Process for the preparation of a selective anorexogenic substance regulating food intake

A process is disclosed for the preparation of an endogenous anorexogenic substance obtained from blood serum. The anorexogenic substance itself is also described having a specific activity of 100 units/mg.

BACKGROUND OF THE INVENTION 
In No. RI-683 Hungarian patent application (U.S. Pat. No. 4,294,825) we 
have expounded a process for the production of an anorexogenic substance 
having specific effect on the central regulation of food intake. According 
to the procedure described in that patent, human and/or animal serum was 
ultrafiltered on a membrane passing molecules under 50,000 daltons (a 
membrane with 50,000 dalton cutoff), the ultrafiltrate was taken into 
dryness, redissolved in water and the solution was chromatographed on a 
gel column having the void volume at 50,000 daltons. The column was eluted 
with an aqueous solution of 0.1-1.0% sodium chloride and the fractions 
containing biological activity were evaporated and the residue was 
dissolved in water and again chromatographed on a gel column which had 
50,000 daltons void volume. 
Fractionation was carried out with water and the biologically active 
fractions were evaporated. In this way we succeded to separate an active 
glycoprotein fraction which contained amino acids at an average of 60%, 
and carbohydrate components at an average of 10% after acid hydrolysis. 
The intact molecule showed specific activity on the central regulation 
system of food intake and did not behave as a depressive agent nor did it 
stimulate the central nervous system. 
DESCRIPTION OF THE INVENTION 
Elaborating this invention it was found that the active fraction can be 
further separated by the procedure described below and a new product 
obtained which is 3 to 4 times more effective than the previously purified 
one; the chemical composition of the new product essentially differs from 
the active fraction described and prepared earlier and it can be 
characterized as a pure homogeneous substance with well defined chemical 
composition. 
We have found that a considerable part of the peptide content of the 
earlier purified active fraction is not covalently linked to the substance 
which is responsible for the biological activity and exists as indifferent 
components which can be eliminated by proper protein precipitation 
methods; at the same time the still retained small peptide-like molecules 
and others can completely be removed partly by electrophoresis and partly 
by affinity chomatography, and the pure chemically substance may be 
obtained this way. 
According to the invention, the new substance which has an activity which 
is a multiple of that of the previously prepared fraction and affects 
selectively the satiety center, is chemically homogeneous, i.e. the pure 
satietin can be extracted from the human or animal serum in the following 
way: 
Human and/or mammalian serum is filtered through a membrane with 50,000 
dalton cutoff, the ultrafiltrate is then concentrated and the insoluble 
fraction is removed by centrifuging (advisable), then to the supernatant 
5-25% (w/v), preferably 10-12% (w/v), trichloroacetic acid is added 
keeping the temperature between 0.degree.-10.degree. C., the precipitated 
proteins advantageously being, removed by centrifuging. The supernatant 
obtained is chromatographed on a gel having a void volume under 4000 
daltons with a solution of 0.5-1.0% sodium chloride or with pH 6.0-7.0 
buffer. The biologically active fractions were concentrated by 
lyophilization and subjected to chromatography on another gel column with 
void volume of 3000 daltons, fractionated by using distilled water and the 
crude product obtained in this way was further purified by electrophoresis 
and the pure material was isolated by means of affinity chromatography. 
According to the process of this invention the execution of the first 
purification step, ultrafiltration, is carried out on Amnicon UM-10 
membrane or on Sartorius membrane, for example, preferably under 3 at 
pressure and with constant strirring. The filtrate was concentrated by 
vacuum evaporation and the insolubles removed by centrifuging. The turbid, 
but precipitate free solution was brought to between 0.degree.-10.degree. 
C. and trichloroacetic acid added till the trichloroacetic acid 
concentration of the mixture reached 5% (w/v)-25% (w/v), preferably around 
10% (w/v). Then the suspension was kept between 0.degree.-5.degree. C. at 
least for 1 h (preferably overnight) then the precipitated proteins were 
removed by ultracentrifugation at the same temperature when optically 
clear yellow supernatant was obtained. The trichloroacetic acid treatment 
removes all the high molecular weight serum proteins including serum 
albumin. Satietin containing high amount of carbohydrate, nevertheless, is 
not precipitated but remains insoluble in the solution in contrary to some 
amount of protein are still existing. It follows that this protein content 
already is covalently bound to the carbohydrate part in the satietin 
molecule. 
