PLASMA ~~~~~~~~~~~~~~~~~~~todm5migu4zj3pb.cloudfront.net/manuscripts/100000/100024/JCI2510… · THEPLASMAPROTEINS IN RELATIONTOBLOOD HYDRATION II. IN DIABETES MELLITUS By JOHNP

THE PLASMAPROTEINS IN RELATION TO BLOODHYDRATION

II. IN DIABETES MELLITUS

By JOHN P. PETERS, HAROLDA. BULGERAND ANNAJ. EISENMAN

(From the Department of Internal Medicine of Yake University and the Medical Serviceof the NewHawn Hospital, NewHaven, Connecticut)

(Received for publication, February 14, 1925)

Rowe (17), in 1917, reported observations of the serum proteins of10 patients with diabetes. The only remarkable thing was the extremevariability of the findings. Both high and low values were obtained.In the majority of instances albumin and globulin preserved theirnormal proportions; but in some instances globulin appeared relativelyhigh. Subsequent observers have confirmed Rowe and the generalopinion. appears to be that there is nothing characteristic about theserum protein level in diabetes.

In table 1 are shown the results of 52 observations of the plasmaproteins of 31 patients with diabetes mellitus of varying degrees ofseverity, studied at different stages of the disease.

EXPERIMENTALPROCEDURE

Whenever there is no note to the contrary, the blood was taken in the morning,before the patient had breakfast and the morning dose of insulin. The blood waswithdrawn in a syringe and coagulation prevented by the addition of enoughneutral potassium oxalate to make an 0.2 per cent oxalate solution in the blood.The treatment of the blood is indicated in column 6 of the table. Those specimensmarked cont. were obtained without stasis and immediately placed over mercuryin a blood sampling tube of the type described by Austin, Cullen, Hastings, Mc-Lean, Peters and Van Slyke (1). Usually venous blood (ven.) was employed, butoccasionally arterial (art.) was used instead. In specimens indicated as cap. theblood was withdrawn without precautions against air contact and brought intoequilibrium with 40 mm. of CO2 in the air at 38°C. by the method previouslydescribed (16) before it was placed in the sampling tube over mercury. Fromthe mercury sampling tube part of the blood was transferred to a centrifuge tube

451

PLASMAPROTEINS IN DIABETES

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J. P. PETERS, H. A. BULGERAND A. J. EISENMAN

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458 PLASMAPROTEINS IN DIABETES

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and the plasma separated. The latter was then removed to a second samplingtube over mercury and from this samples were taken for analysis. The remainderof the blood was utilized for the determination of cell volume and for other purposesof no immediate importance in this connection.

For the estimation of cell volume an hematocrit of the Daland type manu-factured by the International Instrument Company for their centrifuges wasemployed. Carbon dioxide was determined by the method of Van Slyke andStadie in a water-jacketed machine calibrated in 0.01 cc. For the oxygen capacitya technique devised by Lundsgaard and Neill1 which permits the saturation of theblood in the Van Slyke apparatus, was used. In order to reduce the amount ofblood required the oxygen capacity was frequently determined not on the wholeblood, but on the cell residue. The centrifuge tubes in which the plasma wasseparated were especially prepared with contracted necks. After the cells hadbeen whirled down the upper level of the plasma was carefully marked. Afterthe plasma had been removed the tube was filled to the mark with 0.9 per centNaCl solution, the cells were well mixed with the saline, and the resulting emulsionwas employed for the determination of oxygen capacity. Values thus obtainedagree with those obtained directly from whole blood. (If water is used for thedilution instead of saline the resulting solution becomes so viscous that it is un-manageable.) For the total nitrogen of the plasma an ordinary macro-Kjeldahltechnique was employed. Non-protein nitrogen was determined on the wholeblood by the Folin-Wu method (7), except that distillation and titration with 0.02N alkali was substituted for direct Nesslerization. In some instances, indicated byan asterisk in the table, non-protein nitrogen was not determined. In these cases0.19 per cent (corresponding to 30 mg. of nitrogen per 100 cc.) was subtracted fromthe values of the total nitrogen in terms of protein. This was only done when therewas a reasonable certainty that the blood non-protein nitrogen would prove nor-mal. In no case can this omission introduce any significant error. It is, ofcourse, not strictly proper to use blood non-protein nitrogen values for plasma.Presumably the latter would have proved somewhat less. The necessity foreconomy of blood made direct determinations on plasma impracticable.

