Studies in hyperproteinemia

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NAME AND ADDRESS

DATE

NORTHWESTERN UNIVERSITY

STUDIES IN HYPERPROTEINEMIA

A DISSERTATION SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE DOCTOR OF PHILOSOPHY

DEPARTMENT OF MEDICINE

BY DONALD HERMAN ATLAS

EVANSTON,

ILLINOIS

APRIL, 1942

ProQuest Number: 10060843

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OUTLINE /; 6 f

> ^

I. Introduction. II. Review of Literature. 1. Conditions reported to be associated with hyperproteinemia. A. Diseases associated with severe dehydration. B. Shock. C. Venous stasis. D. Multiple myeloma. E. Lymphogranjiloma venereum. F. Liver disease. G. Infections. H. Parasitic infestations. I. Miscellaneous conditions. 2. Hyperproteinemia and the source of the plasma proteins. 3. Fractionation of the proteins in hyperproteinemia( nature of the protein increase) 4. Pathological physiology of hyperproteinemia (associated physical chemical phenomena) A. Increased sedimentation rate. B. Increased rouleaux formation. C. Takata^Ara reaction. D. Formol gel reaction. E. Anti-complementary and biologic false positive serologic reactions for syphilis. F..Miscellaneous phenomena. 5. Incidence of hyperproteinemia (statement of problem). III. Procedure and Material. IV. Methods. 1. Possible sourcew of error of the falling drop method. 2* Experimental investigations on the possible sources of error of the Kagan method. A. Alteration in the viscosity of the oil. B. Evaporation effect. C. Effect of temperature extremes. D. Spontaneous separation of serum. 3. Conclusions. V. Results. 1.Incidence of hyperproteinemia. 2. Takata-Ara reaction, formol gel reaction, and sedimentation rate. 3. Nature of the protein increase. 4. Biologic false positive serologic reactions. 5. Conditions found to be associated with hyperproteinemia. VI. Comment. VII. Conclusions. VIII. Bibliography. Vita Acknowledgement•

I. INTRODUCTION The phenomenon of hyperproteinemia has received scant attention in this country *

It has been thought that the condition does not occur

with sufficient frequency in our climate to be of any great diagnostic significance except in multiple myeloma and lymphogranuloma venereum.

In

regions where schistosomiasis and similar parasitic conditions are fre­ quent, its diagnostic significance is of such importance that the routine preliminary tests for the presence of these diseases are merely tests for increases in the concentration of globulin.

Recent literature, however,

suggests that hyperproteinemia is not as rare in the temperate regions as commonly believed, and may have considerable diagnostic significance in a number of varied conditions.

In addition, hyperproteinemia may be of aid

in clarifying many physico—chemical phenomena heretofore obscure.

Until

recently, the complexity of the available methods for the determination of blood protein concentration had discouraged routine examination for this value, and

requests for its determination were usually limited to conditions

known to be associated with bypoproteinemia such as edema and malnutrition. The work presented here is an attempt to determine the incidence and sig­ nificance of hyperproteinemia in this climate. II. REVIEW OF LITERATURE 1. Conditions reported to be associated with hyperproteinemia A* Diseases associated with severe dehydration 1 Since the report of Schmidt in 1850 of high blood protein values in severe cases of cholera, it has been

known that extreme states of de­

hydration may readily produce an elevation of the blood protein concen­ tration regardless of the underlying condition or mechanism producing the dehydration.

Peters and his associates in 1925 stated " In conditions of

severe diabetic toxemia with ketosis a loss of water.

the blood becomes concentrated by

Under those circumstances the plasma proteins appear

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2

i

2 relatively high" ♦-8- Gamble

and his co-workers were among the first to

study experimental dehydration in animals and observed,in addition to an increased plasma protein concentration, a reduction of the ionic content of the ! j to be

body fluids by withdrawal of sodium. He believed the loss of sodium ^ 5 the significant factor in dehydration. Talbot in his studies on

I

j heat cramps in human subjects observed high concentrations of total proj

teins up to 10.6 grams per cent.

He stated that "such values are seen clini­

cally in only one disease not associated

with dehydration.

This is in

multiple myeloma". 4 Mandelbaum described practically all of the conditions that may be associated with an increased

concentration of the plasma.

He felt that

the term dehydration was not adequate to describe the group of symptoms that become manifest when the plasma sodium drops

below the physiological

normal and suggested that "Syndrome of Hemoconcentration" was more applicable. The to

term hemoconcentration has since been applied the

almost exclusively

blood changes observed in certain types of shock.

At present, extreme degrees of dehydration are seen most frequently in diseases of infancy such as diarrhea, pyloric stenosis, fections.

and severe in­

Consequently, most of the studies on serum protein concentration

in dehydration have been done on infants.

Increased concentration of serum 5 protein in dehydrated infants was first observed by Reiss in 1909, using 6 the refractometric method. Schloss and Marriott found the serum protein level to be a helpful guide in the treatment of dehydration, and their work

* Peters, J.Clin. Inv. 1:470, 1925. ** Talbot, Medicine, 14:342, 1935.

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7 was continued and substantiated by Brigge and associates. A contrary view 8 was expressed by Moon, who stated that vomiting and diarrhea alone will not produce hemoconcentration. B. Shock It is not

within the scojje of this work to review the extensive and

conflicting literature and studies on hemoconcentration In various types shock.

Suffice it to state that both increased and decreased concen­

trations of plasma proteins have been reported,depending perhaps on the underlying mechanism or cause.

Comprehensive studies on this subject have 9 been presented by Moon and Scudder and associates. They emphasized the fact

that

impending shock may be recognized

of hemoconcentration, and symptoms of

shock

in its Incipiency by the presence

can thus be treated properly before the actual

develop. Scudder has shown that fluid therapy may be

more properly controlled by repeated

hematocrit, specific gravity, and

plasma protein determinations* C. Venous stasis It has been shown by Rowe

10 11 and Peters and associates that venous

stasis such as that produced by the application of a tourniquet for even a few minutes may result

in an increase of

serum proteins in the affected

part due to a transfer of water from the blood to the tissues. plication of a tourniquet for five minutes,

By the ap­

the latter workers produced

an increase of total protein from 6.62 to 9.17 grams per cent, and an in­ crease in the hematocrit from 41.5 to 52.1 per cent. D. Multiple myeloma It is at present generally accepted that multiple myeloma is fre12 quently associated with hyperproteinemia. Reimann, in 1932, reported a

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case of myeloma with hyperproteinemia without Bence—Jones proteinuria in which the clue to the underlying condition was obtained by an inability 15 to count erythrocytes because of* immediate rouleux formation, Foord in 1934 collected 18 cases from the literature and added three of his own* That the association of nyeloraa with hyperproteinemia was not previously 14 known is shown by the statement of Sweigert that in only 35 of over 500 reported cases of myeloma were adequate quantitative determination of plasma protein completed*

However, of the 55 cases in which the blood proteins

were determined, twenty showed values of over 8*0 grams per cent* Gutman 15 and Gutman in 1936 cited 55 cases of hyperproteinemia among 57 published cases of multiple myeloma*

16

Bing

added 14 cases of multiple myeloma associ­

ated with hyperglobulinemia (as ascertained by a positive f ormol-gel reac­ tion) *

He states that hyperglobulinemia which may give rise to hyperprotein—

emia in

Denmark is ”above all found in patients suffering from multiple * 17 myeloma or chronic infections”* Gutman and his associates more recently reported another series of 38 cases of multiple myeloma of which twenty had total protein values above 8.0 grams per cent.

These studies show

that hyperproteinemia probably occurs in from 50 to 60 per cent of multiple 18 nyeloma cases. According to Jeghers and Selesnick it is of greater aid in the diagnosis of this

condition than Bence-Jones proteinuria*

They

noted that seven cases were discovered in two Boston clinics merely by routine determinations of

blood proteins on suspected cases*

E* lymphogranuloma venereum. The common association of hyperproteinemia with lymphogranuloma 15,17,19,20, venereum was demonstrated by the extensive and pioneer studies of Gutman * Bing, Acta Med.

