OBSERVATIONS ON THE INORGANIC BASES AND PHOS- PHATES IN RELATION TO THE PROTEIN OF BLOOD

AND OTHER BODY FLUIDS IN BRIGHT’S DISEASE AND IN HEART FAILURE.

BY HARALD A. SALVESEN AND GEOFFRY C. LINDER.

(From the Hospital of The Roclcefeller Institute for Medical Research.)

(Received for publication, November 12, 1.923.)

It has been shown by several writers during the last few years that the inorganic bases of blood serum are remarkably constant in normal individuals,’ whereas changes have been demonstrated in certain pathological conditions. It has always been anticipated that a decrease in inorganic bases would occur in severe acidosis and it has been demonstrated that there actually is a loss of sodium from the blood in this condition in children (Kramer and Tisdall, 2) ; this element, as the principal inorganic base, is obviously used for neutralizing acid and excreted as the corresponding salt. Changes in the inorganic bases have also been demonstrated in Bright’s disease. Sodium has been found increased or decreased (Denis and Hobson, ll), potassium and magnesium more or less constant (Myers and Short, 4; Denis and Hobson, ll), and cal- cium decreased, particularly in the uremic stage (Marriott and Howland, 12; Halverson, Mohler, and Bergeim, 5; Denis and Hobson, 11). The cause of these changes found in Bright’s disease is not known; retention is generally accepted as a probable cause of an increased concentration of sodium in serum, while a decreased concentration might be due to water retention. The question of the relation of sodium (and chlorides) to water retention is intimately connected with the problem of edema formation and

1 Sodium: Doisy and Bell (l), Kramer and Tisdall (2). Potassium: Kramer and Tisdall (3), Myers and Short (4). Calcium: Halverson, Mohler, and Bergeim (5), Meigs, Blatherwick, and Cary (6), Marriott and Howland (7), Kramer and Howland (S), Kramer and Tisdall (9). Magnesium: Kramer and Tisdall (9), Denis (10).

617

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618 Inorganic Bases and Phosphates

is as yet unsettled. Marriott and Howland (12), who discovereu the calcium decrease in uremia, pointed out the coincidence of high phosphates and low calcium in this condition. That there might possibly be a relation between the rise in phosphates and the fall in calcium was suggested by the experiments of Binger (13).

As the inorganic bases of the plasma are partly bound to the proteins and the plasma proteins often are low in certain types of kidney disease, it seemed of importance to determine whether there is any parallelism between the changes in the proteins and inorganic bases in Bright’s disease. This has been done and we have further, in one case of Bright’s disease and in several cases of cardiac decompensation, analyzed different body fluids with various concentrations of protein. Also the relation of the phosphates has been investigated because of the possible connec- tion between phosphate retention and the decrease in calcium.

Methods.

The inorganic bases were determined in serum by the methods of Kramer and Tisdall (2, 3, S), the phosphates by the method of Tisdall (14), and the plasma proteins by that of Howe (15). Blood for the determination of the inorganic bases and the phos- phates was always taken in the morning before breakfast; no stasis was used, and the blood was collected under paraffin oil, centrifuged at once, and the serum pipetted off. Blood for the determination of plasma proteins was usually collected at the same time unless otherwise stated. Edema fluid was obtained from the subcutaneous tissues of the legs by means of Southey’s tubes; narrow rubber tubing filled with sterile oil was attached to the cannula and the fluid was collected in large test-tubes under oil.

Normal Values oJ’ the Plasma Proteins, the Inorganic B&es, and the Phosphates of Serum.

In a previous work from this clinic Linder, Lundsgaard, and Van Slyke (16) found the average normal values of the plasma proteins per 100 cc. of plasma to be as follows: total protein 6.73 gm., albumin 4.12 gm., globulin 2.61 gm., and the albumin: globulin ratio 1.6. The highest value for total protein was 7.45

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H. A. Salvesen and G. C. Linder 619

gm. and the lowest accepted was 6.22 gm., although in one of the normal individuals a value of 5.62 gm. was found with an albumin: globulin ratio of 1.5. A determination made 6 weeks later on the same individual gave 6.34 gm. with the same ratio. The albumin: globulin ratio varied between 1.4 and 2.