In some of the earlier experiments no special effort to avoid venous stasis waspractised in the collection of cap. specimens. Partly this was due to a failure toappreciate the importance of such precautions; partly because the blood wasintended more particularly for other purposes and the studies of plasma proteinswere only incidental. This invalidates none of the important experiments. Inthe earliest observations the plasma used for protein determinations was made upfrom all the remnants left from other work on the blood specimens and portions ofit were even derived from blood that had been exposed to the air. This probablyintroduced no serious errors but must be taken into consideration in at least onecase, no. 15096.

l Personal communication.

459


Blood sugar was determined by the method of Folin and Wu(8) except in a fewof the earlier studies when Benedict's modification of Lewis and Benedict's method(2) was used.

OBSERVATIONS

The 13 observations in the first section of table 1 are from mild andfor the most part uncomplicated cases of diabetes. The majoritywere made before breakfast the morning after the patients were ad-mitted to the hospital, and, therefore, while glycosuria still persistedand before dietary treatment had been well started. None of thesepatients showed evidences of serious dehydration or any considerableloss of weight. The plasma proteins are, in all but two of the twelveobservations quite normal. The two exceptions, nos. 15733 and 20387both had rather low proteins. The reduction in 15733 is slight, 5.98,and there were so many complicating factors in the case that it isimpossible to ascribe the fault to diabetes. It is unfortunate thatonly one observation was made on no. 20387. The low protein wasquite unexpectedly encountered in the course of an experiment inwhich the blood was being used for control purposes only. In review-ing the case afterwards it was found that on the day of the bloodexamination the patient had a transitory glycosuria and an extraor-dinary diuresis that persisted for 3 days. Of course it is impossibleto connect these phenomena with the low proteins directly in theabsence of other examinations of the same individual. It may beof some significance that the lowest proteins in this series were asso-ciated also with the lowest hemoglobin.

The 10 observations of section 2 represented single examinationsof patients with diabetes of inherently greater severity or aggravatedby some complicating condition. Although no. 10572 was studiedon two occasions it is impossible to compare the two observationsbecause an interval of almost a year had elapsed between them.On the first occasion he came to the hospital extremely malnourished,on a low diet. Extreme dietary reduction was necessary to renderhim aglycosuric and to overcome an initial acidosis. Under thesecircumstances, as is so often the case, the early part of his recoverywas attended with the development of some edema. It was duringthis edematous period that the low proteins were found. On the

460


second occasion he had been taking insulin for some time, with ex-cellent results, and was admitted to the hospital only because hehad developed a cold and a slight pleurisy that was attended by anexacerbation of diabetic symptoms and the appearance of largeamounts of sugar and acetone in the urine. With this he lost weightvery rapidly and, at the time of the examination, appeared distinctlydehydrated. It will be noted that the plasma proteins are near theupper limit of normal. The hemoglobin does not, however, appearelevated.

With one other exception the values observed vary only withinnormal limits and the variations bear no relation to the level ofblood sugar, carbon dioxide or hemoglobin. No. 18067 was anunusual case in every respect. She was admitted in diabetic comawithin 48 hours after the onset of an acute attack of abdominalpain attended by persistent vomiting. No previous history ofdiabetes could be obtained. Blood was taken immediately after sheentered the hospital when she was in deep coma, with typical respira-tions of acidosis. She appeared extremely desiccated, her pulse wasweak and rapid and her systolic blood pressure only 80 mm. Withinsulin she was brought out of coma, and the respirations weretemporarily quieted; but she sank back into her original state withextreme rapidity. After her admission to the hospital she passedvery little urine and the administration of fluids by mouth and byhypodermoclysis resulted only in the development of edema. Shedied within 72 hours after her admission to the hospital.

