBBaBaiaaB 



B BBBaaaaaa'iaBBaaaaBBBBBBBaaBBBBBi 

laaag agir.aBaaaaBaaaBaaaaaMiKaeaagi 



BBBBBaaa aaaaaaaaaaaaaaaBS 

a ■■'iBiBaaaiiaMMMHHMHagi 



8888 ■■rBB BBBBaaaaaaaaaaaaaaal 

^^^MiaaiaaaaaaaaaiBair'iarrnTn’T] 



■BBaaraaBaaaBaaBBBBaBaaaairaarrnrcnT'al 
IgBag'JBBaaaaBaaBBBBBBaxi^Me'JB&sgsa^il 
aiSB^aBflBgaBBaaaaBBP^aaaaaBBaaaiBial 
gBBrgaBBBaBBBaaBBB^aBBBBaBfe.r*'' bbbbb 
B aa^aaaa aaaaaaaar^aaaaaaaaag-j%aaaaa| 
m^aaa aaaga r aaaa aaaaa aaaaa bbbbb 

iBflflBflBPi BBBBBBBBBB BBBBB BBBBB 
■■■|BB^|aaaBaBBBaBc..ia7<En!Tn'iB 
naaiar.aiaBBBBBa^auaBxiz^a^al 
■aaaar < aaaaaBBi'^aaaBBBaaaBBBBB| 

■■■Bar.aaaaaa^aBflBBBBaBBBBaBBBflal 



rgaaaaaaai 

igaaaaaaai 

lilflBBBBBI 



■nHMarjgBB 

raaggBgaaaaraHgBBHHHHH 

aBaiEaaBaaraaaaap^aBBaBaaaai 



lairfBBBBB aaaaa aaaia bbbbb I 



gaa>iBtL»i!i*S|s.B| 

Baaiaal 
■ bB’J^BBBBBBI 
iBa^pawaaBBaBBl 

ik»aaii r ;nr¥a“i 



ai ■BBaaaaaBBgBBBBaaaBBaaBBagB: 

bbb-Ibbbbi BaBaaBBaiBaaBaaaBBaBaiBaBaaHHHHHHMHl^H^HHH^BHHMHBH^^^^^^^^^H 

aaa -bbbbbi bbbbb bbbbb bbbbb BBBBBBBBBBaBBBiBBB'iBBBBBBp'.iBBBirBBgiBBBaBBBBBraBBr*BBBBBaBBBBi«aaB 

Saaai aaa laaaaai - mr.mt.'Szmiu- • '.•■■ai' *<■ • «ra* • -bbbbb.. 

B iaaiaaaaai iaSSl ■■ ia ibi isr,t m iaaa ip^bbbi ibbj ■> 

lB:H:ss:::ss:»ssss:ssKssss:ssas»ass^ssHa^8:B;dasssss:Bs^»%sBss:ss:sHSBs:Bs 

^■B 8 USSMMttHSnntti 55 '»«S”»«""»"S»S 8 a^!» 8 SS 8 SSS 8 aS 8 SS 88 SS 888 aS 

laaaaBar,aaaar < aaBa«BBaaBB ] r;p^aaaaaBaaBaaaaaaaaaaBaaaaaa| 



■■■■■■■■■aaBiaa 

liiiiliii laaaaifl: 

ISSS'S"""”! 8888888888881 



■«bm: 

■zaaai 



ismi aaaai 



BBBBBBBBBBB: 

aaaaa laaaa 
■bbbbbbbbbbh 
aaaaaaaaaaa 



laaaaa 

■■aar. 



aaaai bbbbb aaaaa iflaBaBaaBBi 
IlSi '*■■■■■■ aBBBBBBBBBBBBBBBBBiil 



I aaa.aBBaai aiBaaBiiaBiniiiiiBinrjiir<ii*iaiBilgaaBgga 

bbi^bbbbbi aaBBaaBaa , aasaa'i 



, -■--■•■■■ ■■■■- - ip^i ibi . aaa iflaaaa!. ib bbbb B 

aaaaaaagaaaar. aaaa^aaaBaaaaaSr^^aaaaaaaaaaaaaaaaaaaaaaaaaaaaai 
^^^^^^^^■aaa^aaBBBa aBBar^aaaaaaaaaaaaaaaaaaaaaaaaaaaaaal 
aaaaaBPo^agBBBaBBBaBaBBaaBBBBBBBaaBaaaaBBl 

gggggBP>;*BBB bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb I 

Baa^iBaaragBP'-aaBaaag aaar-;aaBagaaaaaaaaaBBaaaaaaaaaaaaaaaaaaa| 
■aaaaaBaaaSMBMMaaiTS'' ^aaaBaaflB' BflaaaaBBBBBBBaBaaBBBBBBBBBl 



a aaaag bbbbb aasaa bbbbb B^iBBBBfiflBfla'^aBBBBBai 

iar.i avasBi aaa 
ibi «aaaBBBB 



iBBr.flBBraaap^BaaaaBBi 



■aaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaal 
iaaaBBSaiaflaaBMMMMawaw||||H| 



i is i® n paaipgaaBBaai ^gfegaag aK s::sss::sss 8 ss 8 sss 8 sss| 18 S 88888 SS 

bbbtbbbbbi BBBBBBBBBBBBBBBBBBBBraBpaBBP4BaBBBBap-..BBBBBBaBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB 

iwbbbbbi bbbbb bbb bb bb bbb bbbbb 'JBP iaBBpaaBaaaBap'gBBa bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb 
laBBBaaa Baaaaaaaaaaaaaaaaaai’jBPgaaraaaaaaBP'.aaBaaaaBBaaBBaaaaaaaaBBaaaBBaaaBaaaBBaaaaBBBB 
mmbi BaaaflaaaaaaaaBBaaarapgaBraaaaaaar .aaaBBaaBBBaBBBBaaaBBBBBBBBBBBBBBBBBBBBBBiiBBBBB 
■an ■■■■■■■■■■■■■ ■■■■paa^aaraBaaagp^-^aaaaaaaaaaaggaaaaaaaaaaaaaagaaaaaaaagaaaggaaiig 
_ . Bfiaiaai muaBBBBUBHMHBiiiiiaaiiiiiHHMMHMIlHHHMHIIMIIIIII 

|Bimw S 8 SS 88 JI 



a aaaaaa (a 

l:i§! 



laaaBBPgpitfaap a BflflflBPii'^BBaBflBiaBBfliaBBBggBBaBaflBBaflBBBa aaaaa bbbbb bbbbbi 

IBBBBa'iP^aaraBBBaBr^aBBBaBBBBBBaagBBBBBaaaBBBflBBBBBBBBBBBBBBBBBBBBBBBl 

iBBBir.p'jBipaBBflBP^^ bbbbbbbbbb bbbbbbbbbbbbbbb bbbbbbbbbb bbbbbbbbbb bbbbbi 



BBBaaaaBBBaai'#iaBr.aaBaar/uaaBBBaaBflfliaBflBiBBBaBBBaBBaaBaaaaaBBBaBBaaaaaaaBaaal 
■■■■■■■■■■■BaBBBflBaflflfla’i^BargaBBP^raBaBBflBaaBBflaBBflBgaflBaBaBBflaaBBBaBaBBaBflaBBflBBBflBBBflBl 
lBBBBBBBBBiBBBBBBBaaBBaBa',^aBr.aaaar 4 ^«BBaaBBBBBBBaaaBBBaaBBBaaBaaaBaaaaaBBBBBaBaaBaaaaaaaaal 