Preparative gel chromatography was applied as the next purification step on 
a gel column with a void volume of 4000 daltons. This purification is 
preferably done on Sephadex G-15, G-10 or G-25 columns. 0.1 M ammonium 
acetate, pH 6.6 buffer was used as eluent, since according to the 
experiments this buffer assured the separation of satietin active 
fractions with best efficiency and a practically salt free product could 
be prepared by means of this volatile buffer. For the chromatography it is 
advisable to use 0.9% physiological sodium chloride solution, or different 
phosphate buffers can also be applied in pH range of 6.0-7.0. The active 
fraction is excluded from the gel column at the void volume (V.sub.o) 
where K.sub.d =0; K.sub.d : distribution coefficient of chromatographed 
substances; this means that the molecular size of the product is larger 
than the inner holes of the gel particle so they are excluded from the gel 
and appeared with the eluent at 1/3 volume of the gel column. In case of 
Sephadex G-15 gel the molecules which are larger than 1500 daltons behave 
like this. The smaller molecules from the serum, on the other hand, 
penetrate the gel and are retarded (this being the case where K.sub.d &gt;0). 
Because the amount of small molecules and salts are incomparably higher 
than the separated satietin content in the serum, it clearly indicates 
that the purification rate is probably several hundred fold compared to 
the amount of satietin active material to the other small molecules and 
salts in the solute which are being retarded on the column. The 
trichloroacetic acid, used for protein denaturation, as a small molecule 
is also retarded on the gel column and so salt and acid free active 
substance is obtained at the void volume 1/3 of the total volume of the 
gel column. These fractions were pooled and concentrated by 
lyophilization. 
The next step of the purification is also gel chromatography which is 
carried out, however, on a 3000 daltons void volume gel, practically on 
Bio-Gel P-2 column in distilled water. As a result of this step the still 
remaining salts and other small molecular weight fragments are removed. 
The fractions having satietin activity are also excluded from the gel 
column, and appeared at an elution volume of 1/3 the gel column volume. 
The active material is obtained after pooling and freeze drying of the 
fractions and obtained as light yellow or white powder. In this way 8-10 
mg lyophilized product is obtained from 1000 ml (2/3 liter ultrafiltrate) 
human serum. Satietin purified by the above described method can be 
considered as standard crude material which, however, can be used for 
practical purposes as anorexogenic substance in this pure enough form, its 
satietin activity is as much as 25-50 units/mg. 
The satietin activity of the product is measured by a bioassay worked out 
by us: 1 unit is equivalent to the anorexogenic activity of the amount of 
a satietin sample which, when given intracerebroventricularly, decreases 
the chow pellets consumption of rats deprived of food for 96 hours, during 
the first day of feeding, from 24.04.+-.0.76 g to 10 g. 
The crude satietin, which is produced according to this procedure described 
in this invention, can be obtained from the sera of different animals, as 
cattle, horse, rabbit, rat as well, although the yield and activity of the 
products obtained can be different i.g. from bovine serum 10-13 mg of 
crude material can be extracted from 1 liter serum with the activity as 
the human product, the specificity of the active substance of different 
sources was alike. 
By the help of above described method 25-50 units/mg activity crude product 
showed an apparent molecular weight of 50,000-70,000 daltons, was a salt 
free substance which practically does not contain albumin and had a low 
protein content (5-25%) and high (60-90%) carbohydrate content in 
lyophilized yellow-white powder which carbohydrate part consists of four 
hexoses, fructose, mannose, galactose and glucose. 
A considerable amount of glucoseamine could be detected in every case in 
the product after acid hydrolysis. Analyzing the product 4-6 protein 
stained bands (components or subunits) could be detected by means of SDS 
gel electrophoresis and analytical isoelectric focusing. By the use of 
high-voltage electrophoresis the product also proved to be still complex 
at pH 6.2 and 1.6 buffer systems. The biologically active major component 
does not move or only slightly moves from the start in the direction of 
the negative pole. The active components were detectable by ninhydrin or 
periodic acid-Schiff staining simultaneously indicating the glycoprotein 
nature of the material. 
The crude satietin obtained by the above mentioned method can be further 
purified by electrophoretic methods, practically with paper 
electrophoresis under laboratory conditions. In the course of the paper 
electrophoresis separation in pH 6.2 buffer, the major product (R.sub.f 
=0.1) can be separated with 75% yield, while the other glycopeptide-like 
components were moved towards the positive pole, thus the procedure 
resulted in highly active substance identically stained with ninhydrin and 
periodic acid-Schiff reagent was nicely separated. (60-100 unit/mg) 