Scand., 103:551, 1940.

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and his coworkers.

5

Twenty-six of the 55 cases they studied had total blood

protein values above 8.0 grams per cent, 11.2 being the highest level reached*

The hyperproteinemia in this series was more marked in those

patients with rectal strictures. lymphogranuloma venereum,the above

8.0.

Jones and

ZZ

Rome studied 79 patients with

majority of whom had total protein values

The mean total protein value was 8.55 and the highest value

obtained was

13.35 grams per cent.

They thought that the activity of the

disease and not the pathologic picture present is the factor that is associ23 ated with the increased globulin* Schamberg studied the course of the plasma protein changes in 20 Negroes with early lymphogranuloma with sulfanilamide*

All of his patients presented an initial hyperglobulin­

emia, which reverted toward the normal manifest.

venereum treated

level as clinical improvement was

Seventeen out of the 20 patients in this series had total pro­

tein values of above 8.0 at one time or another during the course of the disease.

Schamberg suggested "that the increase in the blood globulin

uniformly seen represents a humoral antibody response against the virus of lymphogranuloma venereum, and

that its reduction to normal in patients

treated early with sulfanilamide demonstrates inhibition of antibody for•jf

mation through destruction of

the virus."

Jeghers and Selesniclc stated that "since this disease is now known to be common in this

country, it must always be considered where high

blood protein values are found

and

confirmed by the

Erei test.

Con­

versely, all persons with inguinal adenitis or rectal strictures should have blood protein studies as part of their diagnostic study". # Schamberg, Am. J. Med. Sc., 201:12, 1941. Jeghers and

Selesnick, Int. Clinics, 47;253, 1937.

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F. Liver disease* Gutman and his coworkers stated that cirrhosis of the liver is one of the three main groups of conditions in which marked hyperglobulinemia and hyperproteinemia occur.

However, it is more commonly thought that al­

though hyperglobulinemia is frequently found in liver disease and especi­ ally in cirrhosis, the total protein concentration is usually low because 24 of the decrease in albumin and reversal of the A/G ratio. Snell suggests that concomittant infection probably is responsible for the increased globulin concentration in liver disease.

Luetscher, using the Electrophoresis

technique,concluded that in the early stages of portal cirrhosis the total protein is normal or occasionally elevated and that the albumin, however, is both relatively and absolutely diminished.

In cirrhosis the serum albumin

is often disproportionately low in relation to the total protein content because of

the frequent

concomitant increase in the globulin concentration.

G. Infections. h y p e r g l o b u l i n e m i a

with a number

a n c j

occasionally hyperproteinemia have been reported

of varied infections,usually chronic.

Schamberg stated

that "I^rperglobulinemia of greater or less extent occurs in most infectious diseases, and

the hyperproteinemia and hypoalbuminemia are considered secon— -ft.

dary to the globulin increase”.

It has been frequently suggested by numerous

workers that specific humoral antibodies to infectious agents are associated with the globulin., fraction of

the blood proteins.

Bing stated that ”The

most frequent cause of hyperglobulinemia is an infection, and the increase of serum globulin has been placed in connection with the formation of anti­ bodies,

it

having been shown that the function of antibodies is associated

■x Schamberg, Am. J. Med* Sc., 201:12, 1941.

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with the globulins”.

He stated further that ”the hyperglobulinemia that

occurs with the ordinary acute infections is only slight in most cases”, and ”in only rare cases is the globulin increase so great that one can speak of hyperproteinemia as well”. n§n the other hand, in chronic spe­ cific or non-specific infections, it is more often the case that hyper■** 26 globulinemia is so excessive that it leads to hyperproteinemia”. Sabin stated ”It has long been known that antibodies are almost invariably as— sociated with the globulin fractions of the serum”. Of the chronic infections, various types of tuberculosis have been most often reported with hyperglobulinemia and hyperproteinemia.

Je&hers

and Selesnick suggest that sub-acute bacterial endocarditis will eventually be included in the group of diseases in which hyperproteinemia is charac­ teristic.

This view has not yet been substantiated. Bing and his col27,28 laborators reported three cases of a syndrome which apparently had never

been published before: ”sepsis lenta with considerable affections of the 29 central nervous system and changes in the spinal fluid”. Salvesen reported three cases of Sarcoid of

Boeck with hyperproteinemia.

The exact cause

of this condition is not known, but it is thought by some to be closely related to tuberculosis.

Leprosy is said to be associated with hyperpro30 teinemia in 80 per cent of the cases. H. Parasitic infestations. In tropical regions where certain parasitic infestations are common,

*Bing, Acta. Med. Scand., 103;549, 1940. Ibid. p.551 Sabin, Jour. Exper. Med., 70;S7, 1939.

hyperglobulinemia and hyperproteinemia are of such frequency that tests for increased protein concentration are as routine as serologic tests for 31 the presence of syphilis • Sia and Wu in 1921 reported hyperproteinemia in 40 per cent of the cases of

Kala-azar which they studied. Their ob32 33 34 servations have since been confirmed by Lloyd and Paul, Ling, Ginandes,

and humerous others. the temperate zones.

Sporadic cases of Kala—azar crop up occasionally in 35 Meleney and Wu in 1924 reported similar protein

changes to be frequent in Schistosomiasis Japonieum, and their obser­ vations have been substantiated.

Elevations in the globulin concen-

30 tration producing hyperproteinemia have also been reported in malaria, 36 37 Filariasis, and Trypanosomiasis. It was the opinion of the earlier workers, that syphilis produces 38 an increase in globulin and total protein. Rowe in 1915 stated that "in syphilis the globulin fraction is definitely increased, while the total 39 protein remains about normal". Lloyd in 1932 wrote "It has of course been known for a long time that the globulins in syphilitic serum are increased".

He studied 11 cases of secondary syphilis, all but three

of which had globulin values above three, but only one of which had a 40 total protein concentration above eight grams per cent. Wu reported two cases of syphilis with hyperproteinemia in which the protein concentra­ tions were determined by the colorimetric method. rence of hyperglobulinemia and

Recently, the occur­

hyperproteinemia in uncomplicated syphilis

has been questioned, and as demonstrated by Jones and Rome, its frequency in syphilis may be attributed to concomitant presence of lymphogranuloma venereum.

The latter workers did not find marked increases in the globu­

lin fractions in syphilis except in those individuals who also showed a positive

Frei test.

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I* Miscellaneous conditions I^rperglobulinemia and hyperproteinemia have also been reported with 41 a number of apparently unrelated conditions, Gildea and his associates showed that serum proteins tend to lie above or in the upper part of the normal range

in hypothyroidism and that the

finding of values over 7.7

may constitute view has not of various

an additional and useful criterion of hypothyroidism. This 42 been substantiated. Kennaway in 1924 reported over 40 cases

types of carcinomas in which the total protein concentration

was over 10.0 grams per cent with inversion of

the

a /G

ratio.

He con­

cluded that some advanced cases of cancer show an increase in the globulin of the serum which is greater than any which has been found in other con­ ditions, but that this increase in globulin is very seldom perceptible sufficiently early to be of use in diagnosis. have

not been confirmed.

These observations also 43 In fact, Peters and Eisenman wrote "the state­

ments of Galehr and Kennaway that globulin is usually increased in car-jfcinoma is hardly substantiated by these data”. They further state that "the results in general lend little support to the views that malignant tumors have any consistent effect on serum proteins or its fractions with the single exception of the globulin increases found with certain peculiar ~A%r

neoplasms, especially myelomatosisV Elevations of the total blood protein with reversal of the a /G 44 45 ratio have been reported in Still’s disease, granuloma inguinale, leukemia, 16 chronic eczema, agranulocytosis, purpura, and erythema induxatum. The find­ ing of hyperproteinemia in these conditions is rare and it may well be that the factor of dehydration may not have been sufficiently considered In many of these reports. ^Peters and

Eisenman, Am. J. Med.Sc., 186:819, 1933.