■ 88 ! 



laaaaai bbbbb bbbbb bbbp'jbb^bb bp 
laeaggi aaaaB aaaaaaat^— aaaaa 
^^^^^■■■aaaaal 



VMHPVPggaaaagaBaaaaBaBaBBBgaaaBaaBaaBBaBBaaBBBBBBaaaBBBBBBBaBal 

aBariPiaaBaaaaBBBMBBBaaBBBaaaaBaaflaBaflafliBBBBBaaBBBBBBBBaaBBBa| 

HiraaflaBBBBaBiBaaiBBaaiBagaBBBaaBaaaBiaBiaaaBaaBaBBaBBBBiiiHl 



| 8 n 8 S 5 ^S 8888 S 888888 r^ 88 » 8 %% 8 S 8 SS 8 S 888 S 88 S 8 S 8 K 888 SK 88 S 888 » 88 S 88 .......a............ 

Ibbbbbbbbbi ■■■■■■■■■■ rjaraarap^aaaBaaaaaaaaaBaaaBaaaaaaaaaaaaaBBaaaaaaaaaaaaaBBaaaBaaaaBaaal 



■aa! 5 ! B 8 SS 88 S 8 S"a?Bp 5 i 



Wbb bbbb bbbbb b bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbbbbbbbI 
bbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbbbbbbbbbbbb bbbbbbbbbbI 



■aaaaaaai bbbbb BBB r.arap^p bbbbbbbbbbbbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbb bbbbbbbbbb| 

I gGflBBiaaai BBBBB BBPJPBP'. 4 P 4 BBBBBBBBBB BBBBB BBBIBBBBIIBBBBBBMBB BBBB BBBBBBBBBBB BBBBB BBBBB BBBBB I 
BBBBB BBBBI BBBBB BB r 4 P 4 P 4 P'.BBBflBBBBBflBB BBBBB BBBBB BBBBB BBBflgBBBBggBBBfl BBBBBBBBBB BBBBB BBBBBBBBBBI 

aaaaa bbbbi bbbbb Br.P'apar.BBB bbbbb a bbb bbbbbbbbbbb bbbbb bbbbb bbbbb bbbbb bbbbbbbbbb bbbbbbbbbb bbbbbI 

BBBBB BBBBI BBBBB PB r iP 4 r . BBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB I 
■MB BBBBI BBBaB'i^li^HHHMaHHHBHaHHaMMgHHHHagMMIHHHailllllgflllglllgllllll 

Iiiiibi mmmmrjmimm 

I HKaaaaBi bbbe m 
BBBBBBBBBI BBBr.J 
BBBBB BBBBI BBP .■ 

■■■BBBaaBi aa' r ^ aaaaa aaBaa aaaaa aaaBaaBBBaaBBl 
BBBBB BBBBI BP / ^BBBBBBBBBBBBBBBBBBBB^^if 



■■■■■■■HainiiiaaiiiiaiiBiifliiiMil 

BBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB I 
■BBBBBBBaaBBBB BBBBB BBBBa BBBBB BBBBB I 
■■BBBBBBgaBBBBaBBBB BBBBBBBBBB BBBflal 
- BBaBaaBaa bbbbb ■bbbbbbbbbbbbbbbbbbbI 

iBBBBaBBBB BBBBB aBBBBBBBBgBBBBB BBBBBBBBBB BBBBB BBBBB BBBBB I 
BBBB BBBBB BBBBB gBBBBBB BBBBB BBB BBBBB BBBBB BBBBB BBBBB BBBBB I 
IgmilllMllllllllIHglllllllglllBBBaBBBBflaaaaflgBBBBBBBBBaBBBBBBBBBBBBBBBBBBaBBBBBBBaBBl 
BBBBB BBBBI f jlBBB BBBBB BB BBB ■ BBBB BBBBB BBBBB ■BBBflaBBBBaflBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB I 



IBBBBBB WZdM II 
■BBBBBBBBBBI 
■BBagiBBgBBI 



BHHpTmi IBBBBBBBBl 
BBB BBBaB BBBBB flailll 



BBBBB BBBBI ’iBaaBaaaaaaaaaaaaaaaaaBBaaaaaaaaBaaaaaaaaBaaaaaaaaaaPBaaBBaaaBBaiBaBPBaaaaaBBBBBB 
BBBBB BBBBB BBBBB BBB B'^r. a BBB BBB nr.tflBBfllflBr'I^BBBBBBflV (l?BBBa*BCV^’?BBBflflBP 1 ^rBBBBBBV i P*PBriaBiaill 
■ BBBBBBBBBBBBBBBBBB 'L<JBBBBBB.,.>IBBBBgaBL‘>l>.aBBBBBfl('>.>BBaBaBP«l •aB||BHBM|a||HMBMB||Miia 
BBBBBBBBBB BBBBBBBBBBBBBBBBBBBBBBBBBBIBBBgBBBBBBflBBBBBBBBBBBagaM 

BBBBBBBBaBBBBBBBBBBBBBBBBflflflBBBBflBBgBflBBBB 7 'rT>BBBPTBflr: 7 ^r-r-|ggB»HHHHBHMHBBaaai ■■■ 

BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBP'P B 11 *BP "IBM V*J # . 

BBBBBBBBBBBBBBBBPBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBaiBBBBBBBBBBBBBBBBB BBBBBBBBBBI 
BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBI gBBBBIBBBg gBBBBBB BBB BBBBB BBBBB BBBBB BBBBB BBB aa 
B a BB 1 BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBB BBBB BBB BBB BBBB BBB BBBBB BBBBB BBBBB BBBBI a ■■■ ■ BBaiB 
BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBBgBBBBaBBBaBBBBB BBBBB BBBBBBBBBB BBBBB BIBBB aaaaa 
BaaaaaaaBaaaaBBaaaaaaaaaaaaaaBaBaBBaaBBBBBBBaaBBBaBaBBBaaBBaaBBaaBBBaBBBBaBaaaaaaaaaBaBaaa 










- 8 - 



It should be emphasized that in preparing the glycerol pectate for 
these solutions, two different batches of Pec tic Acid No. 75 were used, 
and that reaction conditions and isolation procedures were gradually 
modified to improve the process and yields. It, therefore, appears that 
differences in the purified glycerol pectate do not affect the prop- 
erties of the final solution, provided the proper degradation time and 
partial saponification are carried out. 