The separated active material is already a highly purified product, 
according to the previously used methods; for example it seems to be 
homogeneous by gel chromatography of gradient polyacrylamide gel 
electrophoresis, with nearly 100 unit/mg activity, and essentially a 
glycoprotein-like material, which, however, as the composition and 
activity values fluctuate, cannot be considered a chemically homogeneous, 
standard substance. In our further studies we tried to isolate a pure 
homogeneous material in every respect and we have found that this problem 
can be solved if the active substance is submitted to a further 
purification step namely affinity chromatography (which is a newly 
developed technique for separation of glycoproteins, e.g. D. H. Swallow, 
L. Evans, D. A. Hopkinson: Nature 269, 261-262; 1977). 
For this step a sorbent is needed which is capable of binding specifically 
a group of substances to be separated; since satietin is a glycoprotein 
and contained covalently linked carbohydrate moiety we apply an adsorbent 
which shows specific affinity for the glucopyranose groups of satietin. Of 
this group of specific adsorbents, especially the Con A-Sepharose gel 
proved to be convenient for tis purpose. This adsorbent-gel is Sepharose 4 
B activated by cyanbromide to which Concavalin A is coupled, and which 
specifically binds the alpha-D-Mannopyranosyl- or alpha-D-glucopyranosyl 
groups while the other substances (proteins, peptides, etc) not containing 
these groups will be excluded from the gel. These excluded substances 
appeared at the void volume in the course of the elution, at 1/3 volume of 
the total volume of the gel column, while the bound glycoprotein-like 
substances on the gel in some cases can be further separated depending on 
the chemical nature of the compounds particularly if gradient elution is 
used. 
According to this step of the invention, in the practical work of affinity 
chromatography the earlier purified satietin product is dissolved in a 
neutral starting buffer. 
0.02 M Tris-hydrochloride 
(2-amino-2-hydroxymethyl-1,3-propanediol-hydrochloride) is preferably used 
as a starting buffer which contains 0.5-1.0 M sodium chloride and the 
mixture should also contain Mn.sup.2+ and Ca.sup.2+ ions (1 mM). The high 
concentration of sodium chloride is required in the starting buffer in 
order to prevent nonspecific protein-binding between the adsorbent and the 
proteins present in the solute. The dissolved substance in the starting 
buffer is then chromatographed on the Con A-Sepharose column which was 
equilibrated with the starting buffer, by 10 column volumes washing at 
least with the starting buffer. 
After feeding the column with the satietin product to be purified the 
elution was carried out with the starting buffer containing 
alpha-D-methylmannoside in increasing concentrations. Practically, 0.5 M 
concentration of alpha-methylmannoside solution is poured in drops 
continuously into the eluent (starting buffer) and the linear gradient is 
formed by keeping the mixture constantly stirring. The 
alpha-methylmannoside concentration of the eluent thus increases linearly 
during the elution; the ion concentration and pH value are not changed 
during the elution. If the elution process is followed by UV 
spectrophotometer at 254 or 280 nm, then an elution diagram is obtained. 
FIG. 2 shows the elution profile, where the unretarded component A 
excluded from the column and appeared at the void volume, whereas bound 
component B is retarded with higher retention value and has an 
approximately symmetrical peak as a result of the increasing concentration 
of alpha-methylmannoside. In this way the biolgically active substance is 
obtained by pooling and concentrating the appropriate fractions eluted 
from the column. Since the substance still contains salts and 
alpha-methylmannoside originated from the buffer it needs a further 
desalting procedure which is carried out by gel chromatography. This step 
is performed on a gel column with 3000 daltons void volume, advantageously 
on Bio-Gel P-2 gel with deionized water. In the course of this 
gelchromatographic step the salts and other small molecules because of 
their molecular size are going inside the gel particles and retarding, the 
pure satietin, however, is excluded from the gel and appears with 1/3 
volume of the total volume of the column. The active fractions obtained by 
the elution of deionized water are pooled and freeze dried; the pure, 
homogeneous active satietin is obtained as white powder. 
The complete isolation procedure of satietin active substance from human or 
animal serum is outlined in FIG. 1. 1.5 mg active satietin can be 
extracted from 1 liter human serum, the specific activity of this 
substance was found to be 100 units/mg. 