Ibid., p. 822

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Hyperproteinemia and the source of the plasma proteins As yet no definite proof is available as to origin of the plasma pro46, 47 teins, with the exception of fibrinogen which has been shown ty Whipple and his associates to be formed in the liver.

Several attempts have been

made to see if a study of hyperproteinemia would aid in the elucidation of

this problem.

Because of the frequent finding of

bone marrow involve­

ment with hyperproteinemia of multiple myeloma, it has been suggested that the bone marrow may be the source of blood proteins.

Some workers believe

that the reticuloendothelial system participates in the regeneration of plasma proteins (globulin in particular).

Bing and his coworkers believe

that they have demonstrated that ,fit is a feature common to the various diseases

with hyperglobulinemia that there is an increase of the plasma

cells and reticuloendothelial cells in the bone marrow or other places in the organism, which makes it probable that the formation of globulin takes place in these cells”. Sabin studied the mechanism of

antibody formation with dye-protein

and presented evidence that serum proteins and especially globulin come from the cytoplasm of the cells of the reticuloendothelial system. liver,

she believed, may be a source of

the

The

blood proteins because of

the Kupfer cells which are an integral part of the reticuloendothelial 47 system. Madden and Whipple question the formation of plasma proteins in the bone marrow by calling attention to the fact that the proteins are unchanged in aplastic anemia, a condition in which the bone marrow is almost completely absent.

* Bing, Acta Med. Scand., 103:567, 1940

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3.

11

Fractionation of proteins in hyperproteinemia (nature of protein increase) Various methods of fractionating the plasma proteins have been de­

veloped, and has been the subject of a vast amount of investigation*

In

general, the methods may be classified ad biological, chemical, and physi­ cal. of

A review of these methods, however brief, is far beyond the scope this work.

The separations obtained by the commonly used ”salting out" 49 methods of Howe are arbitrary and empirical, and consequently have been the object of considerable criticism of late, especially since the frac­ tions thus obtained did not fulfill the criteria required to establish them as chemical individuals.

The opinion has been voiced that the frac­

tions thus isolated from serum may have been the results of treatment of the serum during their isolation and may therefore even be considered as artefacts. 5° Block recently expressed the view that serum does not contain several independent proteins. 11The fractions isolated by plysical-chemical * methods are not pre-existent in serum but result from the technique employed”* Much valuable information regarding the nature of serum proteins in normal and pathological conditions was obtained by the ultra-centrifuge method 51 of Svedberg. By far the most accurate of the recently developed methods 52 53,54 55 is that of electrophoresis as described by Tiselixas, Longsworth, Abramson, 56 25 Shedlovsky, Luescher, and numerous others. This is based upon the fact that diluted serum placed in an electrical field separates into zones or boundaries of different electrical mobilities, which may be observed and recorded by a complex optical system.

The rate of mobility and the con­

centration of the components can be calculated and the component fractions separated into homogeneous fractions. the greatest mobility.

* Block, Jour.

Albumin has been observed to have

Three components of globulin,alx^ha,beta, and

Bio. Chem., 105*455, 1934.

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gamma, in order of their mbilities are constantly observed.

12

Similar

patterns have been recorded in experimental animals. A comparison of the protein composition of normal and pathological sera has been ms.de by a number of workers. been located in the gamma globulin.

Certain antibodies have

Whereas gamm^globululins are usu­

ally nearly colorless, beta globulin fractions are often highly colored (as a result, perhaps, of prosthetic groups such as lipoids).

Febrile

conditions have been reported to be associated usually with an increase in gamma globulin concentration.

Abramson and others have shown that

the antibody to rag weed is contained only in the gamma globulin. Excluding hemoconcentration, practically all of the cases of hyper­ proteinemia reported were associated with an increase in the concen­ tration of globulin, and usually an absolute diminution of the concen­ tration of albumin.

Thu3, hyperproteinemia implies hyperglobulinemia

and hypoalbuminemia.

In rare cases, Bence-Jones protein, fibrinogen,

or some unusual unidentified protein has constituted the protein in­ crease.

Inasmuch as the separation of fractions by "salting-out" pro­

cedures such as Howe’s method is considered imperfect, particularly with respect to the subglobulin fractions, the huge amount of litera­ ture dealing with fractionation of serum proteins in hyperproteinemia will not be considered here.

Moreover, the composition of fractions

precipitable with the same concentration of salt may be quite different in various conditions*

Gutman states, for example, that "the large"

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euglobulin fraction in some cases of multiple myeloma doubtless contains proteins not present in the corresponding euglobulin fracnormal serum and may be different in composition from the ®t*globulin fractions in infections'*•

However* he has demonstrated

a fairly consistent increase in the Howe euglobulin fraction which accompanies any marked rise in the total globulins*

He concluded

that the Howe method* despite its deficiencies* gives reasonably uniform results when applied to fractionation of serum proteins in chronic infections and cirrhosis* but that multiple myeloma represents a special case in the distribution of serum protein fractions in hyperproteinemia* with anomalies that are without precedent in their experience with any other disease*

He further stated that "when the

results of electrophoretic and Howe fractionation are compared* the correspondence between the distribution of protein fractions by these two methods is found to be imperfect"* 4* Pathological physiology of hyperproteinemia (Associated physical chemical phenomena) Unusual phenomena associated with or perhaps due to the presence of hyperproteinemia have been observed frequently* one or more being present in practically every case*

The under^i|ng mechanisms of these

phenomena are little understood and consequently most of them are em­ pirical reactions which* nevertheless, have considerable diagnostic

* Gutman, Jour. Clin* Inv.* 20:768* 1941. ** Ibid.* p. 777

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significance•

14

In most instances* these phenomena are dependent upon

an increased globulin concentration* but occasionally an increased con­ centration of fibrinogen may be the significant factor* A* Increased sedimentation rate The exact underlying mechanisms of the increased rate of sedimen­ tation of the red blood cells in various pathologic conditions is not as yet known* although the literature on this subject is voluminous* Moreover* in spite of these extensive investigations* the present day knowledge of the exact cause of the increased rate is not much greater 58 than the information contained in the pioneer work of Fahraeus. He stated that although there was no correlation between the sedimenta­ tion rate and the total plasma protein* an increased globulin and fibrino­ gen concentration frequently occurred coincidentally with the increased sinking velocity*

He did not believe* however* that a direct cause and

effect relationship exists* 59,60,61,62 Since that time numerous workers have demonstrated a fairly consis­ tent correlation between an increased concentration of fibrinogen and an increased rate of sedimentation.

However* extremely rapid sedimentation

rates have been frequently observed in the presence of normal fibrinogen 61 values, thereby indicating that other factors may be of equal importance. Most workers seem to agree with the opinion of Gilligan and Ernstene that "the rate of settling bears an inverse ratio to the volume percen­ tage of red blood cells: the fewer the cells* the faster the sedimentation * 50 62 rate". Cutler, on the other hand* believes that the factor of anemia * Gilligan and Ernstene, Am* Jour* Med. Sc., 187:554, 1934.

15

should be disregarded*

63

Lucia and associates concluded that the sedimen­

tation time is accelerated as the globulin content of the serum increases and is retarded as the albumin content increases*

They believed the cor64

relations, though low in value, to be statistically significant*

Davison

contended that although there was no relation to the A/G ratio or total protein, globulin increase m s always proportional to the increased sedi­ mentation rate*

Shedlovsky and Scudder compared the rate of sedimenta­

tion with the electrophoretic patterns of normal and pathological human blood, and found a significant correlation between the sedimentation rate and alpha globulin,which they believed to be at least as good as similar correlation involving fibrinogen levels*

They observed no satis­

factory correlation with either beta globulin, gamma globulin, or the A/G ratio* Ropes

61 and associates concluded that there is not a quantitative

correlation between the sedimentation rate and the concentration of 18 fibrin, globulin or total protein*

It has been frequently observed

that hyperproteinemia is almost invariably associated with markedly in­ creased sedimentation rates, even in the absence of fever or infection. In fact, the most rapid sedimentation rates on record have been in as­ sociation with hyperproteinemia.