As already pointed out, glycerol pectate shows an optimum stability 
at a pH of about 4, the natural pH of pectin. It has therefore been 
necessary to store the degraded solutions at this pH and to neutralize 
them just prior to administration. As this was thought to be incon- 
venient, attempts were made to develop a neutral product of satisfactory 
stability. These studies led to a reasonably stable solution, and a dry 
material suitable for extended storage as described in the following: 

The initial procedure of neutralizing the pasteurized and degraded 
solutions of glycerol pectate with sodium hydroxide just prior to use, 
caused considerable handling difficulties. When the glycerol pectate 
solution was not adequately^agitated the alkali was used up locally be- 
fore it had a chance to become evenly distributed. Substitutes for 
sodium hydroxide were then investigated among which sodium bicarbonate 
appeared most promising. When an equivalent amount of bicarbonate 
(equal to the amount of NaOH calculated for neutralization and partial 
saponification of excess ester to 85?0 was added, the pH remained be- 
low neutrality. It could be shown that the slightly acid pH was due to 
dissolved carbon dioxide. The latter could be removed by aeration with 
nitrogen whereupon the pH increased to about 7*3 to 7.4. Solutions 
neutralized with NaOH showed an initial pH rise beyond neutrality fol- 
lowed by a gradual decline to a pH of 5 t-c 5*5 (after 2 weeks); sodium 
bicarbonate differed from NaOH in that it exerted an obvious buffering 
action. 

As it seemed advantageous to have a colorless parenteral solution, 
a small amount of chlorine dioxide wa3 added to the degraded solution of 
glycerol pectate before buffering with sodium bicarbonate. This measure 
was found of great importance as it prevented (presumably due to chem- 
ical sterilization) any visible contamination of the solutions. Since 
the chlorine dioxide completely disappeared in the first 48 hours, and 
since its decomposition products were hydrochloric acid and oxygen, it 
was regarded a. safe means of destroying the last traces of proteinaceous 
matter and impurities other than polysaccharides. Table III shows the 
course of the pH of a solution of glycerol pectate treated with ClOp 
and neutralized with bicarbonate. Since its preparation in November 1953, 
no visible contamination has been detected, and the buffering action was 
still effective after 70 days of storage at room temperature. Similar 
shelf-life tests, on solutions in sterile containers, have confirmed this 
result to date; they are being continued to determine the practical 
shelf-life of bicarbonate solutions. Sterility tests will also be made 
to establish the effectiveness of chemical sterilization with chlorine 
dioxide. 



v 



- 9 - 



f 



TA3LE III 



£H 



Time 


Buffered with 
NaHCO^ 


C02 Removed 


1 Hour 


6.5 


7.4 


2-i Days 


6.0 


7.4 


3| Days 


6.0 


— 


10 Days 


5.7 


7.4 


18 Days 


5.4 


6.4 


38 Days 


5.2 


6.7 


70 Days 


5.2 


6.6 



Another approach to developing a stable product was seen in a dry 
product of glycerol pectate which would only require sterile water for 
reconstitution. Several methods of drying properly degraded 4$ solution 
of glycerol pectate in normal saline have been investigated and found 
effective. Spray-drying, drum drying and lyophilization gave nonhy- 
groscopic products with good to excellent dissolving properties. The 
dry materials, upon reconstitution with water, had substantially the same 
characteristics as their original solutions before drying:. At present, 
lyophilization is the preferred drying method as all equipment required 
is readily available or can be easily assembled from standard laboratory 
equipment . 



Discussion 



The present status of the work on glycerol pectate allows some very 
interesting deductions and speculations with regard to its action as a 
plasma volume expander. The pectin, solutions (sometimes referred to also 
as pectin sols) used by Hartman and others have consisted of a 1 ^fo so- 
lution of degraded Pectin N.F. with a relative viscosity of around 4-5. 
They have shown a number average molecular weight of 18,000 ( 6 ) and a 
degree of esterification of about 6 C$. When we tried to prepare a suit- 
able parenteral solution from highly esterified Pectin N.F. , obtained by 
esterification of the free carboxyl groups with glycidol, we found we 
had to resort to higher concentration levels in order to attain oncotic 
pressures equal to plasma. It was quite evident that the loss of on- 
cotic pressure on esterification was not due to the slightly higher aver- 
age molecular weight and thus not due to a smaller number of molecules 
at a given concentration, but. that it was in direct proportion to the de- 
crease of free carboxyl groups. As pointed out before, the same was true 
for glycerol pectate where relatively minor variations in the degree of 
esterification brought forth significant changes in oncotic pressure in 
vitro as well as in response in vivo, whereas variations in molecular 
weight, as affected by various degrees of degradation, were without sign- 
ificant effect. As a. consequence, the number of free carboxyl groups in- 
cluding the sodium ions associated therewith was a contributing factor 

6 t.H. Schultz et al. J. Am. Pharm. Assn. 4l, 251 (1952) 



% 




for oncotic pressure. This is illustrated in Graph III where oncotic 
pressure is plotted against number of free carboxyl groups expressed in 
milliequivalents per liter. It happens from the curve shewn that between 
24 and 26 meq/liter of unesterified carboxyl groups are necessary to 
attain oncotic pressure equal tc 6 $ dextra solution. This would in- 
dicate that there is a practically unlimited number of combinations of 
concentration and degree of esterification that will fulfill these re- 
quirements. So, for instance, do the earlier pectin sols at 60$ ester- 
ification and at 1.5$ level. The possible compositions of solutions of 
pectic substances having a constant total free carboxyl content of 24 
meq/liter are represented by the calculated curve shown in Graph IV. 

Any combination of degree of esterification and concentration of pectic 
substances which falls on this curve should show equivalent oncotic 
pressure. However, the useable combination range is limited by two 
factors, (a) the threshold for calcium precipitation (at about 70$ ester- 
ification) and (b) the maximum permissible relative viscosity of the so- 
lution (at about 5$). While the 4$ level at 85$ esterification was stud- 
ied more extensively and found satisfactory in animals, other combin- 
ations falling on the curve of Graph IV, around the 4$ level, seem 
feasible to produce adequate oncotic pressure and may be also satis- 
factory as a plasma replacer. Compared with other plasma expanders such 
as dextren or FVP, glycerol pectatc has as an additional degree of free- 
dom, the degree of esterification, which allows a broader concentration 
range, and thus, in turn, also a broader- molecular weight range. 

Arrangements have been made with the National Bureau of Standards 
for molecular weight determinations on glycerol pectate solutions. In 
lieu of these measurements predictions can be made of the approximate 
magnitude. Based upon the determination of 1.5$ pectin sols of around 
4 viscosity, it is conceivable that at 4$ concentration and 4 viscosity, 
the average molecular weight will be about one-half of 18,000. Thus, 
assuming an average molecular weight of 10,000 for glycerol pectate, a 
molecule would be composed of 40 glycerol anhydrogalacturonote units. 

At 85$ esterification there would be 6 free carboxyl groups per molecule 
surrounded by an equal number of sodium ions . Both the carboxyl and 
the sodium ions are' likely to be hydrated . The arrangement of hydrated 
sodium ions around the polymeric ester acid may contribute to an appar- 
ent molecular size greater than expected from the true molecular weight, 
thus being retained in the circulatory system for a sufficient length 
of time to restore and maintain plasma volume in shock. 

