A PHYSICOCHEMICAL STUDY OF BLOOD SERA. I1 ANALYSESOF FIVEHUNDRED CASES
JOS8 ZOZAYA Gladwyne Research Laboratories, Gladwyne, Pennsylvania Received April $0, 1997
The change in the total protein of the blood serum under different conditions has been studied by Peters, Eisenman, and Bulger (18) in normal individuals and under miscellaneous conditions. Rowe (20) studied the effect of venous stasis on the proteins of human serum. The same author (21) studied the effect of muscular work, diet, and hemolysis on the serum proteins. Holman, Mahoney, and Whipple (11) have studied the change in plasma proteins caused by starvation, diet, and intravenous injection of plasma proteins. Bruckman, D’Esopo, and Peters (1) have reported the same changes in malnutrition. Wiener and Wiener (27) have made a general study of the plasma proteins in many conditions. Marriott (15) has observed and reviewed the literature of blood changes in anhydremia. Peters and Van Slyke (19) have discussed and further reviewed the general literature on the subject. The author (29) has described a physicochemical method for the study of blood serum and has suggested the direct application of that method in differentiating various pathological conditions. This differentiation can be made with exclusive physicochemical data; d e h i t e groups can be formed. The present paper includes the analyses and grouping of five hundred consecutive cases from over fifteen hundred mental and nervous cases that have been studied at the Gladwyne Research Laboratories. The normal amount of total protein in human serum or plasma has been studied by many investigators. Salvesen (22), Linder, Lundsgaard, and Van Slyke (14), Bruckman, D’Esopo, and Peters (1) are among those who have reported data comparable to ours, for they have used the method of Howe (12) for the fractionation of the albumin and globulin and determined the nitrogen by the Kjeldahl method. The average of all these determinations gives 7 per cent total protein, 4.39 per cent albumin, and 2.61 per cent globulin, including fibrinogen. The figures which we have used as our normal average are those of Handovsky (8), which are similar to those obtained by ourselves in normal average people, namely 7.00 per cent total protein, 4.15 per cent albumin, 1.60 per cent pseudoglobulin, and 1.25 per cent euglobulin, making albumin 60.0 per cent of the total protein, pseudoglobulin 22 per cent, and euglobulin 18 per cent. 191
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The grouping of our cases was based on the deviation of a normal average distribution of the proteins and total amount of protein. Group I1 contains those cases with a euglobulin per cent of 21 or more; group 111, those with a pseudoglobulin per cent of 25 or over; group IV, those cases with an albumin percentage of 65 or over; group I, those cases that did not fall in any of the other groups and which imply an average normal distribution of the proteins with a pliis or minus of 3 per cent for the euglobulin and pseudoglobulin and of 5 per cent for the albumin. We have further subdivided each group accoiding to the amount of total protein in each case. In subgroups A we considered the cases with a total protein between 6.70 per cent and 7.49 per cent as a normal average amount; in subgroups €3, rases with a total protein higher than 7.49 per cent; and in subgroups C, cases with a total protein lower than 6.70 per cent. This
FIQ.1. Chemical chart of the averages of groups I, 11, 111) and I V
selection has been arbitrary, but seems logical, considering the normal average variations that we have observed. In figure 1 we have plotted in a three-dimensional diagram the chemical distribution of the total average of the different protein fractions in the four main groups. This plotting gives an approximate equilateral cube which shows the average normal distribution. The quantitative and qualitative mixture of these protein fractions gives different colloidal prcperties t o the serum. These in turn change the general physiology of the organism, especially in its relation to the water interchange between blood and tissues, for there is no doubt that the interstitial fluid is produced from the blood serum. Krogh, Landis, and Turner (13), studying the movement of fluid through the human capillary wall in relation to colloidal osmotic pressure, support the view that there is a gradient of pressure which permits filtration a t the arterial
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end and reabsorption a t the venous end of the capillary, thus producing a continuous circulation of fluid between capillaries and tissues, Direct measurements of capillary pressure and serum colloid osmotic pressures definitely provide the circulation of fluid, which has been anticipated by theoretical considerations. Thus the colloidal osmotic pressure of a serum, which is usually directly proportional to the quantitative and qualitative distribution of the protein fractions of a serum, becomes of direct importance in the study of movement of water between blood and tissues. This subject has been treated in detail by Peters (17). There also exist changes in the protein fractions and their amounts in immune reactions. There seem to be sufficient data concerning the two fundamental types of immune reactions (28). One type has to do with tubstances which are essentially active poisons and whose toxicity is neutralized by direct chemical action. To this group belong the antitoxins from bacterial toxins, such as diphtheria, tetanus, botulinus, etc., snake