Thus it can be seen from all of these

conflicting reports that variations in sedimentation rates cannot be ascribed to any one known factor*

The situation has been well summarized

by Ropes in the statement '*The only concept which explains all of the findings is that variations in sedimentation rates are due to variations in the colloidal state of the plasma with consequent changes in the

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electric charges on the proteins and red cells*

16

Variations in the con­

centration of fibrinogen* globulin* and other constituents affect the rate through their effect on the colloidal state of the plasma" —

"the

changes represent a direct response of all factors (fibrinogen* globulin* and sedimentation rate) to the stimulating agent rather than a response *

of one indirectly because of a change in one or the other factors"* B. Increased rouleaux formation Abnormally rapid and marked rouleaux formation (auto-hemagglutination) of red blood cells has been observed frequently in cases of hyper­ proteinemia.

It is generally accepted that one of the factors in the

rapid sedimentation of red blood cells is the formation of large ag64 gregates or rouleaux of the red cells. The rouleaux once formed settle at a given rate of speed* more or less in accordance with Stokes* law of hydrodynamics — and vice versa*

the larger the aggregate the more rapid the settling

The relation of the plasma proteins to this phenomenon

was considered in the previous section.

The attempt that hasboen made to

differentiate true auto-agglutination from false or pseudo auto-agglutination by temperature variation and dilution procedures will not be considered here*

The phenomenon has also been noted on hyperproteinemic

patients by inability to (1) make satisfactory blood smears; (2) perform an erythrocyte count; (3) type blood for a transfusion*

When such diffi­

culties arise*hyperproteinemia should be considered and may often be a clue to the diagnosis* In addition, Foord suggested the possibility of intra-vascular auto-hemagglutination*

In one case reported by him* this phenomenon was

* Ropes, J. Clin. Inv*, 17:523, 1938.

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observed in the retinal veins during opthalmoscopic examination when the circulation was slowed by firm pressure on the eyeball.

Large, slow moving

granules were observed in the veins similar to the aggregates seen in the tubes of rapidly sedimenting blood. C. The Takata-Ara reaction The extensive and conflicting literature available concerning this

66 recently popularized test was carefully reviewed and evaluated by Magath. 18,22 It has been generally thought that the test is positive in any disease associated with an elevation of the globulin fraction above 3.00 grams per cent and a reversal of the A/G ratio.

Magath, however, cited as

many observers who denied this relationship as those who supported it. 67 For example, Bowman and Bray obtained maiiy positive Takata-Ara reac­ tions with serum in which the level of protein was low and in which the A/G ratio was not reversed.

Magath concluded that "the test is correla­

ted to a great extent with changes in the ratio of serum albumin to globu­ lin, but the correlation is not absolute and the mechanism and cause of * the reaction remain unexplained"• He further concluded that the test is empiric, not very sensitive, and may give positive results in a wide variety of diseases, for none of which it is specific.

However, he does

concede that the test usually gives positive results in cirrhosis of the liver, especially when the disease has progressed to moderate severity. A study of the literature reveals that the Takata-Ara reaction is so frequently positive in hyperproteinemic conditions that it has been used as a simple means of detecting the presence of hyperproteinemia * Magath, Jour. Lab. and Clin. Med., 26:170, 1940.

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22,68,69 and hyperglobulinemia.

18

68 Jeghers

noted markedly positive Takata-

Ara reactions in each of five cases of multiple myeloma associated with hyperproteinemia.

Jeghers and Selesnick reported the reaction to be

positive in nine out of the ten cases of hyperproteinemia in which it was performed.

Salvesen reported positive reactions in his cases of

Boeck's sarcoid associated with hyperproteinemia, and it has been re­ ported positive by numerous workers in lymphogranuloma venereum associ­ ated with increasd protein concentration.

Inasmuch as in hyperprotein­

emia it is globulin that is usually increased, and the albumin that is lowered, it is understandable why a strongly positive reaction occurs in this condition. D. Formol-gel reaction The formol-gel reaction (known also as the Gate and Papacosta reac­ tion, Fox and Hackle*s reaction and Kurten's reaction) has been shown to be positive usually under the same conditions that produce a posi­ tive Takata-Ara reaction and consequently has been used as a simple 20,71,77 method of detecting hyperproteinemia and hyper-globulinemia. The reaction depends upon the formation cf a gel when 36 per cent neutral formalin is added to blood serum.

Bing and others have conclusively

shown that the reaction is positive whenever the globulin concentra­ tion is above 3.5 grams per cent.

The test has been used almost rou­

tinely in tropical countries in the diagnosis cf the parasitic infes­ tations previously described. The reaction is believed by some to be correlated with an increase in the euglobulin component of globulin. 71 Hassan and Salah mad^k comparative study of the formol-gel and TakataAra reactions in relation to serum proteins.

In 85 per cent of 600 sera

-

there was similarity between the two tests.

19

They concluded that a

strongly positive formol-gel reaction is always associated with hyper­ globulinemia (usually euglobulinemia),and that such sera contain four per cent or more of globulin.

The reaction, however, cannot be applied

as an absolute quantitative method for determining the degree of hyper­ globulinemia, but because of its simplicity and reliability, can be used routinely to detect the presence of hyperproteinemia and hyperglobulin­ emia. E • Anti-complementary and biologic false positive serologic reactions for syphilis Numerous reports of anti-complementary Wa3sermann reactions in the various conditions associated with hyperproteinemia have appeared in the 27,28,70 literature especially with multiple myeloma and lymphogranuloma 73 venereum. Gutman and Williams noted anti-complementary Wassermann reac­ tions in 22 per cent of 24 positive Frei test (for lymphogranuloma venereum) patients as compared with one per cent anti-complementary reac­ tions in the general hospital population.

They also reported anti-com-

plementary reactions in two of three cases of multiple myeloma with hyper­ proteinemia, but negative serologic reactions in four cases of myeloma not associated with hyperproteinemia.

They stated that there is evi­

dence, presumptive and Experimental, to indicate that increased serum globulin, particularly the euglobulin fraction, is one of the most im­ portant factors associated with non-specific fixation of complement in man.

-

20

Jeghers and Selesnick suggest that hyperglobulinemia may be a fac— tor in the production of false positive Wassermann reactions commonly found in sub—acute bacterial endocarditis and Boeck*3 sarcoid* and that all doubtful Wassermann reactions in cases of hyperproteinemia be checked* The relationship of increased globulin concentration to false positive serologic reactions has not been investigated*

Moreover, the mechanisms

of the serologic tests for syphilis are themselves little understood and consequently are entirely empirical* F* Miscellaneous phenomena Sporadic reports of a number of other physical chemical phenomena said to be related to hyperproteinemia and hyperglobulinemia have appeared in the literature. Many of these reports are conflicting, and sufficient data on these phenomena has not as yet bden accumulated from which definite conclusions can be drawn* A partial list of the changes reported are; 72 13,14 (1) rapid coagulation of blood, (2) impaired renal function, (3) fail72 ure of blood clot to retract, (4) increase in pH of serum with altered 19 74 iso-electric point, (5) spontaneous precipitation of blood proteins, 75,76 and (6) increase in the total blood calcium* 5. Incidence of hyperproteinemia (statement of problem) Jeghers and Selesnick summarizing the total plasma protein deter­ minations made by the clinical laboratories of the Boston City Hospital, reported an incidence of hyperproteinemia (taking 8*0 grams per cent as the upper limits of normal) of 0.2 per cent of 557 determinations in 1934, 1*19 per cent in 1935, and 2*4 per cent in 1936. The increased incidence of elevated values in 1935 and 1936 reflect a greater number

21

of determinations made specifically for hyperproteinemia where multiple myeloma was suspected*

This data represents presumably determinations

made only when some abnormality was suspected* and as such does not re* veal the true incidence of hyperproteinemia in the general run of hos­ pital cases*

Schuman and Jeghers* in 320 consecutive determinations of

total serum protein concentrations by the Kagan falling drop method* found only one case of hyperproteinemia on their medical service in the Boston City Hospital.

This was a case of multiple myeloma.