Our observations have shown that at a given concentration, vis- 
cosity and degree of esterification, the oncotic properties of glycerol 
pectate vary depending upon the amount of electrolyte present. For ex- 
ample , solutions in pure water have 0 5-4 times greater oncotic pressure 
than in saline. In the circulatory system the oncotic pressure of gly- 
cerol pectate will also be influenced by the concentration of elect- 
rolytes and presumably also by the presence of plasma proteins. It is 
therefore conceivable that glycerol pectate is excreted more rapidly 
when administered to normal dogs since high electrolyte and plasma, pro- 
tein levels would be expected to depress the degree of hydration and 
thus reduce the apparent molecular configuration. In hemorrhaged dogs 



K«a» M M l »a«««»M«MBaMM*S« 9 «»!!fiSeBS"SSS*i»Sa§S™^ 



!■■■■■■■■■■■■■■■ 

■■■■■■■■■■■■■■■a 



iBBflflaaBBBflBBaaaaaflflflBaaBaaflBaBBflBBBflaaflflBflBflBaflBflaaflBBBaaBBaaBaBBBBaBBl 
■■■■■■■■■■■flBBBflflflBBflflflflBBBflflflBflflBflfl BBBBB BBBBB Bflflflfl BBBBB BBBBB BBBBflflBBBfl I 



.... ... H 5 SS 5 !!R 53 S 555 H 555 i 5555 T 55 !! 5 r 5 ?r! 5555 ! 555 ?rS 5 SB 5 ! 5 !SSH*l 

ii rm- 1 »:. i-i.r-ijpmpp *.._i mbbbbbbbbbbbI 

BrSiSiHi 5 ip«iHiiiiir?i 5 iiiniSiiiSiii 5 EEESSSSSSy 

IBBiBflSHTflflBflifiiflrBflBflBrrflBMaflnBBflTMEflBTBiBHBBBBBPBBB'rBrTjBriflBBBBBBBBBBBBBBBBBl 
EliSlBI.I tvi:K|l»»;HW ■«■»># iMSflBMiM'J'-UMNflira-MllllC-M^liUt •BBflnPfllBflBBBBBBflBflBBBflBBl 

MillllBmilll illll IlflXlt! I !!•':» U:i|l»B’/;)B|(i>fi;ik»; I lU -I IBHK ) JBBBBBBBBBBBBBBBBaBaBBBBaBa 

BBBBBBBBBBBflBBBBBBBBBBBBBBJIBPIBBBBBBBBPBBBBBBBBPBBflMBBBBBBBBBflBBBBBBBBBBBBBBBBBBB 
■■■■■■BBBBBBBBBBI * rt i.': InlBBKA CiflF.:* >!ilBi;.1 l«' , «Tl IB il f: I.< 1 1 L' )BB BBBBBBBBBIBBBBIBBBBIBBBBB 
iSSSSSSMiilMWiJMgJg^gJgglgjgjjjggJgJJJggJj^ggBBgggBBBMJBM 

glHESHMaMMBBHHMaMj 



WESSSESSSSSBSSfiSSSSSSBSSSSSSSSSSSSEESSSSSl 



|s 3 isS 3 SS! 2 £y 5 j]?!!”j«Ei«f 

■SSSSSESSBaiiiiiiiiiiifl iiiiiiiifliiiiii iiiiiiiiiiiiiiiiiiiiiiiiiiii 



■BBBBBBBBBBBBBBB' 

■MB BBBBBBBBflnifil 



BBBBBBBBaSMlBBBBaBBBraBBB 

ESSSSSSSSSSSSESBESSSSESSE 



ESS 



BBBBBBBBBBBBBBBBBflBBBnBBnaBBBBBIBBBaBBBl 
■BBBBBBBlElBBBBBlBaaBBBflaaBBBaHiBaMj 
■BHBBflliBBIIB lllflflHIBBIII 
a*BB BBBBBBBBBBBBBBBBBBBBBBBBj^^^^^^^M 

I ^HBaBBBflflBaBBBaaaBBBaBBBBaaaBBaBBBaaBB 
HflBBBBB BBBBBBBBBBBBBBB BBBBBBBBBBBBBBB 
. iHBBflB BBBBBBBBBBBBBBB BBBBB BBBBBBBBBBBBBBB 
BBbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb 

■Bfl BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBBBBBBB 
BBB BBBBB BBBBB BBBBBBBBBBBBBBB BBBBBBBBBBBBBBB 
■BlBflBBBiBBBBBBBBBBBBBBBBBB BBBBBBBBBBBBBBB 
BBBBBBgBBBBB BBBBB BBBBBBBBBBBBBBBBBBBBBBBBB 
■B BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBI 
IflBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBBBBBB 

S HBggBBflBflBBBBBBBBflBBBBBBBBBBBBBBBBBBBBMBiMl 
. : Bfl BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBBflBBBBi 
IBB BBBBB BBBBB BBBBBBBBBBBBBBBBBBBBBBBBBBBBBB I 
■BiaflBBBBBBBBBBBBBBaBflBBnBHBBBBBBBMBBBMM 
IflBBBiBBBBBBBBBHBBBBS 

mmaaaaamaaamaaaaaaaama iiiiiiiil 



ESSSSESSSSSSSbSbEEbbEES 

WWBbbbbbbbbbbBbbbbbbBbbbbI 

IbBBB iBBBfl BBBBBBBBBBBBBBB BBBBB Bl 
r'?lp v piB BBBBBBBBBB BBBBB BBBBB BBBBB B 



IBB BBBBB BBBBB B 



^^^^■bbbbbbbbbbbb 

BflBBBBBBBBHBBBBBi 



■bbbbH .. 

IBB BBBBBBBBBB BBBBBBBBP^BBBBI 
■iggBBBBBBBagBBBBBBBrBBBBBBI 
■BBiBBaBaBBBaBBBBBBOBBBBBBBI 



EESSSEEESEEESSEE 

BBBBBP fk-t'l J'J'U-ll 



EembbSSSESSSESESESEESEEBSSSSESSSSSESESEE 

I BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB 
| iBSBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB 
PPHHiBBBBBBBBBBBBBBflBBBBBBBBBBBBBBBBflflBinn 
E BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBB 
R^BBBMiBBBBBBBBBBBBBflBBBBBBBBBBBBBBflBBBBBBBBBHHI 

y£BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB Bl B— — j— — j— — i | 

S«t < iB> ■■ “ta . IBBBB^' 'f'V' . . .. . 

■SrSIlCB BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBr..-:iBfc.»BBr.aB--E l » *» 3 .-P'li l ».J* l BI'>iH.»:«'I"Hi^BBBBBBBBB«am"K,j| 

■ =BMPa»J BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BP^.BBBBBBBBBB«>r;r'JBBBBBBBBaBBBBBBBBBBBfli^B^BBB^H^ri 
■B BBBBB BBBflfl BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB ^BBBBBBaBBBBBBaiBH 

PlHBHBBMMHIBHBBBBMMH 

■! 