venoms, vegetable toxins, etc. The substances in this group are similar in that, while having a chemical structure more simple than that of a true protein, they resemble more a colloidal aggregate. The protein change that occurs in this form of immunity reaction is a definite increase in the pseudoglobulin fraction, for it is in this fraction of the protein complex that we find the greatest amount of the antitoxic substance. This fact is recognized, for it is used in the concentration of the antitoxins for commercial use. The other group of immunity reactions consists of those which are concerned with the defense of the body to definite foreign proteins, polysaccharides, or lipoid complexes, whether toxic or non-toxic in themselves. To this group belong the whole protoplasmic bodies of bacteria, their different lipoid or carbohydrate fractions, animal or vegetable proteins, etc. In this type of immunity reaction we find a definite increase in the euglobulin fraction of the whole plasma protein of the immunized animal, and the majority of the antibodies produced are found in this fraction. This fact is used in the preparation of purified therapeutic bacterial antibodies. The difference in chemical response in these two types of immunity Tractions by these two groups of substances suggests the direct effect of the chemical structure of the antigen on the reaction of the body to the administration of these substances. In the separation of our groups we h a w taken this fact into consideration; in group I1 we have included the cases with a euglobulin increase (second group of immunity reactions) and in group I11 those with a pseudoglobulin increase (first group of immunity reactions). The chemical difference between the pseudoglobulins and the euglobulins suggested by Chick (4) and confirmed by Sorensen and some THE
JOURXAL OF PHYSICAL CBEMISTRT, \ - O b 42, S O ,2
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of his coworkers (24) is that euglobulin in serum is a complex material, formed from pseudoglobulin by association with some serum lipoid, to the presence of which it owes its phosphorus content. Hardy and Mellanby (9) made studies many years ago on the solubility of serum globulin in salt solution, and more recently Sgrensen (24) did likewise. The last author believes that in the serum itself, as well as in the special globulin fractions prepared from it, the globulins do not occur as mixtures of two or more globulins but as readily dissociable combinations of the same. As a simple and general term for the composition of such a combination the formula EpPq was utilized, where E indicated a euglobulin complex and P a pseudoglobulin complex, and where previously the probable difference between the individual euglobulin and pseudoglobulin complexes was disregarded. The study of serum as a readily dissociable component system of protein and the differences observed in our cases give us a fundamental physicochemical point of view which is of general practical and theoretical value. Sgrensen (24) says that in serum an interaction must be presumed to occur between all the protein systems present, an interaction which niay lead to the formation of new systems possibly containing components from albumin systems as well as from globulin systems and perhaps capable, to a limited extent, of giving rise to the formation of systems even larger than those known as albumins and globulins. Not only proteins, which as ampholytes have a particularly marked tendency to mutual interaction, but also t o a greater or less extent, dependent on concentration conditions and other circumstances, many of the other substances present in biological liquids must be supposed to be more or less closely knit to the, in all probability, highly hydrated protein systems. The views here advanced apply not only to serum but also to plasma and blood, for the blood corpuscles and other form elements in the blood,readily dissociable complexes consisting of proteins,-and also, to a greater or less extent, other substances present in the blood have to be taken into account. For these large complexes which condition many of the physical properties of the blood plasma, for example, its great viscosity and its comparatively low colloid osmotic pressure, must be imagined in continuous reversible dissociation, dependent on the ever-changing composition of the blood. The marked changes in amount of pseudoglobulin and euglobulin in serum give the serum different physicochemical properties. The lipoid complex in euglobulin gives this substance marked hydrophilic properties, as Chick (3) has found in the study of the hydration numbers of the different serum fractions, establishing for the euglobulin 5.8 cc. per gram, for pseudoglobulin 3.8 cc. per gram, and for albumin 2.1 cc. per gram. These differences in hydration of the various protein fractions affect their hold-
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ing power for water. We are aware of the work of Grollman (7), Sunderman (B), and Greenberg (B), who do not believe that there is any appreciable amount of “bound water” in serum. But we agree with Gortner TABLE 1 Averages of physicochemical observations of the blood serum i n 600 mental and nervous cases
Group I: Distribution of protein fractions average. Group 11: Percentage of euglobulin 21 or more. Group 111: Percentage of pseudoglobulin 25 or more. Group IV: Percentage of albumin 65 or more. Subgroup A: Total protein between 6.70 and 7.50. Subgroup B: Total protein higher than 7.49. Subgroup C: Total protein lower than 6.70.