Table I shows the values of the inorganic bases and phosphates found in seven normal men. The blood was taken in the morning before breakfast when they were still in bed. The average values of the bases correspond to those of Kramer and Tisdall (2, 3, 9) except that of magnesium, which is a little lower. The values of the phosphates correspond to those found by Tisdall (14).

TABLE I.

Content of Inorganic Bases and Phs

NO.

Average. High.. . . Low.....

Na

320 347 335 335 333 331 339

?nM.

zz”,

139 150 146 146 145 144 147

.-

n&M.

it%

1

19.7 5.0 20.4 5.2 20.1 5.1 19.2 4.9 19.3 4.9 19.1 4.9 21.8 5.6

334 145 19.9 5.1 347 150 21.8 5.6 320 139 19.1 4.9

T

K

-

,hates in Serum of Normal Adults.

Ca

2”; ::

10.2 10.3 9.7

10.0 10.4 10.1 10.0

10.1 10.4 9.7

- n&M.

z%G

2.6 2.6 2.4 2.5 2.6 2.5 2.5

2.6 2.6 2.4

I --

1

--

-

Mxg Inorganic P.

1.7 1.8 1.8 1.8 1.7 1.7 1.9

1.8 1.9 1.7

-- 9nM. &$ %z 0.70 3.6 0.74 3.8 0.74 3.4 0.74 3.6 0.70 4.6 0:70 3.7 0.78 4.3 -- 0.74 3.8 0.78 4.6 0.70 3.4

?nM. z%z 1.2 1.2 1.1 1.2 1.5 1.2 1.4

1.2 1.5 1.1

DISCUSSION.

In the classification of Bright’s disease we have followed the direction of Volhard and Fahr (17). The material consists of fifteen cases of Bright’s disease (three cases of nephrosis, ten cases of glomerulonephritis, and two cases of nephrosclerosis) and five cases of heart failure. The observations in the cases of Bright’s disease with normal plasma protein are found in Table II, the results in cases with low plasma protein are recorded in Tables III to VII, and those in two cases of uremia are found in Table VIII. In patients with heart failure determinations of the proteins, calcium, and phosphates were made upon the blood and

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620 Inorganic Bases and Phosphates

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TABL

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Jan.

29

Se

pt.

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624

2

f i : :

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Inorganic Bases and Phosphates

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12

13

TABL

E VI

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Seru

m

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gani

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ma

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ein

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tent

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cium

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rally

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3. L

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ale.

14

Date

.

toss

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23

Fe

b.

8

Mar

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Oct

. 26

Feb.

1

“ 14

“ 26

Mar

. 27

June

13

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. _

-

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1 K

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ma*

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cc.

liter

315

315

137

137

6.9

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2.0

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332

1442

1.4

5.5

8.2

332

1442

1.4

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2.1

2.3

0.5

2.3

0.5

320

320

1392

2.0

5.6

7.8

1392

2.0

5.6

7.8

2.0

2.0

2.4

2.4

l.f

l.f

311

311

1352

2.7

5.8

7.7

1352

2.7

5.8

7.7

1.9

1.9

2.2

2.2

0.E

0.E

316

316

137

137

8.0

8.0

2.0

2.0

2.1

2.1

0.E

0.E

. .

347

1502

2.5

5.8

8.8

347

1502

2.5

5.8

8.8

2.2

2.2

331

1442

3.2

5.9

8.1

331

1442

3.2

5.9

8.1

2.0

2.0

1.7

1.7

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6.3

321

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1.6

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6.3

6.3

1.6

1.6

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2.6

4.52

2.

77

1.75

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6

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5.15

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4.87

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to

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lcium

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5

per

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ily.

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26

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alciu

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lact

ate

treat

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.

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14

15

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Age:

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Male

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T -I-

-

TABL

E VI

II.