The molecular weight of pure satietin isolated according to the invention 
was estimated by SDS-gel electrophoresis and showed an appearent molecular 
weight of 60,000-70,000 daltons, When the sample was examined by 
isoelectric focusing in polyacrylamide gel in the presence of ampholines 
with pH range of 5-7 and 7-9 we found that it exists in two distinct 
isoelectric forms in the pI range of 7.00-7.05 providing further support 
that the isolated glycoprotein was homogeneous. The analysis of product 
after acid hydrolysis showed the next composition: 
______________________________________ 
protein content 15% 
carbohydrate content 75% 
glucose amine 4% 
water 5% 
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Amino acid content: 
Asp 1.21%, Thr 0.66%, Ser 0.71%, Glu 1.87% Pro 0.45%, Gly 0.42%, Ala 1.58%, 
Cys 0.13% Val 0.55%, Met 0.12%, Ile 0.26%, Leu 0.90% Tyr 0.31%, Phe 0.44%, 
NH.sub.3 0.24%, Lys 3.79% His 0.15%, Arg 0.49% 
Sugar components: 
fructose 19%, mannose 21%, galactose 11%, glucose 24% 
The water content of the product vary depending upon the condition of 
lyophilization. 
The homogeneity of satietin product isolated according to this invention is 
proved by the evidence of affinity chromatography, polyacrylamide gel 
electrophoresis in presence of sodium dodecyl sulfate (SDS), gradient gel 
electrophoresis in slab gel, analytical isoelectric focusing, and end 
group analysis by means of the dansylation technique.

The isolation procedure of this invention is illustrated by the next 
example: 
EXAMPLE 
(a) 3,000 ml of human serum was processed by ultrafiltration on Amicon 
UM-10 membrane under 3-4 at. and constant stirring. The ultrafiltrate, 
about 2,000 ml, was then concentrated to 60 ml under vacuum evaporation. 
The mixture was centrifuged at 9,000.times.g for 30 min, the clear 
supernatant was brought to 0.degree. C. and 0.2 volume of 55% (w/v) 
trichloroacetic acid was added keeping the mixture between 
0.degree.-5.degree. C. and constant stirring during this procedure. After 
1 h the suspension was centrifuged at 30,000.times.g for 30 min at 
5.degree. C. in order to remove the precipitated proteins. Further 
purification of the active material present in the completely clear 
supernatant was achieved by gel chromatography on a column (5.0.times.90 
cm) of Sephadex G-15, equilibrated and eluted with 0.1 M ammonium acetate, 
pH 6.6 buffer. The fractions between 500-600 ml were pooled and 
lyophilized, dissolved in 10 ml of distilled water and applied to a column 
(2.5.times.90 cm) of Bio-Gel P-2 (100-200 mesh) and eluted with the same 
solvent. The fractions between 130-180 ml were pooled, lyophilized and 
yielded 25-30 mg of salt free crude satietin product as white powder. 
(b) 30 mg of the previously purified substance was dissolved in 6 ml of 
distilled water, applied to Whatman No. 3M paper (1 mg loaded on the 1 
cm), and subjected to high-voltage electrophoresis at a voltage gradient 
of 30 V/cm for 3 h at pH 6.2 (10% v/v pyridine; 0.5% v/v acetic acid). 
After drying the 1 cm wide paper strips were stained by ninhydrin and 
perjodic acid-Schiff reagents, respectively, in order to visualize the 
separated components. According to these double stainings four distinctive 
bands (Nos. 1,2,3,4) were ninhydrin positive, while only No. 1 and No. 2 
were stained by periodic acid-Schiff reagent. Each band was eluted with 
water and lyophilized to dryness. Fractions No. 1 and No. 2 proved to be 
biologically active (FIG. 1) and yielded amounts 20 to 23 mg of freeze 
dried powder with 50-100 units/mg satietin activity. (R.sub.f values of 
Nos. 1 and 2 were 0.00 and -0.11, respectively; mobility relative to 
Phe.fwdarw.Lys=1.00). 
(c) 20 mg of electrophoretically purified substance was dissolved in 4 ml 
of starting buffer (0.02 M Tris-HCl, 0.5 M NaCl, 0.001 M MnCl.sub.2, and 
0.001 M CaCl.sub.2, pH 7.0) and subjected to a column (1.7.times.37 cm) of 
Con A-Sepharose. Elution of the bound substance was achieved by using a 
linear concentration gradient (0-0.5 M) of alpha-D-methylmannoside (as a 
result of mixing 100 ml of starting buffer and 100 ml of the same buffer 
containing 0.5 M alpha-D-methylmannoside under constant stirring). Elution 
was followed at 280 nm by using an UV monitor. The active fractions 
(between 75-100 ml) were pooled and concentrated to 10 ml by 
lyophilization. 
The salt free substance separated by this procedure was obtained by simple 
gel filtration on Bio-Gel P-2 column (2.5.times.90 cm) as it was 
previously applied. Deionized water was used as eluent and the separated 
fractions (between 140-180 ml) were pooled and lyophilized. The pure 
satietin appeared as dried white powder in the amount of 4-5 mg. The 
specific activity of this substance was found to be 100 units/mg.