The high hemoglobin at once suggests a concentration of the bloodfrom loss of water and this seems reasonable in view of the vomitingand the overventilation. On the other hand concentration can offerno explanation for the low proteins. In fact no mere alteration ofblood water can explain the paradoxical findings. There was noevidence of any preexisting condition that could have determinedsuch extremely low proteins. The immediate disease might havecaused a specific destruction of the plasma proteins, but the durationof symptoms was so short that this seems unlikely. It is far easierto believe that proteins had passed from the blood. White andErlanger (19) found that in surgical shock and allied conditions,although the cellular elements of the blood became concentrated by

461


loss of water to the tissues, the plasma proteins fell. The low bloodpressure and the clinical picture in this case is quite suggestive of"shock" and it is not surprising, therefore, to find an inspissation ofthe blood with low plasma proteins. If this explanation of the find-ings is correct the protein change can not be referred directly to thediabetes but must be ascribed to the condition that precipitated theacute crisis. No clue to this underlying condition could be foundand autopsy was refused.

On each of the 9 cases in section 3 two or more studies were madeat comparatively short intervals and opportunity is therefore givento study the mechanism of some of the variations that occur indifferent stages of the disease. The first 2 patients, nos. 26461 and29754, had diabetes of moderate severity, in the latter case compli-cated by an otitis media. Both had typical diabetic symptoms, andon admission appeared dehydrated. They had been losing weightrapidly and showed marked glycosuria and ketonuria. In each casethe administration of insulin and adequate amounts of fluids resultedin a striking increase in weight with a simultaneous fall in bothhemoglobin and plasma proteins. In table 2 the relative plasmavolumes, calculated from hemoglobin and cell volume and fromplasma protein, are compared. In both instances the changes calcu-lated by the two methods are in the same direction; in the secondcase they are also of the same magnitude. One can not escape thefeeling that water exchange is the predominant factor behind suchalterations and that the preliminary dehydration and subsequentrestoration of the normal fluid content of the body are reflected inthe protein values. The fourth case, no. 15120, presents a differentpicture. The patient, an elderly womanwith diabetes and a compli-cating pneumonia was in a desperate state at the time of admission.Dehydration was quite noticeable and the administration of fluids bymouth difficult. Although with iletin it proved possible to over-come the glycosuria and ketonuria, the ovetventilation, temperatureand inadequate fluid intake resulted probably in a further negativewater balance which is associated with an increase of plasma pro-teins, hemoglobin and cell volume. The changes in nos. 29061,15096 and 22350 seem to be of the same nature and in the first andlast of these three cases the high initial values, 8.37 and 8.02 areindicative of a very definite concentration of the blood.

462


It is hard to avoid the feeling that these rapid alterations of plasmaprotein that occur in simple cases are merely expressions of changesin the state of hydration of the blood. The fact that the calcula-tions based on hemoglobin and cell volumes do not agree exactlywith those based on plasma protein values does not refute such atheory. Perfect agreement could hardly be expected in view of theprofound disturbances of circulation that must accompany condi-tions of diabetic intoxication and may affect the number of cellscirculating in the peripheral blood. Regeneration and destruction

TABLE 2

Relative plasma volumecaic3ilated fromCase number Remarks

02-apacity and Plasmacell volume Plsaproteins

26461 1.00 1.00 At time of admission1.10 1.03 2 days later


15120 1.00 1.00 At time of admission0.89 0.96 The next morning

29061 1.00 1.00 At time of admission, January 281.14 1.04 The next morning, January 291.30 1.35 One day later, January 301.39 1.43 One week later, February 6


15096 1.00 1.00 At time of admission1.09 1.26 2 days later1.38 1.16 1 month later

of the proteins themselves must gometabolic disturbance that exists.portance of hydration as a factor,

on and may share in the generalWith the recognition of the im-however, little has been gained.

The production of appreciable changes in the water content of theblood in normal individuals is difficult. To effect a plasma dilu-tion amounting to 30 per cent is almost impossible.

Widal, Abrami, Weill and Laudat (20) have noted similar changesin the concentration of the proteins in the serum of diabetic patientswith acidosis after the administration of iletin and they also inter-pret the diminution of protein as an expression of blood dilution.

463


They could not, however, connect this directly with the water balancenor with the glycemia, the acidosis nor the assimilation of carbo-hydrates. In those of our cases in which rapid dilution occurredthere is a definite retention of fluids and increase of weight as theprotein falls. That the administration of fluids alone is not capableof producing such an effect is, however, well illustrated by case no.15096 who was admitted with an edema. It is also clear from thiscase that edemas which occur in the course of diabetes are not in-variably associated with low plasma proteins and blood dilution.