Seru

m

Inor

gani

c C

onst

ituen

ts

and

Plas

ma

Prot

ein

Con

tent

in

Tw

o C

ases

of

Ur

emia.

Date

.

19FB

May

7

“ 20

“

24

“ 26

Sept

. 18

NEI

K I

I CS

152

153

$11

135

19 .:

S.2lW

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9.0

7.3

1.8

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1.

1

4.9

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ic 4.f

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6.i

7.;

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aSDl

a.

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ein.

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mo.

m*,

om.

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gi

cc.

7.07

29

182

6.15

6.

87

3249

1.26

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0 5.

95

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.

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1.6:

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!.94

i-

s.24

1.1

$.30

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10.9

$

R%UX

WkB

.

Nep

hros

cler

osis

. D

efin

ite

sign

s of

ur

emia

. Sa

lt so

lutio

n ad

min

is-

tere

d su

bcut

aneo

usly

an

d pe

r re

ctum

. Co

ma.

D

ied

May

27

.

Chr

onic

gl

omer

olun

e-

phrit

is;

urem

ia.

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TABL

E IX

.

Inor

gani

c C

onst

ituen

ts

and

Prot

ein

Con

tent

of

Diff

eren

t Bo

dy

Flui

ds.

Cas

e 16

. M

. D

. M

ale.

Ag

e 46

yea

rs.

Dia

gnos

is

: Sy

philis

;

aorti

c in

suffi

cien

cy.

x

Date

.

Iov?S

Mar

. 29

Apr.

6

May

14

Body

flu

id.

Seru

m.

Asci

tic

fluid

. Ed

ema

“ Se

rum

.

Che

st

fluid

. Se

rum

.

- I -

Prot

ein.

A:G

Re

mar

ks.

360

1562

2.0

5.61

0.4

2.6

1.70

.70

3.6

1.2

6.12

36

0 15

622.

0 5.

610.

4 2.

6 1.

70.7

0 3.

6 1.

2 6.

12

7.5

1.9

1.30

.53

3.7

1.2

2.66

7.

5 1.

9 1.

30.5

3 3.

7 1.

2 2.

66

351

1531

5.9

4.0

6.3

1.6

1.10

.45

3.6

1.2

0.29

35

1 15

315.

9 4.

0 6.

3 1.

6 1.

10.4

5 3.

6 1.

2 0.

29

334

1452

1.8

5.6

9.0

2.3

334

1452

1.8

5.6

9.0

2.3

3.4

1.1

5.72

3.

4 1.

1 5.

72

6.9

6.9

1.7

1.7

3.4

1.1

1.72

3.

4 1.

1 1.

72

330

1432

1.2

5.41

0.0

2.5

1.50

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3.9

1.3

7.87

33

0 14

321.

2 5.

410.

0 2.

5 1.

50.6

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9 1.

3 7.

87

- _ .

. 6

-

Albu

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in.

vn.

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100

GC.

2.78

2.53

0.87

4.

01

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-

m. p

el

uwcc

.

3.34

3.19

0.85

3.

86

0.83

Pr

otei

n de

term

ined

in

P

plas

ma.

L ?

0.80

Pr

otei

n de

term

ined

in

G

plas

ma.

F

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C.

1.1

Prot

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dete

rmin

ed

in

&

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8

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TABL

E X.

Inor

gani

c C

onst

ituen

ts

and

Prot

ein

Con

tent

of

Scr

una

(Pla

sma)

an

d Ed

ema

Flui

d.

Cas

e 17

. E.

G

. M

ale.

Date

.

19.33

May

9

Seru

m.

June

8

Body

flu

id.

-I

Edem

a flu

id.

305

1321

8.8

4.8

5.8

1.5

0.24

Se

rum

. 31

4 13

720.

0 5.

1 8.

5 2.

1 1.

40.5

7 3.

9 1.

3 5.

94

2.57

3.

37

-

Age

41 y

ears

. D

iagn

osis:

Sy

philis

; va

lvul

ar

dise

ase.

L._

AL

Y..

--D

r.