Bing

found seven hyperglobulinemic sera out of 3*697 sera as determined by routine formol-gel tests.

None of these series represents random sam­

pling of all cases admitted to a hospital.

It is the purpose of this

work to determined the incidence of, and conditions associated with, hyperproteinemia in the general run of hospital patients by doing rou­ tine total protein determinations on large numbers of sera selected at random. In addition* an attempt is made in this work to demonstrate the relationship of hyperproteinemia to the sedimentation rate, the Takata-Ara reaction, and the formol-gel reaction. III. PROCEDURE AND MATERIAL In order to determine the incidence of* and conditions associated with hyperproteinemia* and to obtain ca3e3 of hyperproteinemia for 78 study* total protein determinations were done by the Kagan filling drop method on 4*370 sera submitted to the serology department of the Cook County Hospital for routine Kahn and Wassermann tests.

This wqs done

by the method of random sampling, but did not include serum samples

-

22

from the Tuberculosis* Contagious Diseases, and Children's Hospitals. The number of patients admitted to the latter three departments consti­ tute only twenty per cent of the total number of admissions.

More than

half of the tuberculosis cases are admitted first to the general hos­ pital and consequently are included in this survey. When a high total protein value was discovered by the falling drop method, the source of the serum was traced so that the patient could be further studied and a fresh sample of blood obtained under proper precautions for chemical verification of the total protein level, deter­ mination of the albumin and globulin ao ncentration, and determination of the sedimentation rate* Takata-Ara* and formol-gel reactions, and hematocrit values. The frequency of lymphogranuloma venereum in the Cook County Hospital*where there is a large Negro population, made it advisable to per­ form Frei tests whenever possible.

This test alone may disclose the

true cause of hyperproteinemia in the absence of clinical evidence for the disease. An attempt was made to establish the state of hydration of each case.

This phase of the problem will be considered later. XV. METHODS

A number of methods of varying complexity and accuracy have been developed for the

determination of serum protein concentration. The

micro and macro-Kjeldahl digestion method*or one of the many modifications, is the most widely used* and has been generally accepted as the standard for comparison. The refractometric

and coloerimetric methods are simpler

- 23

79 but of leaser accuracy* especially in the presence of lipemia. Of the many physical methods available* the specific gravity method by 80 81 was deemed most satisfactory^Peters and Van Slyke. Moore and Van Slyke* using the pyknometer*established a linear correlation between the

pe—

3

cific gravity and the total protein content of human heparinized plasma* as expressed by the straight line formula P a 343 (G - 1.0070).

P rep­

resents the plasma protein in grams per cent and G the specific gravity. 82 Weech et. al verified this* and demonstrated a similar linear relation83 ship for serum. Working on the same premise, Bing, and later Simeone 79 and Sarris* presented an ingeneous yet simple method for serum pro­ tein determination by the use of standardized glass beads.

In twenty

serum samples they obtained an average difference of 0.25 grams per cent between the bead and micro-Kjeldahl methods. The most popular by far of the specific gravity procedures is 84 the falling drop technique first described by Barbour and Hamilton in 1924.

It is based on Stokes* law that the specific gravity of a

spherical substance as it falls through a viscous fluid with which it is not miscible is a function of the rate of fall* the radius of the sphere* the viscosity of the fluid* and the accelerating jtffect of gravity.

Moore and Van Slyke compared the accuracy of the method with

that of the more time-consuming procedure of weighing bottles and al­ though their results of fifteen determinations agreed to three decimals* they felt that the chances of error were greater in the falling drop because of the precautions required in the use of standard solutions*

-

the timing of the drops and the reading of hemograms,

24

85 Terry and Seib,

using the apparatus of wh'ole blood specific gravity determinations* 86 found the method reliable and exceedingly sensitive, Spencer compared the falling drop method of determining specific gravity as an index of dehydration with the determination of total solid content of plasma* weight of the plasma* and total protein content by refractometric analysis.

He concluded that the falling drop was the most accurate 87 of the several compared, Leake et al, found the procedure sufficiently

sensitive to detect significant diurnal variations in blood specific 88 gravity of men and women, Beilis stated that the Barbour and Hamil­ ton apparatus is fully as accurate as the Kjeldahl method*and prepared tables for use with the apparatus which eliminate the use of nomograms. The technique enjoyed its most extensive use in the well known work of 17,89 Scudder, Recently Kagan simplified the original apparatus by the a use of standardized mixture of methyl salicylate and mineral oil for the purpose of eliminating the use of standard solutions of known spe­ cific gravity,

Shuman and Jeghers found the method to be accurate and

simple in 320 determinations*

They also compared the values obtained

by the Kagan technique with those obtained by the micro- and macroKjeldahl methods on twenty sera.

The maximum variation was ,49 grams

per cent with a mean deviation of 0,14, They concluded that for clini­ cal purposes such small deviations are irrelevant.

Bridge et al, state

that there was satisfactory correlation of the method of Kagan with the other methods they employed* including the use of the photoelectric

- 25

colorimeter•

The Kagan proteinometer being the simplest and most rapid

to operate was chosen Tor our work* and it is the purpose of this sec­ tion to present the results obtained by carefully checking the validity of the method. 1« Possible sources of error of the falling drop method Kagan demonstrated that the linear correlation of specific gravity to total protein of serum is not significantly affected by alterations in the cholesterol or A/G ratio values.

Increases in the lipoid con81 centrations have little influence on the specific gravity of serum. Simeone and Sarris demonstrated that "abnormal concentrations of blood

glucose may introduce a significant error in the protein concentration computed from the specific gravity"* but that "sufficiently accurate corrections may be made by deducting 0.1 grams per cent protein for *

each increment of 100 mg. per cent glucose". Venous 3tasis* as mentioned on page three* may result in an in­ crease of serum proteins in the affected part* due to a transfer of water from blood to the tissues.

To obviate this error* the blood

samples were drawn with a minimum of stasis by having the tourniquet applied for as short a time as possible. Kagan states that the oil supplied with the proteinometer is stable indefinitely* and is not miscible with serum* blood, or plasm.

*Simeone and Sarris* J. lab. and Clin. Med., 26;1051, 1941.

- 26

2# Experimental investigations on the possible sources of error of the Kagan method A. Alteration in viscosity of the oil. In order to determine whether or not the proteinometer undergoes oil changes from prolonged use* the total protein values obtained with oils that had been in use for varying periods of time were compared on identical serum samples with the values obtained with fresh unused oils. It was observed* that after U3ing the same oil over a period of weeks or months for hundreds of determinations* the viscosity decreases, the oil di scolors* and values may be obtained that deviate as much as 1.9 grams per cent when checked against a fresh unused ssraple of oil. It could not be determined whether the degree of alteration of the oil was correlated with the duration of use or number of determinations made.

This change in viscosity is shown in Table 1. on three dif­

ferent speciments of oil; specimen A in use four months* specimen B in use two and one half months* and specimen C in use one month. To reduce this error* the viscosity of the oil was checked periodi­ cally with a standard salt solution of known specific gravity.

In ad­

dition* the construction of the apparatus was modified by attaching a stop cock to the center tube to eliminate the pouring off of the oil to remove the serum which ordinarily collects at the bottom of the tube. The modification is based upon the assumption that the viscosity change results from either diffusion of oil soluble substances*such as lipoids* from the serum into the oil, or from evaporation of the more volatile component of the oil* or both.

- 27

TABLE 1. Total serum protein concentration determined by used and fresh samples of oil. Specimen A. Oil in use four months compared with fresh oil. No.

4 months

1. 2. 3. 4. 5.

8.60 8.32 8.07 8.00 8.87

Fresh

Difference

6.83 6.56 6.18 6.11 7.04

-1.77 -1.76 -1.89 -1.89 -1.83

mean deviation 1.83 gm. per cent

Specimen B. Oil in use two and one half months compared with fresh oil. No.