Gamble, Ross and Tisdall (9) have shown that fasting results ina loss of water from the body and that this dehydration is one of theeffects of acidosis and depletion of base. The well known diureticeffects of ammonium chloride and calcium chloride have been shownby Haldane (10) to depend also on the production of an acidosis,It has repeatedly been demonstrated (5) that the edemas which occurin the course of diabetes can be rapidly delivered by the administra-tion of these acid producing salts and that edema is prone to developafter the administration of sodium bicarbonate. Oehme (14) hasmade the statement that all conditions that tend towards the pro-duction of acidosis result in diuresis, and, vice versa, any increase inthe alkalinity of the blood leads to a retention of fluid. At firstsight it seems as if in these studies, also, dehydration was associatedwith acidosis and retention of fluid with the restoration of the normalalkalinity of the blood.

No. 18067 of section 2, mentioned above, is apparently an excep-tion to the rule; but this must not be given too much weight because,as we said above, she was exceptional in every respect. When fluidbalance is considered the case seems comparatively clear. From thestandpoint of blood hydration, however, frequent inconsistenciesappear. For instance, no. 15096, at the time of admission, had anormal plasma CO2and an edema; at the same time both hemoglobinand plasma proteins were high, indicating a concentration of theblood. Apparently the hydration of tissues and blood may be disso-ciated in diabetes just as they have been found to be in nephritis andin cardiac disease.

Although tissue dehydration is in most instances associated withacidosis and retention of fluid with the restoration of the normal

464


alkaline reserve, the state of hydration apparently bears no relationto the absolute level of plasma CO2. In only one or two cases wasthe CO2 of the plasma above the upper normal level and edemaoccurred frequently in the presence of a normal CO2. From a clin-ical standpoint only, the conditions that usually result in edema indiabetes can be fairly well defined. It usually occurs in poorlynourished individuals who are receiving an inadequate diet, but showno reduction in the plasma bicarbonate. It is especially apt todevelop during or just after recovery from a severe attack of diabetictoxemia. Before the advent of insulin it was more common thanit is at present. The development of acidosis or the administrationof acidosis producing drugs results in rapid diuresis. In the absenceof such therapy the edema disappears when the diet is increased tomeet the demands of the individual. This is well iustrated by caseno. 15670. At the time of the second blood examination he haddeveloped general anasarca. At that time he was receiving only1340 calories without insulin and had moderate glycosuria. After theaddition of enough insulin to clear the glycosuria, the edemadisappeared as rapidly as it had come. No. 10572 delivered hisedema as soon as he was given a maintenance diet and insulin.

This nutritional factor plays a part in the development of edemathat appears to be independent of the acid-base equilibrium, or atleast of the level of the plasma or blood carbon dioxide. It may besome of the acid-base elements other than bicarbonate that is atfault. Peters (15), Stillman, Van Slyke, Cullen and Fitz (18),Cullen and Jonas (21) and most recently Bock (3) have noted thatafter recovery from diabetic acidosis the plasma bicarbonate risesrapidly, while the alveolar carbon dioxide tension remains low. If thealveolar carbon dioxide is the same as that of the arterial blood sucha discrepancy can only result in an uncompensated alkalosis. ThisBock and his coworkers found in some of their cases. Wehave alsonoted a tendency on the part of some of these patients to develop amild grade of uncompensated alkalosis during the period of recovery.

Gamble, Ross and Tisdall (9) suggest that the water balance ismore strictly determined by the level of the alkaline metals in thebody and that a retention of these elements is attended by a dimin-ished fluid output. To test this hypothesis we have analyzed the

465


serum of some of the patients for total base by a method devised byCullen (4) similar to the Fiske (6) method for the determination oftotal base in urine. In conjunction with this method the chlorides,carbon dioxide, inorganic phosphates and proteins have also beendetermined. In this manner the total inorganic anions and kationscan be balanced against one another. In table 3 are presented theresults obtained on one patient during a period of edema and afterdiuresis. The edema was very slight. There is no evidence of anychange in the level of the total base of the serum in this case and an

TABLE 3

Case 34295

August 23....

w

so0gn10

m

mgm.per

100 cc.