Tota

l. “;F

z-

tknt

A:G

I I

ppy-

__---

-

;g

Wm

. ;g

7n

Y.

ym-

7nM

. 10

0 cc.

izr

;y pe

r. ;z

2%

zits

30

5 13

223.

6 6.

0 8.

5 2.

1 1.

40.5

7 3.

7 1.

2 5.

82

2.48

3.

34

RelllS

&S.

Prot

ein

dete

rmin

ed

in

plas

ma.

Prot

ein

dete

rmin

ed

in

plas

ma.

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18

19

20

TABL

E XI

.

Cal

ciuna

, Ph

osph

ates

, an

d Pr

otei

n C

onte

nt

of D

iffer

ent

Body

Fl

uids

in

Th

ree

Cas

es

of H

eart

Failu

re.

“&Fe

. A&

F.

K.

Male

. 51

H.

R.

Male

. 67

L.

P.

Fem

ale.

10

Date

. Bo

dy

fluid.

102s

Apr.

JO

“ 17

ac

t. 5

May

19

June

9

Seru

m

(pla

sma)

. As

citic

flu

id.

ma.

yg

.;g

ec

. lite

r

9.3

2.3

6.9

1.7

3.0

1.0

6.24

3.

1 1.

0 2.

24

Edem

a.

5.1

1.3

0.35

Se

rum

(p

lasm

a).

8.4

2.1

4.1

1.3

5.17

“

I‘ (

> 9.

7 2.

4 3.

6 1.

2 6.

87

‘I C

‘ (

> 9.

8 2.

5 4.

4 1.

4 6.

57

Che

st

fluid

. 6.

8 1.

7 4.

0 1.

3 1.

76

Seru

m

(pla

sma)

. 8.

4 2.

1 2.

9 0.

9 5.

95

Che

st

fluid

. 5.

9 1.

5 3.

1 1.

0 1.

88

-

CS

lnoP

c - To

tal.

_ Q

-

Pm

tein

. T

Alb

u-

min

.

m. p

m

100 c

c.

2.94

1.

04

2.61

3.

64

-

22-

-

rm.

pm

100

cc.

3.30

,

1.20

,

2.56

3.

23

3.35

3.

22

1.02

0.

74

3.25

2.

70

1.02

0.

86

A:G

0.8!

0.

8:

1.0

1.1

1.0

1.4

1.2

1.2

R~I

lW&

S.

Nep

hros

cler

osis

; m

yoca

rdia

l in

- su

ffici

ency

.

Pneu

mon

ia;

sept

icem

ia.

Rec

over

ed

from

pn

eum

onia

an

d bi

late

ral

empq

ema.

Seni

le

hear

t.

Valv

ular

di

seas

e.

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630 Inorganic Bases and Phosphates

fluid obtained at the same time from the pleura or peritoneum; edema fluid also was obtained in three cases and similarly analysed. In two of the cases the other inorganic bases were determined. The results in these tw’o latter cases may be seen from Tables IX and X, while the observations in the other three cases are recorded in Table XI. The relationship between calcium and protein content of different body fluids is seen from Fig. 1.

It seems obvious, from all the observations in Bright’s disease, that there is a parallelism between the changes in plasma protein and in calcium content of serum in Bright’s disease. In every

FIG. 1. Relation between calcium and protein per 100 cc. of various body fluids in patients with cardial hydrops.

case in which low protein was found, there was also a decrease in the calcium content. A low plasma protein concentration was ob- served in some of the heart cases, and here, too, the calcium content of the serum was diminished. Subsequent determinations showed that when the protein concentration returned to normal, the calcium content rose to normal also. This is seen from Fig. 2 (nephrosis case) and from Fig. 1, Cases 16 and 18 (heart cases). Likewise, whenever a lowered serum calcium was demonstrated the plasma protein was found to be decreased. In two cases of uremia, however, the calcium decrease seemed out of proportion to the drop in protein when compared with the other cases in which renal function was relatively unimpaired. This is seen especially well in Case 14 (Table VIII). However, in these

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H. A. Salvesen and G. C. Linder 631

cases of uremia there was an enormous retention of phosphates and in Case 14 (Table VIII) the decrease in calcium was propor- tional to the increase in phosphate. In repeating Binger’s experi- ments (13) upon the effects of intravenous injections of phosphates on the serum calcium, Tisdall (18) found that when the decrease of calcium was established the phosphates increased to about the same degree as observed in our uremia cases. It seems very likely that the phosphate retention is responsible for the excessive drop of serum calcium in uremia.