2g- months

Fresh

Difference

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

6.04 8.63 6.11 7.77 8.56 8.04 8.35 8.87 6.59 7.80

5.59 8.35 5.59 7.22 8.07 7.56 7.94 8.21 6.32 6.94

-0.45 -0.28 -0.52 -0.55 -0.49 -0.48 -0.41 -0.66 -0.27 -086

mean deviation 0.50 gm. per cent

Specimen C. Oil in use one month compared with fresh oil* No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

1 month 7.70 8.39 8.25 7.45 8.60 6.07 6.30 5.31 6.59 6.87

Fresh 7.22 7.63 7.45 6.55 8.18 5.59 5.63 4.62 6.07 6.25

Difference -0.48 -0.76 -0.80 -0.90 -0.42 -0.48 -0.67 -0.69 -0.52 -0.62

mean deviation 0.63 gm. per cent

-

28

i

Table 2 shows the constant values obtained with the modified apparatus over a period of months* using a standard solution of potassium sul­ phate* specific gravity 1.0306 at 24 degrees centigrade kept under oil to prevent evaporation.

TABLE 2. Specific gravity values obtained with same proteinometer oil with stan­ dard solution of potassium sulphate* using the modified proteinometer. Date

IO/9/4 I

2/18/42

3/27/42

4/3/42

Spec.gravity 1.0305 Standard 1.0306

1.0301

1.0303

1.0303

Deviation

0.0005

0.0001

0.0003

0.0003

4

/10/42

1.0304 0.0002

B. Evaporation effect. Inasmuch as blood samples frequently remained loosely covered with cotton plugs for several days before the 3erums were obtained for the routine total protein determinations, it seemed of value to determine the day to day affect of evaporation on the total protein values* both at room temperature and in the ice box. Table 3. shows the effect of evaporation on the total protein content of uncorked serum samples using the falling drop method. Table 4. demonstrates that this alt­ eration from evaporation is minimized in tightly corked serum samples.

- 29 TABLE 3. Effect of evaporation on the total protein content of uncorked serum samples*

No.

1st day

1. 7.22 2. 7.63 3. 7.45 4. 6.55 5. 8.18 6. 5.59 7. 5.63 8. 4.62 9. 6.21 10. 6.25 mean increase

2nd day 7.31 7.66 7.84 6.94 8.21 5.59 5.77 4.94 6.49 6.42 -0.18

3rd day 7.45 7.91 7.94 7.01 8.35 5.63 6.00 4.97 6.59 6.49 -0.12

4th day 7.70 8.11 8.00 7.31 8.42 5.71 5.95 5.02 6.71 6.57 -0.12

5th 7.63 8.11 8.07 7.52 8.60 5.93 6.18 5.21 6.83 6.69 -0.13

mean daily increase -0*14

B. Serums storedin ice box. 8.21 1. 2. 7.56 3. 8.04 4. 7.01 5. 7.87 6. 7.42 7. 7.31 8. 7.18 9. 6.18 10. 6.53 mean increase

8.42 7.66 8.28 7.35 8.07 7.63 7.56 7.49 6.35 6.62 -0.21

8.73 8.04 8.83 7.77 8.42 7.94 8.11 8.00 6.59 7.08 -0.40

8.53 7.91 8.91 7.80 8.32 7.91 8.32 8.32 6.76 7.08 -0.03

8.69 8.21 9.02 8.11 8.80 8.18 8.42 8.53 6.97 7.11 -0.22

TABLE 4. Daily total protein determinations on corked serum samples. No.

1st day

4.46 1. 6.87 2. 3. 7.52 4. 5. 31 5. 6.76 mean deviation

2nd day

3rd day

4th day

5th day

4.35 6.90 7.63 5.17 6.59 0.06

4.21 6.94 7.66 5.21 6.66 0.01

4. 39 7.01 7.84 5.17 6.66 0.08

411 7.01 7.52 5.21 mean daily deviation 0.08 6.42 0.16

30

C* Effect of temperature extremes As the temperature rises* the viscosity and specific gravity of the oil decreases*

The specific gravity of the

decreases as the temperature rises*

3

erum to be tested also

If the temperature of the serum

/

to be tested is kept at the same temperature as the oil* all the vari­ ables can be accounted for at one time*

Kagan prepared a chart for the

correction of the specific gravity obtained from the falling time at any temperature between 20 and 30 degrees centigrade*

He states that

"when drops take longer than 30 seconds to fall* the variations in time * of fall of individual drops i3 greater”* At this point* however* the curve of the falling time to specific gravity becomes more flattened so that the error does not mean as much in specific gravity*

From this

he concludes that the accuracy over the entire range remains about the 3ame* The data in Table 5* however* indicates that at extremes of tem­ perature* where the drops fall more rapidly or slowly* the error is greater than at room temperature ranges*

The mean deviation obtained

on identical sera at temperature extremes was 0*20 grams per cent as compared to 0*08 obtained at roam temperature range* (from Table 4) TABLE 5. No*

29.0 to 29*7 degrees C*

20 to 22*5 degrees C.

1.

7*45 7.04 6.80 7.25 6.90 7.52 7.11 7.38 6.56 7*38

7.15 7.04 6.62 6*97 6.94 7.15 6.94 7.01 6.46 7*15

2*

3* 4. 5. 6* 7* 8* 9* 10

.

* Kagan, J. Clin. Inv., 17s371, 1938

Deviation 0.30 0.00 0.18 0.28 0.04 0.37 0.17 0.37 0.10 0.23

MEAN DEVIATION 0*20 GM. PER CENT

-

31

When large numbers of determi nations were done daily , it was found “to be unnecessary "to place each individual

3

©rum in the w^ter bath pro*

vided all of the sera and the apparatus were allowed to stand for a time at room temperature*

Xn this way* over 100 determinations could

be made daily by a single operator* TABLE 6* Comparison of total protein values obtained from the same sera kept at room temperature and in the water bath at room temperature* No*

Room temp.

in water bath at room temp.

deviation

1. 2. 3. 4. 5. 6* 7. 8. 9. 10.

6.53 6.66 6.32 7*73 7.45 5.87 6.62 8.28 6.69 7.91

6.62 6.76 6.49 7.66 7.28 6.04 6.69 8.28 6.94 7.91

0.09 0.10 0.17 0.07 0.17 0.17 0.07 0.00 0.25 0.00

mean

D. Spontaneous separation of serum. The spontaneous separation or precipitation of serum i3 an exceeding­ ly rare phenomenon, and only two reports of this change could be found in the literature*

Both were in cases of myeloma.

Only one example of

this phenomenon was observed during the course of this investigation. It was noticed accidentally in the serum of an acutely ill patient with liver disease*

The exact nature of the patient's ailment was not known

because he expired before a definite diagnosis could be established, and autopsy permission was not obtained*

32

At 18 degrees centigrade an opalescent gel began to form in the lower one—third of the tube of fresh serum. became complete at 15 degrees centigrade.

Gelation of this layer The process was reversible*

for the serum spontaneously returned to normal color and viscosity at room temperature.

The upper strata of serum was decanted off and gave

a total protein value of 4.97 grams per cent,whereas the total protein concentration of the lower gel forming layer was about 13 grams per cent by the falling drop method.

The latter fraction also gave a strong­

ly positive TaJkata-Ara and formol-gel reaction which was not present in the upper fraction*

This strange phenomenon was observed on two subse­

quent samples from the same patient.

It can readily be seen that the

total protein value obtained in such a serum by any method would depend upon the layer into which the pipette is inserted and upon the tempera­ ture of the serum at the time of analysis. 3. Conclusions. A. The literature of the devlopment and use of the falling drop method of specific gravity determination of total serum protein concen­ tration is reviewed. B. Possible sources of error of the Kagan falling drop method are considered* and the results of experimental investigation of the method are presented. C.

Several sources of error were observed* the greatest being

that which resulted from alteration in the viscosity of the oil* D.

Modifications are suggested in the procedure and construction

which serve to obtain a constant accuracy within 1x10 ^ in specific gravity determination.

- 33

S. The rare phenomenon of spontaneous separation of serum from a patient with liver disease into two layers of different protein content is described.