200

Z.

co

1o.perC6#

18.0

August 30....I 152 116.7

_

voas.percent

41.5

0

co

percent

6.03*

6.45*

a-

038

Ce

OISWits.percent

70.3

68-.0

Ud

gramsper

liter

5.89

sC

4in60.0

grams

perliter

0 .0351

.0Ca0.iZ

134.0

Ce.0

0

mM

154.3

.00I

mM220.

5.9010.0371133 .21154.9121.7

insignificant alteration of the total inorganic acid. If these observa-tions are typical of diabetic edematous conditions it appears thatsome factor other than the acid-base balance must be active in theproduction of the edema. It is possible that undernutrition withits attendant loss of body protein may render these subjects more

susceptible than normals to the effects of alkali. In this connectionone can not but be reminded of the famine edemas that were so

prominent in the War (13).The fact that it is really the acidosis of diabetes and not the ketosis

Remarks

Slight edemaof ankles.Weight 147pounds, in-creasing

No edema.Weight 144pounds, di-minishingpH of plas-ma 7.39

J. P. PETERS, H. A. BULGERAND A. J. EISENMAN 46

or the other metabolic disorders associated with diabetic toxemiasthat determines the tissue dehydration is well illustrated in no.15096. This patient had received large doses of alkali before ad-mission and had also vomited large amounts of food and fluid, pos-sibly with the loss of considerable chloride. The consequence wasthat in spite of the presence of an enormously high blood sugar,marked ketonuria and all the symptoms of impending diabetic comaexcept the hyperpnea, her plasma CO2 content proved normal.Under these circumstances she developed an edema during the dia-betic intoxication.

Although it is possible to explain the changes that occur in theplasma proteins over short periods of time on the basis of alterationsof blood hydration in the majority of cases; in a few the facts do notseem to permit such an explanation. For example in no. 18467 theoxygen capacity rose in the course of a few hours from 15.3 to 16.6,while the proteins fell from 6.72 to 6.26. Cell volumes were notdetermined in this case, but it is inconceivable that the cells couldhave shrunk enough to account for such a discrepancy. Such occa-sional exceptions do not seem to us to invalidate our explanation.It would be extraordinary indeed if hemoglobin, cells and proteinswere always proportionally distributed in all parts of the circulatingblood. The matter can be definitely decided only by the simul-taneous application of some other method for the direct determina-tion of plasma volume.

Comparisons of proteins and hemoglobin made at intervals of morethan two days in most instances show no parallelism. The methodsare inapplicable to studies over long periods because of the play ofthe forces that determine regeneration and destruction of proteins andblood cells. In case 15096, for instance, at the time of the secondexamination hemoglobin, cell volume and plasma proteins had allfallen together; one month later at a third examination the hemo-globin and cell volume were still lower, but the plasma proteins hadrisen to the normal level. No. 22350 shows a similar course ofevents. One gains the impression that the high proteins found inthe first examination were due to the extreme dehydration of ablood that really contained less protein than normal. Relievingthe acute condition results in the restoration of the normal plasma

467


volume and reveals the true level of the proteins, which is low. Asimprovement in the general condition progresses the total amount ofprotein in the body increases, possibly because of an increased pro-duction. In favor of such a theory is the general nature of the con-

TABLE 4Case 22350

September 6, 11 p.m.....

4ir.

C02 .2

.9 ,O 0

I 'N u0w (5 Remarksa a 0 im=1

co 9310 ? I 1

to8 -,, Cs 4m u N l:w

mgm.per

100 cc.

462

September 7, 9 a.m ...... 370

September 8, 9 a.m ...... 233 |

September 10, 10 a.m..... 257

September 16, 8 a.m ...... 270

September 24, 8 a.m......

October 7, 8:30 a.m.......

Wols.percent

Wis per

cent cent

vols.percent

19.0

5.06127.7

3.22 47.0

4.75128.0

6.70159.8

462 16.5 37.6 6.14 57

130 16.5

. 7

40.9 6.98 65.1

At time of admission. Patientvomiting continuously. Largeamounts of glucose and ace-tone in urine

Still vomiting continuously, buthas received insulin, carbohy-drate and fluids. Acetone hasdiminished

Vomiting has ceased. Improve-ment continues. Fluid in-take larger

Vomited again this morning,with recurrence of all symp-toms

Greatly improved. Receivingadequate diet with large dosesof insulin

Improvement continues.Weight has increased steadily,without occurrence of edema.pH of plasma 7.48

Just before discharge from hos-pital

ditions associated with low plasma proteins in diabetes. In thisseries, with the exception of no. 20387, no protein value below 6 percent was found except in the presence of a severe diabetes withmarked evidences of wasting. The same patients invariably hadnormal plasma proteins when the disease was under control and they

468


were receiving adequate diets. (See, for examples, nos. 10572, 15096and 22350.)