:; 2 f”

FIG. 2. FIQ. 3.

FIG. 2. Relation between calcium and protein in 100 cc. of eerum (plasma) in a case of acute nephrosis at the time of admission and when recovered (Case 5).

FIG. 3. Relation between calcium and protein per 100 cc. of serum and edema fluid in Case 7.

The observations on the calcium content of blood and different extravascular body fluids obtained from one case of nephrosis (Fig. 3) and from five cases of heart failure with anasarca show a close parallelism between the calcium content and the protein content. It seems justifiable to conclude that the cause of the drop in serum calcium observed in Bright’s disease without uremia is the decrease in the plasma protein. The other inorganic bases

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632 Inorganic Bases and Phosphates

show an irregular behavior and may vary in either direction.2 The deproteinization of the blood does not seem to affect these bases in any characteristic way.

It is still an open question in what form calcium exists in the blood serum. From dialysis and ultrafiltration experiments in vitro it seems to be certain that 50 to 70 per cent of the blood calcium is diffusible (Rona and Takahashi, 19; Cushny, 20; van Meysenbug, Pappenheimer, Zucker, and Murray, 21; Neuhausen and Pincus, 22). How much of this diffusible calcium is ionized is unsettled. The experiments of Neuhausen and Marshall (23),

10 8

6

4

2

0

FIQ. 4. Relation between diffusible (lower part) and non-diffusible (upper part) serum calcium in Cases 7 (nephrosis), 16, 17, and 18 (heart failure).

who worked with calcium amalgam electrodes, indicate that only 15 to 25 per cent is ionized. In one of our cases of nephrosis (Case 7) and in three of the cases of heart failure, in which the calcium was determined in blood and in the practically protein- free edema fluid, the dialysis “experiment” is performed in tivo by nature, and it shows that 55 to 70 per cent of the serum calcium is diffusible and is found in the edema fluid (in Case 7, Fig. 3, 70 per cent; in Cases 16, 17, and 18, Fig. 1, 60, 68, and 55 per cent, respectively). The diffusible quota of the total serum cal- cium in these cases is seen from Fig. 4, which is charted from the data of Figs. 1 and 3. Whether the other 30 to 45 per cent are

2 The magnesium method is not accurate enough to detect smaller variations. The potassium values are given with reservation as we often have encountered difficulties with the method. Sometimes a precipitate, which is not potassium cobalti-nitrite occurs, especially in ascitic or chest fluids.

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H. A. Salvesen and G. C. Linder 633

bound to protein or to other organic material is not known; but the fact that blood calcium is reduced in cases of Bright’s disease in which a low plasma protein concentration is found, and the parallelism between the calcium and the protein content of dif- ferent body fluids indicate that the non-diffusible calcium of the blood probably is bound to protein. This protein-bound calcium is apparently not ionized to the same degree as the diffusible calcium; if the results of Neuhausen and Marshall (23) are correct, most of the protein-bound calcium must be unionized. In the following paper we have discussed the biological significance of the protein-bound calcium. A loss of blood calcium through loss of blood protein will probably have no immediate consequence in regard to lack of specific calcium action; as the amount of ionized calcium remains unchanged, tetany will probably not occur from that cause.

It was observed also that potassium and magnesium were markedly lower in the edema fluid than in the blood serum. We do not feel justified in drawing any conclusion from these few observations for reasons stated above.2 It will be noted that administration of large doses of calcium by mouth had very little effect in raising the calcium content of the serum (Cases 6, 12, and 13).

SUMMARY.