( 5 ) and Bull (2), who point out that the concept of “bound water” has a substantial theoretical basis, and it is with this viewpoint that we have used the term here.
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The methods used in this work are the same as those described in our previous work (29). The mean average for each observation in each group is shown in table 1. PHYSICOCHEMICAL CHARACTERISTICS OF THE DIFFERENT GROUPS
The physicochemical characteristics of the four main groups can be observed from the average figures for each group. Group I, the average normal group, can be used as a comparison for the other three groups. In group I we get the general average of the average normal distribution of the protein fractions, as seen in figure 2, where the three-dimensional diagram of each of the subgroups shows a cube with its sides fairly equal. Subgroup A appears, as expected, midway between subgroups B and C. The general physicochemical characteristics h
-1
FIG.2. Chemical chart of subgroups I .4,I B, and I C
of this subgroup are those which are typical of a normal serum with, the variations expected due to dilution of the total blood. For example, rve find in subgroup B a more concentrated serum than in subgroup C. The concentration of the blood usually occurs through some physiological process, such as anhydremia, malnutrition, etc. The variation of “free water” in each of these subgroups is of interest. The difference between subgroups B and C is 5 cc. of “free water” in 100 cc. of serum. We take subgroup A as our standard of comparison, for it shows the general averages found among normal persons. In group I1 we find higher viscosity, higher specific gravity, higher total protein, increase in euglobulin per cent, a large amount of “bound water,” lo~v“free water,” and a low albumin-globulin ratio. This type of colloidal mixture is a more definite hydrophilic one. With this type of protein system is usually associated a certain definite physiology.
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To this whole group we have given the name “hyper-reactive.” This hyper-reaction, which is based on the increase of the euglobulin fraction, suggests an immunity response to a stimulation of some sort. This response is similar t o that found in immunity reactions produccd by a protein complex. The larger the amount of euglobulin in the serum, the more phospholipins there are bound t o the protein fraction. This union alters the general physicochemical properties of the serum. With this kind of protein complex (euglobulin), we find changes in the water interchange between the blood and tissues, thus disturbing one of the most important of body functions. We find the same condition in subgroups B and C, to a more marked degree in subgroup B, and to a less extent in subgroup C. The main difference between these two subgroups is in the amount of total protein, which suggests in the last group (C) a definite
1 FIG.3. Chemical chart of subgroups I1 A, I1 B, and I1 C
dilution, as is indicated in changes in specific gravity and viscosity. For the chemical-fractional plotting of the three subdivisions of group 11, see figure 3. Group I11 is characterized by an increase of above 25 per cent in the pseudoglobulin fraction in relation to the total protein. To this group belong the general reactions that from immunological experience are expected to come from stimulation by toxic substances of a simple chemical composition. These substances may come from any by-product of digestion (absorption through the intestine), from some substance that is not properly assimilated by the liver (as we often find the pseudoglobulin fraction markedly increased in diseases of the liver), or from any other substance which is absorbed and has the characteristics mentioned above. In subgroup 13 we have only two cases out of the five hundred. In these two cases there is an increase in the pseudoglobulin fraction as well
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as an increase in the euglobulin fraction. This double increase makes the colloidal properties of this subgroup an exceptional one. The specific gravity and the “bound water” are high, while the “free water” is low. From the immunological point of view this shows a double reaction, which suggests a definite liver involvement with a probable generalized reaction to an infection. The low albumin-globulin ratio is typical of this subgroup. Subgroup C has definite characteristics: low total protein, low “bound water,” low specific gravity, and high “free water.” This suggests a marked dilution of the blood. For the chemical plotting of the subdivisions of group 111, see figure 4. Group IV, the high albumin percentage group, is of general interest because of the low pseudoglobulin and euglobulin in the mixture. With A
FIG.4 . Chemical chart of subgroups I11 A, I11 B, and I11 C
a preponderance of the albumin fraction, we expect this group to be proportionately less hydrophilic than the others, so we find high “free water” and a high osmotic pressure. Chemically it is the direct opposite to group 11, and, owing to the low concentration of the pseudoglobulin and euglobulin fractions, we have termed it, from an immunological point of view, the %on-reactive” group. It seems that the individuals in this group do not react immunologically. Probably the reticulo-endothelial system in this group of cases is blocked or markedly deficient. The chemical distribution of the subgroups is seen in figure 5. Subgroup B has the general characteristics of the group except that it has a high total protein. Subgroup C is directly opposite to subgroup B. It is between these two opposite groups that we find the most marked differences. The main characteristics of this subgroup (IV C) are a low total protein, low ‘(bound water,” high “free water,” high albumin-globulin ratio, and a relatively high osmotic pressure.