Case 22350, was again admitted to the hospital recently afteranother attack of vomiting, in a desperate condition. The findingson the second admission are shown in table 4. The same rapid fallin the protein level during the first few days is noticeable. Thistime, however, the initial protein level is much lower and the sub-sequent depression proportionately greater. But again, after aninterval and with the establishment of proper metabolic conditionsthe proteins rapidly rise to the normal level. The low proteins andblood concentration in this instance are quite similar to the findingsin case 18067, mentioned above. It may be that both are due tothe same cause and that the explanation previously advanced for18067 is unnecessary.

It is needless to point out that without insulin these studies couldhave led to no more satisfactory conclusions than those of previousobservers. The miraculous rapidity with which the clinical andmetabolic features of a case can be altered by the use of this drugaccelerates the blood changes to such an extent that blood dilutioncan be studied with less danger from the introduction of complicatingfactors.

As to the underlying cause of the low proteins in severe diabetesonly speculation is as yet permissible. It does not seem absurd toconnect them with the general malnutrition and protein wastagethat these patients exhibit. Kerr, Hurwitz and Whipple (11) have,to be sure, shown that short periods of starvation do not affect theplasma proteins. It is, however, axiomatic that starvation does notresult in nutritional disorders similar to those produced by the pro-longed use of unbalanced diets. The same authors (12) have shownthat the regeneration of protein after plasma depletion is retardedby inadequate diets. One is tempted to conclude that the low pro-teins in the plasma in severe diabetes are due to an insufficient pro-duction or regeneration which is augmented when the normal nutri-tion and metabolism are restored.

This again opens the question of the diabetic edemas. Usually inthe presence of edema the proteins are low. That this is not in-variably the case has been demonstrated. This is to be expected if

THE JOURNALOF CLINCAL INVESTIGATION, VOL. I, NO. 5

469


both low proteins and edema are characteristics of severe diabetesand if edema is provoked by alkalosis, which seldom exists when theblood is dehydrated.

SUMMARYAND CONCLUSIONS

In mild diabetes unassociated with ketosis or malnutrition theplasma proteins are usually normal.

In severe diabetes associated with chronic malnutrition the plasmaproteins are usually found reduced. When proper nutrition andmetabolic conditions are restored the plasma proteins resume thenormal level.

In conditions of severe diabetic toxemia with ketosis the bloodbecomes concentrated by loss of water. Under these circumstancesthe plasma proteins appear relatively high. When the toxemia isovercome a rapid dilution of the blood and a reduction of the plasmaproteins occur.

The tissue dehydration which is so striking a part of the pictureof diabetic toxemias appears to be closely related to the acidosis.Restoration of the alkaline reserve is attended by retention of fluidin the body.

True diabetic edema seldom if ever occurs in the presence of acido-sis. A definite alkalosis is, however, not essential to the productionof edema. A nutritional factor apparently plays a part in deter-mining the excessive accumulation of fluids, as edemas are charac-teristic of malnourished patients with severe diabetes who are usinginsufficient food, and can be eliminated by the administration andutilization of adequate diets. The fact that diuresis can be inducedby the production of acidosis indicates that the acid-base equilibriumalso plays its part in the etiology of the edema. It is suggested thatthe state of malnutrition may render the subject peculiarly suscep-tible to the effects of alkali.

The common association of low plasma proteins with diabeticedema is probably not one of cause and effect but is a result of thefact that both phenomena are characteristic of severe diabetes with-out acidosis.

470

J. P. PETERS, R. A. BULGERAND A. J. EISENMAN 471

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PLASMA ~~~~~~~~~~~~~~~~~~~todm5migu4zj3pb.cloudfront.net/manuscripts/100000/100024/JCI2510… · THEPLASMAPROTEINS IN RELATIONTOBLOOD HYDRATION II. IN DIABETES MELLITUS By JOHNP