V. RESULTS 1. Incidence of hyperproteinemia Fifty—four sera with total protein values above 8.5 grams per cent were found in 4370 sera examined by the falling drop method, giving an incidence of hyperproteinemia of 1.2 per cent. a group of 18 cases could be adequately studied.

Of this series, only In addition to these

18 cases obtained by random sampling, attention was called by staff members of the hospital to 18 cases associated with hyperproteinemia* thus making a total of 36 cases of hyperproteinemia observed.

Our

special interest in the relationship of hyperproteinemia to liver disease was known to some of the staff members and is reflected in the large numoer of cases of hepatic diseases in this series. 2. Takata-Ara Reaction. Formol-gel Reaction, and Sedimentation Rate. Twenty-eight Takata-Ara tests and 30 formol-gel tests done in twenty cases of hyperproteinemia were positive regardless of the clini­ cal condition causing the hyperproteinemia.

In only two instances was

there disagreement between the Takata-Ara and the formol-gel tests.

Of

the 20 instances in which the values for the globulin fraction were available, the lowest concentration of globulin to give a positive Takata-Ara or formol-gel reaction was 3.5 grams.

Twenty-six sedimen­

tation rates were done by the Westergren method on 17 cases of hyperproteinemiajof which 23 were corrected for anemia by hematocrit (.Vintrobe)

-

34

90 according "to "the method of Plass and Rourke. The mean of 26 uncor­ rected sedimentation rates was 150 mm, per hour*(normal does not ex­ ceed 20 mm. per hour*)

Some of the values obtained were among the

highest recorded in the literature * and were seen even in the absence of fever or infection. 80 mm. per hour* 42.

The mean of 23 corrected sedimentation rate3 was

The lowest corrected sedimentation rate recorded was

No linear correlation could be established between the concen­

tration of total protein* albumin or globulin* and the sedimentation rate* but one may conclude that in the presence of true hyperprotein­ emia the sedimentation rate is invariably increased to a marked degree. These results also substantiate the observations of other workers that the formol-gel and Takata-Ara reactions are not specific for any particular diseases*but are-invariably positive in any condition producing an increased concentration of globulin above 3.5 or 4.0 grams per cent.

The correlation of the Takata-Ara with the formol-gel test

shows that these reactions are practically interchangeable* and since the formol-gel reaction is of greater simplicity* it may conveniently be substituted for the Takata-Ara test* 3. Nature of the protein increase As described by other workers* the increase in total protein concentration in hyperproteinemia was found to be correlated with an increase in the globulin concentration and a decrease in the albumin level. Fig. 1. demonstrates this relationship of total protein to globulin in 46 determinations on hyperproteinemic sera from a variety of conditions*

The "best" line drawn according to the method of

- 35 * least squares*, shows the direct proportionality. The formula for the "best" straight line ia ys a t bx.

t

O

(f)

7

-J O

■7.

cD o

-J o

ID

cc

Ui if)

-J

cc ho

TOTAL

SERU/n

P R O T E IN S

GAV

Fig. 1. Relation of globulin content to total protein content of serum in diseases presenting hyperproteinemia.

*Mellor, "Higher mathematics for students of chem. and physics", Longmans, Green and Co. N.Y. p.326, 1916.

% .

- 36

Biologic false positive serologic reactions Eight of thirty-six cases or 22 per cent gave what was considered to be biologic false positive serologic reactions*

This type of false

positive reaction occurred with greater frequency in the Kahn floccula­ tion test than in the complement fixation test (Wassermann, Kolmer) for syphilis*

This suggests that one of the factors in the production of

the false positive reaction may be hyperproteinemia.

Moreover, sire e

the increase is almost always in the globulin fraction, it may be one of the components of globulin that produces the true as well as the false positive serology reactions*

Anti-complementary Wassermann and

false positive serology reactions have been reported with the hyper­ proteinemia of lymphogranuloma venereum and multiple myeloma.

Further

study of the relation of the globulin fractions to the biologic false positive serologic reaction for syphilis is being carried out by the author and associates* 5. Conditions found to be associated with hyperproteinemia All of the conditions found to be associated with hyperproteinemia in this investigation are listed in Table 7.

When one of the conditions

known to be a frequent cause of hyperproteinemia was present in a patient, it was assumed that this was the main or only cause in that particular patient, regardless of the presence of other concomitant pathological conditions.

TABLE 7 Cjigj3-3i.fication of 36 cases of hyperproteinemia Number of patients In group Individual HEPATIC DISEASE Cirrhosis Cirrhosis with primary carcinoma of liver Acute hepatitis Catarrhal jaundice

9

LYMPHOGRANULOMA VENEREUM

6

CHRONIC INFECTIONS Calculus pyelonephritis Calculus pyonephrosis Pelvic cellulitis Moist gangrene of leg with diabetes mellitus

4

SCARLET FEVER

2

ERYSIPELAS

1

TUBERCULOSIS Pulmonary with tuberculous peritonitis Pulmonary tuberculosis

2

BACTERIAL ENDOCARDITIS

2

MALARIA

1

SYPHILIS (with hepatitis)

1

MULTIPLE MYELOMA

1

EXTREME DEHYDRATION from vomiting and diarrhea from diabetic coma

2

MISCELLANEOUS CONDITIONS Generalized adenopathy (cause undiagnosed) Alcoholism with mental deterioration Non-specific ulcer scrotum Transverse myelitis

5

6 1 1 1

1 1 1

1

1

1

1 1

2 1 1

1

38

Data on representative cases of hyperproteinemia studied is pre sented in Table

8

.

TABLE Diagnosis

8

.

Sex Ago; Race Date[Total Albu­ Globu-! Takata-I Formol-’ Sedimen-j Hema­ Frei tocrit tesi :pro­ min tation lin Ara gel i tein rate ! i

........

~-fc■

2.05

6.73

♦++4.

•4+4+

39'' N

3/19 9.44 2.0 4/1 10.13 2.47

7.44 7.66

4 44

--+

4444 ♦♦•44*

M

44

W

4/16 11.44 4.37 7.07 4/21 9.50 3.85 5.65 5/13 8.74 2.95 ,5.79

Pulmonary tubercu­ losis

F

27

N

3/30 8.52 5/12 8.16

2.9 3.16

5.62 5.0

Luetic hepatitis

F

31

4/17 8.98

4.86

Pelvic abscess

F

18

6/16 6/20 6/24 6/26

9.38 10.04 12.76 10.1

3/21 3/25 4/9 4/10

8.53 10.15 6.64 7.46

Cirrhosis

M

47

Lympho­ granuloma venereum

F

Lympho­ granuloma venereum

Chronic pyelo­ nephritis

F

49

W

* I

N

M Calculus pyonephro** sis

55 , W

Non­ specific ulcer of scrotum

13

M

* American Indian

N

4/25 8.78

87

30

Neg*

86

25

4+4 +

4++ +

80

25

++♦♦

4444 444+

79 82

34 31

Neg.

++++

4.12

44*4

*.4-4-4

90

27

Neg.

3/65 3.50 4.68 3.45

5.73 6.54 8.08 6.65

4444

-4-4-1-4

90

30

Neg.

3.65 3.61 3.16 2.16

4.88 6.54 3.48 5.3

*4*44.4

89

33

♦44+

*4 4+

4/29 8.53 6/20 7.43

2.81 3.68

5.72 3.8

+44 + *4

+4 +4 *-4

92 96

3/19 9.16 3/24 8.79

3.75 3.61

5.41 6.54

4++4 4444

4444 44 ++

78

4-4+4

Neg.

110

19

Neg.

22

40 36

Neg.

- 39

Legend for Table 8 Standard laboratory methods were used throughout: sedimentation rate 92,93 90 by the Westergren method, corrected according to Plass and Rourke, hematocrit usihg the Wintrobe tube on oxylated plasma, Takata-Ara reac— 94 tion as described by Ragins, formol-gel reaction as described by Klein 95 96 et al*, Frei test using Squibb*3 Lygranum, and total protein, albumin, and globulin determinations by the standard micro-Kjeldahl method. VI. COMMENT Two major difficulties arose in the classification of these cases. We were first faced with the as yet unsettled problem of eliminating hemo— concentration as cause of increased serum proteins.