.... IflflBBBK „ 

Esssssssssssssssssssssss 

EESSESS SSSSgESSSSSSSSSBSEESSSSBSSl 





■MBHMbbbbbbbbbbbbbbbbbbbbbbbI 

IBBBBBBBBgBBBB BBBBB BBBBB BBBBB BBBBB I 



BBBBBBlIBBBBBBBBBBBaBBBBBBBBBBBBa 



■bbbbbbbb 

■bbbbbbbbb 

. _ HtfBMUBBBBBBBBBBflBaBBBflBBBBBB BBBBBBBBBB 

W BBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBBBBBBB BBBBBBBBBBBBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBBBBBBB 
BBBBB BBBBB BBBBB BBBBBBBBBB BBBBBBBBBBBBBBBBBBBBBBBBBBBBBB BBBBB BBBBB BBBBr. Bl "I'BI •iln »BBBBBBB ri^rSfl 
BBB BB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBBBBBBBBBBBBBBBB" 5 B 3 F^a«BLBJJBBWBBiIBL!J 
BBfififi BBBBBBBBBI BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB IBIBI BBBBB BBBBB BlflBBIBBIfl BBBBB BBIBB BBBBB 
I BBBBBBBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBBBBBBB BBBBBBBBBBBBBBBBBBBB 
BBBBB BBBBBBBBBBBBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBBBBBBBBBBBBBBBflflBBBBBBBBBB 
I I V*I^B BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBBBBBBBBBBBB BBBBBBBBB ?hB?V?^BB 7 iir?PT <,M * 4 BBBBBBBn 7 r«B 
h.A^£fl BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBaBBBBBBiMC 2 ^wCBB^«^ 3 UXLl»BBMIIBBBiJZiL!!B 
BBBBB BBBBBBBBBB BBBBBBBBBBBBBBBBBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBBBBBBBBBBBBBBBBB 
BBBBB BBBBB BBBBBBBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BflBBBBBBBBBBBBBBBBBBBBBBB 
BBBB B BBBBB BBBBB BBBBBBBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBBBBBBBBBBBBBBBBB 
flj^BBB BBBBB BBBBBBBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBBBBBBBBBBBBBBBBBBBBBB BBBBB 
BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBBBBBBHBBBBBBBBBB 
BBBBB BBBBBBBBBB BBBBB BBBBB BBBflfl BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBB BBBBBBBBBBB 



knBB 

■bbH 

E BBBBB 

EfMWfll 

t®'B>i*,.»B 

bHhBBBBl 

gss; 

mu 



^^^^^|iBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBBBBBBBBHHBI^H^^HBi^^^l^^^H 
[BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB SBBBBIBBBB BBBBB IBBBfl BBBBB BBBBBBBBBBBBBBB I 
^^^^^|bBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BflBBBl 
BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB I 
^^^^^|BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBBBBBBBBBBBBBBBBB BBBBBBBBBB BBBBB I 
^^^Bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb bbbbbbibbbbI 
^^^^^B|BflBa BBBBB BBaaaaBBBBBBBBB BBBBB BBBBBBBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBaBBBBm| 
[BBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBB BBBBB BBBBB BBBBBBBBBBB 
IBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBB BBBBBBBBBBB BBBBBBBBBBB BBBBB I Bfl BBBBBBBBBBB 
IBBB BBBBB BBBBflflBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBBBBBB 
^^^^HaBBBBBBBBBBBBBBBBBBBBBBBBBBflBBBaBBBBBBBBBBBBBBBBBBBBBBBBBBaBBaBBBBBBBBBBi^^^^^^^H 
ILASaPB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB 
HBjHB BBBBB BBBBB BBBBBBBBBBBBBBB BBBBB BBBBB BBBBB BBB BBBBBBBBBBB BBB BBB BBBBBBBBBB BBBBB BBBBBBBBBB 
I BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB 
I BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB 
I BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB 
IB BBBBB BBBBB BBBBB BflBBBBBflBB BBBBB BBBBB BBBBB BBBBB IB BBB BBBBB BBBBB BBBBB BBBBBBBBBBBBBBB BBBBB 
IB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB 
HMiBBflflBBBBBBBflaflBaflBBBaBBBBBBBBBBBBflBBBBflBBBBBBflBflBBaBBBBBBBBBBBBBBBBBflBBBBBBBflBBBaaBBBB 
BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB 
EB^B BBBBBBBBBB BBBBBBBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB 
BBBBBBaaflBBBBBBaBflBB BBBBB IBBB BBBBBBBBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB 
BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBBBBBBBBBBBBBBBBBBBBBBBBBBB BBBBB BBBBB 
BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBBBBBBB IB BBB BBBBBBBBBB 
BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBBBBBBB BBBBBBBBBB BBBBflflBBBB 
BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB 
BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB 
BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBBBBBBB I Bfl BBBBBBBBBBII 
BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBBBBBBB 
BBBBB BBBBBBBBBB BBBBBBBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB 
BBBBB •••■■•■•••■•■•••■■•••••■■■■•■•■■•■■■•■•■••■■■••■■■■■■■■■•■■■■■■■■BHUMMauMaBBBH 

BBBBCr 4BBBBBBLT IBBBBBBBr;T.'lBBBaaBarT.-<BaBBBBar.'1 nBBflBBBBP^^aBBBBBBBT’.IBBBBBBarPBBBBBBB-rBBBafl 
BBBBP V’JBBBBBBP y'JBBBBBBar-^’JBBBBBBBrilBBBBBBBr -4. iBBBBBBBB*! •BBBBBBBBB. •IBBBBBIBBH l lBBBBBBI>r:flBBBB 
BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB 



BBBaiBBBBBBBaBBBBBBBBBBBBI'j-’VJ*'i»l<aL , l’VH'L-Z[.^BF*l l l ! .• l'l r iP V ^. r - I 'BBBBBBBB BBBBB BBBBB BBBBB BBBBB 



! 




£:s: 5 :ss&s:ssss»ss:BasB 3 as:ssassss 5 sssr^sM?Hi^Hrii(jmBs:as 5 S 5 S 3 aass 85 S 5 S 5 »BBSSBSssl 

>iBi i , iRBBiilliiHEaBBBEaBBaBBBBBiBiBaiBaaaBaaBB| 
■ BBBB BBBBB BBaBBBBBBBBBaBaBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBIBBBBBIBBBBBBBBBgggggBBBggl 

BaaaiBBaBBaaaaBaaaaaaaaaaBaaaaaaaBaaaBaaaaaaaaaaBBaBaaBaBaBaaaBaBBaaaaaBaaaaBBBaaSg 

BBBBBBBBBB-BBBaBBBBBr^airBrBBBrBririBBBBriBBBBTBPBBBBBBnBBBBBBrBBBnBBBBBBB^BrBBBnrBBBi 