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I n order to understand further the physicochemical differences between the various groups, we study the relation between specific gravity and the total protein per cent. The results are shown in figure 6. The total average of each group falls lowest for group I and highest for group 111. Groups I and I1 follow a straight line. Groups I11 and IV are askew,
I LZf FIG.5. Chemical chart of subgroups IVA, IV B, and IVC
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FIG.6. Relation between specific gravity and total protein in all the groups
subgroup I11 A being out of line as well as subgroup IV C. The corresponding subgroups of all the different groups fall within reasonable expectation. The study of these cases in groups shows a different relation between specific gravity and total protein, for in our previous work (29) we found
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the index of correlation between these two measurements to be 0.282 f 0.062. From this chart we can assume that in the present distribution we would expect a much higher correlation when the cases are grouped by chemical characteristics. The heavy solid lines are drawn to show the apparent band of distribution, expecting greater discrepancies between specific gravity and total protein in the cases of lower total protein with increased albumin percentage than in the other groups. In studying further the relation of specific gravity to some of our other measiirements or calculations, we used “bound water” as the other relation, because by the method we use in calculating it we get a quantitativequa1itati-i.e evaluation of the protein fractions in the particular mixture.
FIG.7 . Relation betnern “bound water” and specific gravity in all the groups
Using this value we could study the effect of this difference and specific gravity. Our results are plotted in figure 7 . As expected from the theory, group I is in the middle, group I1 below (plus euglobulin), and group IV above (plus albumin) Group 111 shows a different trend, but group I I I B returns to line. (In group I I I B we have only two cases, n hich we believe are definitely abnormal.) The total average of each group falls rlose within the line of general trend. Chick’s studies (3) on the volume occupied by 1 g. in solution of the different protein fractions used Hatschek’s (10) formula, modified to apply to emulsoids of t h r protein type. The formula as given by Chick is as follows:
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PHYSICOCHEMICAL STUDY OF BLOOD SERA
wheren = viscosity of the protein solution, d = specificgravityof the protein solution, and c = concentration of the protein in per cent. The relation A’/A is the expression of the volume occupied by unit weight of the dissolved substance. We are aware that this formula of Hatschek’s does not directly apply to our systems, because the formula appears to hold true only when the disperse phase occupies more than one-half of the total system. In TABLE 2 T h e value of A ‘ / A to express the volume occupied by unit weight of f h e dissolved substance in each one of the groups GROUP
I A B
C I1
A B C
c’
d
n
A
7.05 7.07 7.89 6.55
1,0272 1,0273 1,0284 1 0266
1.70 1.70 1.81 1.67
14.3 14.3 12.2 15.6
7.24 7.12 7.94 6.36
1,0276 1,0274 1,0283 1,0266
1.74 1.72 1.81 1.66
11.1 15.8
6.81 7.04 7.80 6.37
1,0276 1,0278 1 ,0285 1,0266
1.72 1.72 1.80 1.72
13.6 13.6 11.2 13.6
I
______ -~
I11
A B
C IV A B
7.00 7.07 7.79 6.40
C
-___
1.0273 1 ,0273 I ,0283 1,0270
__.__
1.68 1.67 1.73 1.67
-_I-.-
15.0 15.6 13.3 15.6
I
1 ~
I ’
~
1
A’
A’/A
13.8 13.7 12.3 14.8
0.96 0.96 1.01 0.95
12.2 15.3
1.04 1.oo 1.10 0.96
14.3 13.8 12.4 15.3
1.05 1.01 1.10 1.12
13.9 13.7 12.4 15.2
0.92 0.88 0.93 0.97
* c = concentration of protein in per cent. d = density of system. n = coefficient of viscosity. volume of 100 g. of system ,
A =
A’ =
A’/A
=
volume of disperse phase ( = v ) -volume of 100 g. of system , weight of dissolved substance ( = e ) volume occupied by 1 g. of dissolved substance (=v/c).