Hemoconcentration

resulting from shock, venous stasis, and dehydration has been given as one of the commonest causes of (apparent) hyperproteinemia.

A study of

the literature reveals that in many of the cases of hyperproteinemia re­ ported, these factors were not sufficiently taken into consideration. This fact may in part account for the wide variety of clinical conditions hitherto reported with hyperproteinemia.

It is generally known that the

state of hydration cannot be determined clinically with any degree of certainty. The laboratory methods recently developed to determine the state of hydration are also fraught with misleading factors.

Red blood cell counts

and hematocrit determinations are only of value in the absence of anemia or polycythemia*

Specific gravity and total serum protein values must be

discounted in the presence of pathological conditions known to be associated

- 40

with hypo-or hyperproteinemia.

In chronic states of prolonged dehydration,

the red blood cell counts and protein values tend to fall as a result of J A 97 destruction of the red blood cells and proteins. Since a depletion of sodium is a significant factor in dehydration, it has been thought that the water loss can be computed from the measurement of sodium concentra­ tion in the blood plasma.

However, Simeone states that the total base of

the blood plasma may remain normal in the presence of moderately severe dehydration, provided renal function is unimpaired.

The determination of

blood volume by the dye methods and the computation of total available fluids by the thiocyanate or sucrose methods were too elaborate and timeconsuming for this work. The McClure—Aldrich intra—dermal wheal absorp­ tion test for the state of hydration has not yet been proven to be of value. It has been assumed by some workers that in hemoconcentration, the relative proportion of albumin ahd globulin remains unchanged.

Thus,hyper­

proteinemia associated with only slight alterations in the A/G ratio,has been considered by most authors to be indicative of hemoconcentration, whereas in tru hyperproteinemia (excluding dehydration) the globulin frac­ tion with rare exceptions is elevated, and the albumin fraction is lowered* Peters and Eisenman stated: "In alterations of protein concentration resul­ ting from changes of heiiioconcentration, both protein fractions are equally *

affected —

Rowe, in his studies on hemoconcentration

produced by 98

venous stasis, found no alteration in the A/G ratio. produced pyloric obstruction in cats.

Gomori and Podhradszky

They observed only moderate altera­

tions in the A/G ratio of five animals,of which only one had an inversion * Peters and Eisenman, Am. J. Med7 Sc., 186:812, 1933.

41

of the ratio of approximately 0*85* ranged between 1*0 and 1*5*

The ratios of the other four cats

Talbot, in studies on heat cramps determined

the concentrations of albumin and globulin in seven instances where a high total protein was observed* tration

He reported that the serum albumin concen­

these cases was invariably below 3*4 grams per cant, whereas

the serum globulin concentration was increased to as high as 7*4 grams per cent*

(The actual figures for each case were not given.)

Jeghers

and Selesnick, in commenting on these conflicting reports suggests '* — that another factor besides hemoconcentration *per se* causes the high protein values found in heat cramps".

They further advanced the concept that in

prolonged and severe dehydration changes in the capillary permeability may occur,allowing the smaller albumin molecule to escape, producing a relative concentration of globulin*

As can be seen from these diverse

reports, the effect of various degrees and chronicity of dehydration on the A/G r^tio is an open question that awaits further study.

The pic99 ture is further complicated by the demonstration of Nadal et al. that two distinct types of dehydration may exist in human subjects.

They showed

that dehydration resulting from simple water deprivation is characterized by thirst and oliguria, and does not lead to impairment of circulation or hemoconcentration,whereas dehydration resulting from abnormal salt loss results mainly in a loss of extra cellular fluid, a reduction in plasma volume, hemoconcentration and distrubances in circulation. Our interpretation of the state of hydration rested on clinical Observations (vomiting,, diarrhea, sweating, water deprivation, thirst, *Jeghers and Selesnick, Int. Clinics, 47;251, 1937.

42

dry mucous membranes, loss of tissue turgor, oliguria) and on a judicious evaluation of the laboratory data (serum sodium, hematocrit, and urinary output)* It should be recognized that dehydration and a disease causing true hyperproteinemia may occurin the same patient* It is known that in hyperproteinemia considerable fluctuations in the blood protein levels may occur, and may even be normal on occasion* Therefore, if a serum sample were obtained at a time when the protein concentratittiwas normal, such a case of hyperproteinemia would be over­ looked* Another difficulty in the analysis and grouping of our cases was the frequency of multiple pathological conditions existing in the same patient. Only one case of what was considered to be pure dehydration (that is, not associated with conditions known to alter the A/G ratio) was observed in this series.

This patient, a 56-yq^pir-old Negress was admitted to the

hospital in diabetic coma.

The total protein on admission was 9.00 grams

of which 6.25 grams was albumin and 2.75 was globulin (normal

a /g

ratio).

When the diabetes was controlled and the patient well hydrated, the total protein dropped to 7.49 gr$ms.

Attempts to obtain other similar cases

of hyperproteinemia by drawing blood on patients that appeared clinically dehydrated was disappointing in that normal total protein values were usually obtained even in several other cases of diabetic coma. for this may be two-fold.

The reason

First, the degree of dehydration necessary to

produce hemoconcentration may be more extreme than is commonly thought, and second, the usual indigent type of patient admitted to the Cook County Hospital frequently suffers from anemia and nutritional hypoproteinemia. in However,^8 of the 36 cases in this study, dehydration as manifested by clinical and laboratory signs was considered to be an important factor in the cause of the hyperproteinemia*

- 43

Post mortem examination of four hyperproteinemic patients and biopsy of the liver of two hyperproteinemic patients fail to reveal any consis­ tent histo—pathologic changes such as the proliferation of the reticulo­ endothelial system suggested by Bing*

VII. CONCLUSIONS 1* Fifty—four patients with total serum protein values above 8.5 grams per cent were found in the random sampling examination by the falling drop method of 4,370 sera,

giving the incidence of hyperproteinemia

in the Cook County Hospital of 1.2 per cent. 2. The Takata-Ara and formol gel reactions are interchangeable and are almost invariably positive in conditions associated with hyperproteinemia and hyperglobulinemia. 3. The sedimentation rate is regularly increased to extreme degrees in hyperproteinemic conditions but no linear correlation exists between the concentration of total protein or globulin

and the rate of

sedimentation* 4* The concentration of total protein

is correlated with the concentration

of globulin in hyperproteinemic sera. 5. Biologic false positive serologic reactions for syphilis may occur in conditions associated with hyperproteinemia. 6. No consistent correlation exists between particular histoppathologic changes and hyperproteinemia. 7. In the Cook County Hospital hyperproteinemia is most frequently found to be associated with hepatic diseases, lymphogranuloma venereum, certain acute and chronic infections, and states of extreme dehydration.

-44 VIII.

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VITA DONALD HERMAN ATLAS Date of birth: September 1,1913. Place of birth: Chicago, Illinois. Education: Carl Schurz High School, Chicago, Illinois. Northwestern University, Bachelor of Science, 1934 Master of Science, 1936 Bachelor of Medicine,1937 Doctor of Medicine, 1938 Positions: Rockefeller foundation Fellow, Institute of Neurology, under Dr. S.W. Ranson, N.U. Medical School, 1934-1936. Assistant in Pathology, Cook County Hospital, under the late Hr. R.H. Jaffe', six months, 1937. Interne,Cook County Hospital, 18 months, 1938-1939. Resident in Medicine, Mount Sinai Hospital, Chicago, one year, 1940. Josiah Macy Jr. Fellow in Medicine, N.U. Medical School, one year, 1941. Associate Attending Physician, Cook County Hospital, Department of Medicine, 1941 — . Instructor in Medicine, N.U. Medical School, 1942 ---•

ACKNOWLEDGEMENT The author wishes to express his sincerest appreciation to Doctor Leonard Cardon and Doctor A. C. Ivy

for their guidance

during the course of this work* He also wishes to thank Mr* Joseph Bunata, Serologist of the Cook County Hospital for his kindness in supplying the serums for this work*