BBBBI 



I ■!![•> rf-! ( 1-i-jiM 7 »; « BI'IJ BB>.( H BBBBB BBBBB I 



ii!»i<71 r"r7S7Ki?“i;ir^B*i"7ri“ii""!IrBBkTi»"iiidrr“i!5ri’f BiaBBSSSSS 

,==:=============::====:^=-r======r-:r:55====d=5======= : ;==r:===~===aBl8BBB3Bt 



! BBBBBBBBBBBBBiBiBiBiiiiBiiiiiiiiiipn7icL9B77f?i?i<?i^ilaBiiSiiiiiiiiiiiiiiiiiiiiBBn 

BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB“-r~“— *“r— 'BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBl 
BBBBBBIBBBBBBBBBBIBB BBBBI BBBBBBBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBlI 
BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBflflBBBBBBflBflBBBBBBBBBBBBBBBBBBBBBBBBBBBflBBBflggBBBBBBBBBBBflBBB| 

bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbI 

!BBBBBBBBBBBBBBB7Vi:i'»B»**->*l>T1B!>PB»l*l , BBB"'r*«IB!"piB™BBBW-PBBnBBB1BBBBB:*BBBBcfiBBBBBBBBBBBBBBBBl 

I BBBBBBBBBBBBBBBttt*^C:B»./IJ^l:UtiBtil.--J»,'ll.-i;-a-J'fll-JBl'?"ML'BL'-i>l»P 1 ' < *-11' ! ' ' tl <1".» BBBBBBBBBBaB BBBBbI 

BBBBBBBBBBBBBBBFBBB"BBB-»BI’BB"BBBBrrBBBiB|iBBnnBBBBBBBBnrBBBnBBBBBBBBBBrBBB»lBBBBgBBBBiBBBBBBBl 

iflBBBBBBBBBBBBBBL'V’JI L'L-^ Mirat'K^' BH»J.»(ML*i" BM :i"l'BlB' IBHI i IB~ lll'l «,'/‘;B( •liBBBgHBBBBBBBBBBBal 

■aa—— a— ——a——— — — wbbbbbb bbbbbbbbbbbbI 

iBBBBBBBaaaaaaaaaaal 



BBBBBBBBaBBBHBIflBBBBBrBBBBBBMBlIBBBMBRflBflBflBBBBBBBBgH 

BBBBB BBBBBBBBBBL.'.? I VBI"MI IHU IlIBBgBBBBfllBBjflHBHHHHHBBHMMHHHMn 

BBBBB BBBBB BBBBBBBBBB BBBBB kflBBB BBBBB IflBBBBBBBB BBBBB BBBBBBBBBBBBBBBBBBBBflBBBBBBBBflBBBBBBBBBBl 



IRBBBBBBBBBBBBBBBBBBBBBI 



BBBB BBBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBgflBBBB BBBBB BBBBB BBBBBBBBBBl 
1^131 BBBBBBB BBBBBBBBBB BBBBB IflflflB BBBBB BBBBB BBBBBBBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB I 
BBBBBBBBBBBBBBBBBBBBBBBBB BBBBB BBBBI BBBBBBBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBflBBBBflBBBBBBI 
bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb bbbbI 
BBBBBBBBBBBBBBBBBBBBBBBBB BBBBB BBBBBIBBBBBBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBfiflBB 
■■■BBaBailBBBBIBBBBBBflBBBBBBIBBaBflBBBIflBaiBflMiflBflBBiaBBBBBflllBBlflmBmiBBBBBniBBB^H 



BBBBI ■BBBBBBaBBBBBBBBBBBaBBBBBBBBBBBBBBBBBBBBBBBBBBBBBaBaBBHBBflBBBBBBBBBBflflBBBBBBBBjBal 
■■■rr.'r^BBBBBBaBBBBBBaBaBBBBaBflflflBBBBBflBflflaBRBBflBaflBflBBflBBBflBBBaaaaBaaaflaaafiiiaaBaBMBBBBM 
BBBBBC.XL4^4B BBBBBBBBBBBBBBBaBaBBflBflflaBBBBBBflBaflBBBflBBBBBBBBBBBBflBBBBBBBBBMBBBBBaBBflBBBBBBBBB| 
■BBBBBBBBB BBBBB BBBBBBBBBBBBBBBBBBBBBBBBB BBBBB BBBBB BBBBB BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB I 
BBBBBBBBBB BBBBBBBBBBBBBBB BBBBB BBBBBBBBBB BBBBB BBB BBBBBBB BBBBB BBBBB BBBBB BBB BB BBBBB BBBBBBBBBBl 
BBBBBBBBBB BBBBBBBBgBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBgBBBBBBABABBaBBBBBBBl 
BBBBBBBBBB BBBBB BBBiBBBBBB BBBBBBBBBB BBBBBBBBBB BBBBBBBBBB BBBBI flBflBBBBBBB BBBBB BBBBBBBBBB BBBBB I 
BBBBBBBBBB BBBBBBflBBBflBBBBBaVnr BBBBBBBnarBBBHBBBBBg BflBgBBBBgB BBBBB BBBBB BBBBBBBflBBBBBBBBBBBB| 
BBB BB B B BBB BBBBBBBBBBBBBBBBBim»^'tH>.Hl«^*BI«<-HHIIN HI ^BHBaiBBBBBiBBBB BBBBBBBBBBBBBBwrarr-BBBBBl 
BBBBBBBBBB BBBBBBBBBBBBBBB BBBBBBBBBB BBBBB BBBBBgC'BBBgBBBBBBBBIflaBBaaaBBBaBBBMBBHMMHBBHBMBH 

BBBBBBBBBB BBBBBBBBBBBBBBBBBBBBBBBaflaBBaBBBBBflBB^BiiflBBBBBBBIB*>=^MaiBafliBBB3i 

bbbbbbbbbb bbbbbbbbbbbbbbbbbbbbbbbbbbbbbBbbBBbbbbl'<bbBbbb»»t:=.mbbbbbbbbbbbbbbbbbbbbbbbbbb bbbb| 

BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBglBBBB BBBB/: -^a^flBBBflflBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBBH 
■BB—BMB— B— — — — BBBBBBBBBBBBiiBBBBBB*;s..' J |BBBBBBBBBBBaBBBBBBBB BBBBBBBBBBBBBBBBBBB| 

HBBBBBBBBBBBBBBBBBBBBBflBBaBBBBflBBBaflflRMBBBBflaBBBB I BBBBB BBBBBBBBB I 

■Bl .JBBBBBfl BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBmd BBBBBfl BBBBB gBgBBBnriBB BBBBB BBBrB IB rBBBBBgBBBBBgl 
BBBBBBl'7nlBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB’:BBBBBBBBBBBBBBSBBaBB'J‘'l-l>l»;*|iBni , /BPr «u«BUBBBBBBBBBb| 
BBBaBBO^B BBBBB BBBBBBBBBBBBBBBBBBBBBBB^.BBBBBBBBBBBBgBBBBBBBBBBBBBBBBgBBBBBBBBBgBBBBBBBBBBB| 
■BB.BBBBBI BBBBBBBBBBBBBBBBBBBBBBBBBBroBBBBBBBBBBBBBBBBBiBBBBBBBBBgBBBBBBB BBBBBBBBBBBBBBBBBl 

