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THE ESTIMATION OF PLASMA PROTEIN CONCENTRATION FROM PLASMA SPECIFIC GRAVITY

BY DONALD D.VAN SLYKE,* ALMA HILLER,t ROBERT A. PHILLIPS,$ PAUL B. HAMILTON,8 VINCENT P. DOLE, REGINALD M. ARCHIBALD,

AND HOWARD A. EDER

(From the Hospital of The Rockefeller Institute for Medical Research, New York)

(Received for publication, July 18, 1949)

In 1930 Moore and Van Slyke (1) reviewed the various physical measurements, refractometric and others, that might serve as measures of the protein concentration of plasma, and concluded that specific gravity was the most reliable if application was required to pathological as well as normal plasmas. For plasma of nephritic patients they found that the relation between protein concentration and specific gravity, Diz, was expressed by the linear equation, P = 343 (G - 1.0070), P representing protein concentration in gm. per 100 ml. and G the specific gravity, 0% The constants 343 and 1.0070 were based on gravity measurements by pycnometer and on proteins calculated from Kjeldahl nitrogen as 6.25 X N.

A number of authors have since published modifications of the equation P = a(G - b), with differing constants based on gravities measured by several procedures, and on micro-Kjeldahl analyses also by various procedures. The procedures and the constants arrived at for the equation are indicated in Table I. The gravity method indicated as CuSO4 is the procedure described in the preceding paper (2), but without the correction of 0.0007 for the difference between 0:: of copper sulfate standards and the plasma drops that balance in them. The gradient column method is that of Linderstrgm-Lang in the modification described by Lowry and Hunter (3). The Harbour method is the “falling drop” method described by Barbour and Hamilton (4). In order to compare calculations by the different equations the protein concentration calculated by each equation for plasma which has a gravity of 1.0270 determined by pycnometer is indicated in the column “P calculated.”

From a recently published experimental review of Kjeldahl procedures (13) it appears probable that the differences in the relation between specific gravity and Kjeldahl-determined protein nitrogen found by different investigators (last column of figures, Table I) may in part be due to the fact

* Present address, Brookhaven National Laboratory, Upton, New York. t Present address, Presbyterian Hospital, Chicago 12, Illinois. $ Present address, United States Naval Medical Research Unit No. 3, care of the

American Embassy, Cairo, Egypt. 5 Present, address, Alfred I. du Pont Institute, Wilmington, Delaware.

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TABLE I

Variations of Equation P = a(G - b) Formulated by Different Investigators

Moore and Van Slyke (1) 9 normal men Nephritic patients

Weech, Reeves, and Goettsch (5) dogs, mostly hy poproteinemic

Cole, Allison, and Boyden (6); rab. bits, normal

Hoch and Marrack (7, 8); human, normal

Lowry and Hunter (3); human

Lloyd et al. (9); human, normal

Meyer et al. (10) ; dogs

Dicker (11); rats

Lloyd et al. (12); human with famine edema

Yf?iU Specific ravity metho used f

Pycnometer ‘I

1936 “

1945 Pycnometer Gradient col-

umn cuso4

1945 Gradient column

1945 ‘( “

1945 Barbour cuso4

1948 Gradient column

Values Value of P calcu- of a and b lated for plasma

used of G = 1.0210 by pycnometer

-I-

I I

Gby a b method

used -I -l---

l I Di 1581.00701.0270 7.16 $43 1.0070 1.0270 6.86

540 1.0069 1.0270 6.83

Q51.00641.0263 6.47

166 1.0070 1.0270 7.30 1611.0070 1.0271, 7.25

164 1.0060 1.0260, 7.30

!48 1.0069 1.0275 7.08

)011.0027 1.0270, 7.32

515 1.0050 1.0263 6.41 325 1.0059 1.0263 6.63

%64 1.0060 1.0263 7.39

354 1.0073 1.0270 6.97

P CalCU- lated

Method used to determine proteins

Micro-Kjeldahl, digested with KPO,, HzSOa, K&%Os until clear

Micro-Kjeldahl, no details

“ ‘I

Micro-Kjeldahl, digested l-3 hrs. with Cu and Se catalysts

Micro-Kjeldahl, no details

Gravimetric

Micro-Kjeldahl, Cu and Se catalysts; digested 10 min.

Micro-Kjeldahl, Cu and Se catalysts; digested 5 hrs.

Gravimctric

* The authors quoted, following the usage of a preliminary report by the present writers, took the 0: of plasma as equal to the 0: of the CuSOd solution in which the plasma drop balanced. As shown in the preceding paper (2), by this procedure plasma Dt is estimated on the average as 1.0263 when the pycnometer value is 1.0270. Meyer et al. (10) in their measurements found the same gravities by the Barbour falling drop method as by the preliminary copper sulfate procedure; hence the same correction of 0.0007 is applied to their gravity measurement by the Barbour method.

t G values by methods other than pycnometer, as found for plasmas with pycnometer G of 1.0270, by the authors cited.