In fifteen cases of Bright’s disease the inorganic bases, the phosphates, and the plasma proteins have been studied. In four non-uremic cases in which the plasma protein was normal, the inorganic bases were also normal. In nine cases in which the plasma protein was diminished a marked drop in the calcium content was found, while sodium was normal or slightly decreased, and potassium and magnesium varied in both directions. In two cases of uremia in which the plasma protein was but slightly decreased, the drop in calcium was more marked than in the non- uremic cases; a great retention of phosphates was found in these two cases.

In five cases of heart failure with dropsy the calcium content of different body fluids (serum, chest fluid, ascitic fluid, edema fluid) decreased proportionately to the protein content.

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634 Inorganic Bases and Phosphates

CONCLUBION6.

The decrease in serum calcium found in non-uremic casea of Bright’s disease without phosphate retention parallels the decrease in plasma protein. It appears that phosphate retention may cause a drop in the serum calcium in Bright’s disease. The dXusible serum calcium is from 55 to 70 per cent of the total calcium. The nondiffusible portion is probably bound to the plasma proteins.

BIBLIOOUPEY.

1. Doiey, E. A., and Bell, R. D., J. Biol. Chem., 1920-21, xiv, 313. 2. Kramer, B., and Tisdall, F. F., J. Bill. Chem., 1921, xlvi, 487. 3. Kramer, B., and Tiedall, F. F., J. Biol. Chem., 1921, xlvi, 339. 4. Myers, V. C., and Short, J. J., J. Bill. Chem., 1921, xlviii, 83. 6. Halverson, J. O., Mohler, H. K., and Bergeim, O., J. Biol. Chem.,

1917, xxxii, 171. 6. Me&, E. B., Blatherwick, N. R., and Cary, C. A., J. Biol. Chem.,

1919, xxxvii, 1. 7. Marriott, W. MoK., and Howland, J., J. Biot. Chem., 1917, xxx& 233. 8. Kramer, B., and Howland, J., J. Bill. Chem., 1920, xliii, 35. 9. Kramer, B., and Tiedall, F. F., J. Biol. Chem., 1921, xlvii, 476.

10. Denia, W., r. Biot. C’hem., 1922, lii, 411. 11. Denie, W., and Hobeon, S., J. BioZ. Chem., 1923, Iv, 183. 12. Marriott, W. McK., and Howland, J., Arch. Int. Med., 1916, xviii, 708. 13. Binger, C., J. Pharmacol. and Ezp. Therap., 1917-18, x, 105. 14. Tiedall, F. F., J. BioZ. Chem., lQ22, 1, 329. 1s. Howe, P. E., ,J. BioZ. Chem., 1921, xlix, 1OQ. 16. Linder, G. C., Lundegaard, C., and Van Slyke, D. D., Proc. Soi. Bzp.

BioZ. and Med., 1922-23, xx, 320. 17. Volhard, F., and Fahr, T., Die Brighteohe Nierenkrankheit; Klinik.

Pathologie und Atlas, Berlin, 1914. 18. Tiedall, F. F., J. BioZ. Chem., 1922, liv, 35. 19. Rona, P., and Takahashi, D., B&hem. Z., 1911, xxxi, 338. 20. Cu&ny, A. R., J. PhyeioZ., 191+2O, liii, 391. 21. von Meyaenbug, L., Pappenheimer, A. M., Zucker, T. F., and Mur-

ray, M. F., J. BioZ. Chem., 1921, xlvii, 529. 22. Neuhausen, B. S., and Pincus, J. B., J. BioZ. Chem., 1923, Iv& QQ. 23. Neuhaueen, B. S., and Marshall, E. K., Jr., J. BioZ. Chem., 1922,

liii, 365.

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Harald A. Salvesen and Geoffry C. LinderFAILURE

BRIGHT'S DISEASE AND IN HEARTBLOOD AND OTHER BODY FLUIDS IN

RELATION TO THE PROTEIN OFBASES AND PHOSPHATES IN

OBSERVATIONS ON THE INORGANIC

1923, 58:617-634.J. Biol. Chem. 

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