■■■■aaBaaBaBBaaaBaaflaBBaBaaaaBaBaBiBaaaaaBaBBaBaaBaBHaifiaMiaBliaaaaBaBPPMBBBBBBBBBBBBl 



B>T==^aaaaBBBBBBBBBBBBl 



BBBBBBBBBB BBBBflflBBBBiaBBBBBBI 



BBRaBBBBIBBBBRBBBBBBBBBBBBBBBBBsflif^BBBBMBBBBBBBBBBBBB^^M > 

BBBBBBBBBB BaaBBBIBaaiaBaBaaBBBBBBr^BMBSIsaBfiSIRMBBBBiaaaSiaiSlgf Biggl 
BBBalflBBBI BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBB^^B 
BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BFBBB BBBBB BBBBB BBBBBBBBBBBBBBgBBBgBBBBB 
■BBwBBBBBB BBBBB BBBBB BBBBB BBBBB FBBBBBBBBB BBBBB BBBBB Bl 
•t> . 1BBBI ' '=r ■■ . CH3 ZBIBBBI IB .■ ■ BBBBBBBI : . IBI..« a 

■BBBBBBBBB BBBBB BBBBBBBBBB BBBBBBBBBB BBBBBBBBBB BBBBBBBBBbSbBBB BBBBB 

SKsasHBsr^^^^Ki^ssssssss^ssssssasssssssssssssasBsassasssssssssasassaHHHBHHHi 



■ BBBBB BBBBB BBBBB I 



■BBBBBBBBB BBBBB BBBBB BBBBB I 



■B BBBBB BBBBB BBBBB BBBBf ( > BBBBBBBBB BBBBB BgBgggBBBBBBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBBBBBBBl 
IflflT'fn BBBBB BBBBB BBBBB i7B BBBBBBB BBBBB BBBB BBB BBBBBB BBBBBB BBB BBB BBBBB BBBBB BBBBB BBB BBBBBBB I 



BBBBBBBBB! BBBBB BBBBBBBBB' JBBB.lt* l' 



■BBB BBBBB BBBBB BBBBB BBBBB BB 



BBB BBBBB BBBBB BBBBB BBBBB BBBBB I 
■BB BBBBB BBBBB BBBBB BBBBB BBBBB I 
BB BBBBB BBBBB BBBBBBBBBB BBBBBfl 



■■■^BBBBBB BBBBBBBRBBBaBr IB BBBBI 
aBFi'BBBBBI BBBBB BBBBBBBBBB B BBBB B 
BBB-BBBBBI BflBBBBBBBflBBIBBBBBBBBH 
BBBBBBBBBB BBBBB BBBBB BBfiBB BBBBB BBBBBBBBBBBB 
BBi:-BBBBBB BBBBBBBBBBBB IBBBBBBBBBBBBBBBBBBB 

BBBBBBBBBB BBBBB BBBBB Bl flBflBBBflBIBfllBIBBBBBBHHHHHHHHHHHHBBHH| 

BBrBBBBBBB BBBBB BBBBB BNBBB BBBBB BBBBB BBBBB BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB^B 
BBBBBBBBBB BBABB BBBBB BBBBBBBBBB BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB B| 
BBBmiB BBBBB BBBBBBBBBB V BBBBBBBBBBBBBBBBBBB gBBBBgBBgWBBBBggBBBBBBBBBBBBBBBBBBBBBBBBBBBBBW 
BBBBBBBBBB BBflaBBBBBBriBBBBBBflBBBBBBBBflBBBHBBaBBBBaBaBBBBBBBBBBBBBBBBBBBBBBBBBaBaBBBBaa 
BBBBBBBBBB BBBBBBBBBB JBBBBBBBBBBBBBBBBBBBMBI 

BBBBBBBBBB BflBBBBflBBBaBBBBflflBBflBBBBBBBBBBBBnMHHMMHBMHHHHBHHHHHHHB 

Baa-*BBirriia bbbbbbbbbv bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbBbbbbbbbbbbbbbbbbbbbbbb^^^^M 

BBBaBBBBUB BBBBB BBBBfJ BBBBB BBBBB BBBBgBBBBBgBBBg BBBBBgBBBB BBBBB BBBBB BBBBB BBBBB gBBBBBBB®*SS| 
BBB- ‘ 1 BBBBB BBBBBBBBB IBBBBBBBBBBBBBBBBBBBBBgBgBlBBiBfiBgBBagg BBSgBlBBBBBglliBBiBB—aa—i 
BBBr.-BBBBBBBBBBBBBBBIBBBBBBBBBBBBBBBBBBBBBiBBBgBBBBiBBBBBBaaaiaaaaaaaaaaaaaaa«ai 



■BBBBBBBI 

■BBBBBBBI 

■BBBBBBBI 



IBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB BBBBBBBBBBBl 

MMMMMMMMMMMMMMMtBBNBIBBBB 

BBBWBBBaaaal 



■■■■■BaHHHaBHBBHHHHBaaBBBaBaaaaHaaBBHapHBB! 

IflBr.-BBBBBB BBBBB BBBIB BBBBB BBBBB BBBBBiBBBaBBBBiBBBBg 
BBIaiLB BBBBB BBBBBBBBIBBBB BBBBBBBBBBBBBBBBBBBBBglBBBij 

bbbbb bbbbb bbbbbbbbi ibbbbb—b— i 

pailB BBBBBBaaBBBBIMBBBBBH 
■BBBBBBBBB BBBBBBBB IBBBBBB 



■BBB BBBBB BBBBBBBB BBBBBBBBBBl 
■BBBBBBBI !■■■■■■■■■■■■■■■■■ 



BBBBBBBI BBIBBH 

BBBBBBBBBB BBBBBBBI BBBBBBBHBHHHB 
BBBBBBBBBB BBBBBBBI MBBBBBBBBBBBBBB 
BBBBBBBBBB BBBBBBBI IBBBBBflBBBaaaaaBMHMMM 
BBBBBBBBBB BBBBBBB' IBBBBBBBBBBBB BBBBB BBBBBBBB 
BBBBB B> full BBBBBBB IBBBBBBBBBBIB BBBBBBBBBaaaB 
|BBflBBBUK^B BBBBBBB IBBBBBBBBBBBB BBBBB BBWIBH 
BBBBBBBBBB BBBBBBB BBBBBBBB BBBBB BBBBB BBBBB BBB 
■BBBBBBBBB BBBBBBI BBB BBBBB BBBBII BBBBB BIIBffBBB^ 



* ■11111 BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB I 
IflBBBB BBBBB BBBBB BBBBB BgBBBBBgaaBHaaai 
^■BBBBBBBBBBBBBBBBBBBBBBiBBBBB^^^^^H 
IgggBBBBBBBBBBBBBBBBBBBBgBBBBBBBBBBBagBBl 
BBB B BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB I 
■■BBBBBBB BBB BBBBBBB BBBBB BBBBB BBBBB BBBBB I 
BBBBBBBBBBBB BBBBB BBBBB BBBBBBBBBB BBBBB I 
^■BBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB | 

■bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbhbI 

H M bbbbbbbbbbbbbbbBbbbb bbbbbbbbb—| 

■■■■■■MaaBaaiiiiaaBBaaaaaaaafl 



I BBBBBBI BBBBBBBB BBBBB BBBBBBBI 
I BBBBBBI BBBBBBBBgflBBBBBBBBBBI 
I BBBBB BIBBBBflBBBiBaBa BBBBBBBI 



MSS ! 