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that most of the Kjeldahl digestion procedures used give low results when applied to plasma protein. Hiller et al. (13) found that nitrogen values equal to those yielded by Dumas combustion were obtained only when mercury and potassium sulfate were used as catalysts, or when digestion with copper and selenium as catalysts was prolonged for several hours. It will be noted in Table I that, for plasma of 0: = 1.0270, the protein concentrations estimated by Hoch and Marrack and by Dicker, who digested for 1 to 5 hours, and by Lloyd et al. (9), who determined the proteins gravimetrically, are higher than the concentrations estimated by authors who used shorter or unstated Kjeldahl digestions.

The work presented in this paper was carried out to ascertain with as much accuracy as possible the relation of plasma specific gravity to human plasma protein concentration calculated from nitrogen analyses.

Methods

Blood for the analyses was drawn and treated with heparin as described in the preceding paper (2). For the data on normal subjects in Table III blood samples of about 20 ml. were drawn; 10 ml. of the plasma were used for determination of gravity by pycnometer as described in the preceding paper (2). Portions were then used for check determinations of the gravity by the copper sulfate method (2), and for macro-Kjeldahl nitrogen determinations. For the data on hospital patients reported in Table II, portions of 5 ml. or less of blood were drawn, and gravities were determined by the copper sulfate method (2). In the determinations by the copper sulfate method, the gravity of the plasma is taken as 0.0007 unit above the gravity of the copper sulfate solution in which the plasma drops balance. (This correction is automatically made when the standard copper sulfate solutions are prepared according to Table II of the preceding paper (2) .>

The methods used for total nitrogen determination are indicated in the headings of Tables II and III. The method of Hiller et al. (13), with Hg and KzS04 as catalysts in the digestion, was found (13) t.o give nitrogen values for proteins equal to those obtained by Dumas combustion. The analyses by the Campbell-Hanna (14) method, with Cu and Se as catalysts, were performed before the more accurate procedure of Niller et al. had been worked out. The Campbell-Hanna method was modified by pro- longing the digestion for 2 hours after the digest cleared. In a series of analyses by both methods, the Campbell-Hanna procedure was found to yield 98.4 per cent as much nitrogen as the procedure of Hiller et al. (13) with mercury catalyst; hence nitrogen values obtained by the Campbell- Hanna method were multiplied by the correction factor 1.016.


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334 PL4SM.4 PROTEIN FROM SPECIFIC GRAVITY

Protein nitrogen was calculated by subtracting non-protein nitrogen (trichloroacetic acid filtrate N determined by gasometric micro-Kjeldahl (15)) from the total plasma nitrogen. The weight of protein was calculated as 6.25 X N, the factor 6.25 being that obtained by Hiller et al. (13) in analyses of fat- and ash-free preparations of normal human plasma protein.

TABLE II

Plasmas from Hospital Patients

G vaIues all determined by the copper suIfate method (2). _. .-_~-__

Miscellaneous hospital patients

No. of plasmas’ ; 50 -

Type of Kjeldahl analysis used to determine Q, . . Macro, with Cu and Se catalysts, 2 hrs. digestion

(14)t -__-- _--__-

Range of PN, gm. per 100 ml. 5.62-12.44 Mean PN, gm. per 100 ml. 7.282

‘< G 1.02697 ‘I value of b in equation PN = 365 (G - b) 1.00702

__-- ----- mz. pm 100 m!.

Comparison of Pa calculated as 365 (G - 1.0070) with PN calculated as 6.25 X N

Mean algebraic difference, PO - PN Standard deviation of PO from P.w Maximal + ‘I “ ‘I I‘ “

‘I _ ‘I “ I‘ ‘1 ‘I

f0.014 f0.22 +0.58 -0.32

Nephritic patients

41

Micro. with Hg and KeSO. catalysts (13)

3.71-8.03 4.732 1.01996 1.00700

------ gm. fx?r 100 ml.

ZtO.000 ztO.26 f0.53 -0.52 ----

* Plasmas with non-protein nitrogen over 100 mg. per 100 ml. are excluded from this table, and reported in Table V. In some cases two or more plasmas were drawn from the same patient on different dates.

t The nitrogen values obtained by the digestion with Campbell and Hanna’s (14) mixture of H&Oh, H~POI, Cu, and Se were multiplied by the factor 1.016, to correct for the fact that this digestion was found to give 1.6 per cent less ammonia than the digestion with Hg and K&SO, (13).

Symbols

The following symbols are used. P = gm. of protein per 100 ml. of plasma. G = specific gravity of plasma in terms of II::, the density of water at the same temperature as that of the plasma being taken as unity (0.9976 = the density II”,“). When it is desirable to distinguish between proteins calculated from nitrogen determinations and proteins calculated from plasma specific gravities, P, is used to indicate the former and P, the latter. VP indicates the “apparent specific volume” of the proteins in


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TABLE III Plasmas of Normal Adults

Range of normal specific gravity and protein concentration. Constants of equation, P = a(G - b). Comparison of protein concentration, PN, calculated from Kjeldahl nitrogen, with concentration, Pa, calculated from plasma gravity. -

Type of Kjeldahl analysis used to determine PN . .