greater dilution it is probable that relatively more “bound water” than that indicated by the formula is closely associated with the protein molecule. Nevertheless we thought it of interest to use it here and test its general theory. In table 2 we find the values of A and A’, and the ratio A’/A, and in figure 8 we show the plotting of these values in relation to cubic centimeters of “bound water” per 100 cc. of serum.
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JOSE ZOZAYA
In the study of the figure we note the normal trend of the subgroups in groups 1and I1 and the marked skewness of the curves of the subgroups in groups I11 and 1V. The abnormal point seems to be in both cases in subgroup C. I t may well be that the relation d'/.4 does not hold true in the lower concentration of albumin and pseudoglobulin, for Chick found that in the lower concentrations of the different protein fractions this ratio increased in abnormal proportion as the concentration decreased. Why should albumin and pseudoglobulin in this particular property behave so differently from euglobulin? Probably the "bound water"-"free water" relationship is markedly disturbed, as is also suggested by the specific gravity-total protein relationship. From Chick's work (3) the volume xcupied by I g. of protein solution in high concentration is for serum
FIG.8. Relation between A ' / A and "bound water" in all the groups
albumin 2.8 cc., for pseudoglobulin 4.5 cc., and for euglobulin 6.5 cc. Therefore, the curve for the different groups as shown in figure 8 follows the general theory, although the concentration is much below the one used by Chick. Viscosity measurements are of great significance in the study of colloids and any change in these measurements suggests change in the internal colloidal system. The relation between specific gravity and viscosity has been used as a general estimate of the relation of total protein and the albumin-globulin ratio. In our previous paper (29) we found that the index of correlation betpecn these two measurements was 0.075 i. 0.06, that is, there was no
PHYSICOCHEMICAL STUDY O F BLOOD SERA
203
correlation. This result was obtained from the ungrouped cases studied then. I n figure 9 we have plotted the observations from our different groups. Here the total average of the whole group plots according to the expected theory as far as viscosity is concerned, in which the albuminous group (group IV) has the lowest viscosity for a given concentration, then group I (average mixture), then group I11 (pseudoglobulin), and the highest is group I1 (euglobulin). The trend followed by the different subgroups is of interest. As in other observations, the subgroups in groups I and I1 follow a more or less straight curve, while the low concentrations of total protein in the subgroups of groups I11 and IV show a marked skewness. From the observed facts we can generalize that groups I and I1 follow in all of our analyses an expected course, which
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FIQ.9. Relation between specific gravity and relative viscosity
agrees with the theoretical expectations, while in groups I11 and IV (specifically in subgroup C in both cases) there is always a marked discrepancy as to the expected course of events. I n the case of the viscosity-specific gravity relationship we observe that groups I11 C and IV C give a higher viscosity than expected. In the higher concentrations of protein (subgroups B), all of the groups show a close similarity in their behavior, with the exception of subgroup I V B , which, as we have already stated, has a lower viscosity than the similar subgroups, as might be expected. There is a similar variation in specific gravity in the comparative groups (subgroup IV C excepted), but the viscosity changes follow the laws of protein behavior rather more closely (subgroup I11 C excepted). To study further the effect of the chemical constitution of the different groups on viscosity, we selected cubic centimeters of “bound water” as
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