S ■BBBBBBBBB 

■bbbbbbbbbH 

BBBBB BBBBB BBBBBBI /BBBBBBBB BBBBB BBBBB BBBBB BBBBgl 
BBBBBBBBBB BBBBBBlIBBBBBBBBBBBBBBflBBBBBBBBflBBBS 
BBBBBBBBBB BBBBBBI IBBBBBBBBflBBBBBBBBBBBBBBBBBBBj 
BBBBBBBBBB BBBBBBI IflBBBBBBBBBBBBBBBBBBBBBBBBflBH 

BBBBBBBBBB BBBBBB' IBHIBIIBIBiaBBBBBBlIBBBBBB^^^^^^^^H 
BBBBBBBBBB BBBBBB IflBBBBBBBBBBBBBBBBBBBBBBBBBBgBBBBBggBBgBBBggB! 
BBBBBBBBBB BBBBBB ■aflBaflBBaBBflaaflBBBBflBBBBRBBaglBgBgBgaBflBBiiiBI 
BBBBBBBBBB BBBBBB IBBBBBBBBBBBBBBBBBBBBBBBBBBB jiBiBgllBBlBljjBgBI 
BBBBBBBBBB BBBBBB ■■■■■■■■■■■■■«■■■■■■■■■■■■■■■■■■■■■■■■■■■■ 



PHHHHBBBfliBBBBB! 

■RiiiiiiiBiiBBBBBB! 

^^^^^^^^^llBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBiB BaBB BBBI 
BBBBBBBBBB BBBBBB BBBaBBBBBBBBBBBBBBBBBBBBBBBBBBiBBBBBBBBBBBBBaBBBBBBBBBBBgBBBBI 



SSibbbbiSSbbS 

SHHKSSK 

Ebbbbbbbbbbbi 



BBBBB BBBBB BBBBB BBBBB* /BBBB BBBBY* IBBBBBBBBC'lBBBgBBBBB BBBBBBI 
■BgBBBBBBB BBBBBBBBBB* 'BBBBBBBBr BBBBB BBBBB ’JBBBBBgBBL 1BBBM 
■BBfliBBBBBBBBflBflBBBaBBBBBBBBBBBBBBBBBBaBBBBaBB^^^^^H 



pNHIIIIfllll 

. ■■BrniBBiBifl D 
BBBBBBBBB IBBBBBBBBBl 

Bbbbbbbbbb bbbbb bbbbb I 

BBBBB BBBBB BBBBBBBBBBl 



|BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB^J! MLia3.L!j.2B- I'rS-. fBf lar.-iBaBM 

■— | gg— ■■■ gggggggggg BBBBBBBBBB BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBl 

PmbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbI 

. ... .... - . .HiB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB I 

BBBBB BBBBB BBBBB BBBBB BBBBI BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBBBBBBB BBBBB BBBBBBBBBBl 
BBBBBBBBBB BBBBB BBBBB BBBBBBBBBB BBBBB BBBBBBBBBBBBBBB BBBBB BBBBBBBBBBBBBBBBBBBBBBBBB BBBBB BBBBBB 



BBBBB BBBBB BBBB BBBB BBBBBBB BBBBBBBBBBBBBBgfll 
BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBI 
BBBBBBBBBB BBBBB BBBBB BBBBB BBBBB BBBBB BBBBBBI 




- 11 - 



the opposite effect should be observed, that is, retention should occur 
due to an increase in hydration until normal plasma protein and electro- 
lyte levels are restored. The same phenomenon may also be explained by 
a charge effect on the membrane, in vitro as well as in vivo, which is 
also dependent upon the concentration of electrolytes. 

Whatever the correct explanation may be, the fact that glycerol 
pectate, at an average molecular weight considerably below that of 
plasma protein, has proven to be an effective plasma replacer is of 
great significance. Once the plasma volume is restored to normal the 
glycerol pectate can leave through the kidney much more easily than a 
synthetic plasma expender with an average molecular weight exceeding 
that of plasma proteins, for example, dextran. Where emphasis is placed 
in avoiding deposition in the tissue an effective synthetic replacer with 
an average molecular weight below the barrier of the normal kidney is un- 
doubtedly preferred. 



Program for Future Research Studies 

Future work and research studies are contemplated on: 

1) Preparation of glycerol pectate for biological studies at 
Camp Lejeune and for molecular weight determinations at the National 
Bureau of Standards. 

2) Further studies on the relationship between the oncotic pressure 
and the percentage of free carboxyl groups present in the glycerol 
pectate . 

3) Continuation of studies on the stability of glycerol pectate 
solutions and on the development of a dry product suitable for extended 
storage. 

X) Improvements on the synthesis of glycidol. 

5) Esterification of pectic acid in a heterogenous system with 
lower quantities of glycidol. 

6 ) Analytical methods for determination of glycerol pectate in 
blood, urine and possibly tissue. 



- 12 - 



Acknowledgment 

This research was carried out under grants from the Office of 
Naval Research and the Office of the Surgeon General through the Office 
of Naval Research. 

Appreciation is expressed for Interest in this project to Colonel 
John R. Wood of the Office of the Surgeon General and to Drs. R. 
Robertson, 0. E. Reynolds, Lewis Larrlck, Paul Lindsay and Captain 
C. W. Schilling, and we are appreciative of guidance in this research 
shown by Commander Dr. W. J. Perry, Head, Clinical Branch of the Office 
of Naval Research. 

Grateful acknowledgment is given to the staff of the Naval Medical 
Field Research Laboratories at Camp Lejeune for providing the bio- 
logical evaluation of the product prepared under this research contract 
and especially to Captain Dr. C. B. Galloway, Commander Dr. S. W. 
Handford, Drs. C. M. Smythe , R. S. Leopold and R. H. Kathan for their 
continued guidance and help in this project. Dr. Handford has accomp- 
lished the biological evaluation of the materials supplied to him in a 
remarkably short period of time due to his undaunted efforts . 

Early evaluation of the products of this research were undertaken 
by Dr. F. W. Hartman and Dr. Vivian C. Behrman at the Henry Ford Hos- 
pital, and it was this work that provided the biological background for 
later research studies. Dr. Hartman has been continually available for 
counsel and advise on this project for which we are greatly indebted. 

Dr. Urs Nager has written this report covering this research work 
in which he was the principle investigator with Drs. E. E. Stahly and 
R. G. Jennen and the undersigned contributing a part of their time and 
acknowledgment is hereby made for their contribution. 

The biological studies relating to glycerol pectate will be re- 
viewed in a. report by Dr. Handford. ~ 



Oliver W. Burke, Jr. 



\