Range of normal PN calculated as 6.25 X protein N

Mean, gm. per 100 ml. S.D. from mean, gm. per 100 ml. Maximum observed, gm. per 100 ml. Minimum “ “ “ 100 (‘

Range of normal gravity Mean G S.D. from mean Maximum observed G Minimum ‘I “

Mean value of a calculated as PN

a = (G - 1.0070)

Comparison of Pa calculated as 373 (G - 1.0070 with PN calculated as 6.25 X N

Mean algebraic difference, Pa - PN S.D. Of PO from PN

Maximal + deviation of Pa from PN “ _ “ “ “ “ “

Group I. 17 subjects

Macro, with Hg and Kn% catalysts, 2 hrs. digestion

(13) ; samples of 2 ml. plasma

7.39 10.34

8.03 6.76

- G by PYC- nometer

G ~etu~04

/

1.02683 1.02681 fO.0099 zto.0091

1.0287 1.0283 1.0247 1.0249

-

372.7 373.0 __..-

:?a. per 100 ml. gm. per 100 ml

+0.007 fO.000 zko.093 fO.106 +0.14 +0.15 -0.15 -0.25

Group II, 20 subjects

MCd’u”rc;dwi;h

(14). 2 hrs. digestion*

7.38 f0.28

8.17 7.05

1.02678 rfo .0086

1.0285 1.0252

373.1

gm. per 100 ml.

-0.002 f0.136 +0.17 -0.31

* See foot-note (I), Table II.

plasma, defined as the volume of solvent (water + non-protein solutes) that is displaced by 1 gm. of protein.’

Estimation of Constants of Equation, P = a(G - b)

The range of protein concentrations in normal plasmas is too narrow to indicate closely the best values for the constants. Thus a and b respec-

1 For a discussion of “apparent partial specific volumes” of proteins in solut’ion, see Kraemer (16).


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336 PLa4SMh4 PROTEIN FROM SPECIFIC GRAVITY

tively may be taken for normal plasma as 360 and 1.0063, 370 and 1.0069, or 380 and 1.0074, without a statistically significant difference in the accuracy of the equation. Consequently we have used the wider protein concentration range of pathological plasmas to estimate the best line (Fig. 1) which is defined by Equation 1 (Table IV).

1.042

1.038

1.036

h 1.032 .3 2 1.030 &

;I.,*8

1 1.026 03

1.024

1.018

“‘163 4 5 6 7 8 9 10 11 12 1 PN = Plasma protein concentration as 6.25 X N

FIG. 1. Relation of specific gravity to nitrogen-calculated protein in pathological plasmaa.

When this equation was used to calculate proteins from gravities for the normal plasmas of Table III, the resulting PG values averaged 2.2 per cent lower than t,he PN values. The difference is indicated by the greater a value of Equation 2 (Tables III and IV), b being kept constant at 1.0070.

Equation 3 (Table IV) is used for computing the nomograms for general


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VAN SLYKE, HILLER, PHILLIPS, HSMILTQN, DOLE,

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337

use in the following paper (17). Equation 3 gives on the average plasma protein concentrations 1 per cent lower t,han nitrogen-calculated values of the normal plasmas of Table III, and 1 per cent higher than the nitrogen- calculated values of the pathological plasmas of Table II.

Factors Determining Constant a in Equation P = a(G - b)-From the equation it is evident that l/a represents the increase in gravity per increase of 1 gm. per 100 ml. in protein concentration. An a of 373 indicates that an increase of 1 gm. per 100 ml. in P causes an increase of l/373 = 0.00268 in G, owing to replacement of solvent (water + non-protein solutes) of gravity b by protein of greater density.

The quantitative relation of a to the specific volume of the dissolved proteins may be calculated as follows: Increase of 1 gm. per 100 ml. in P displaces VP ml. of solvent of density 0.997b. The weight of solvent displaced is 0.997bVp (density X volume). The resultant increment of weight per 100 ml. of plasma is 1 - 0.997bVp, and the increment in weight per

TABLE IV Equations for Calculating Plasma Protein Concentration, P, from Specific Gravity, G

Equation No. Type of plasma Formula to calculate protein, P, from gravity, G

1 Pathological P = 365(G - 1.0070) 2 Normal “ = 373(“ - 1.0070) 3 “ and pathological “ = 369(“ - 1.0070)

1 ml., or in OS”, is (1 - 0.997bVP)/100. Since 0:” = 0.9970:: = 0.997G, the increment in G caused by an increase of 1 gm. in P is (1 - 0.997bVp)/99.7. This increment is l/u. Hence

(4 99.7

a = 1 - 0.997bVp

Rearranging to calculate VP gives

(5)

From the a and b values of Equation 2, for normal plasmas, one calculates VP = 0.730. Hence an increase of 1 gm. per 100 ml. in P involves the displacement of 0.730 ml., or 0.730 X 1.0070 X 0.997 = 0.7329 gm., of solvent, and an increase of 1 - 0.7329 = 0.2671 gm. in the weight of 100 ml. of plasma. There results an increase of 0.002671 in the density Di”, and an increase of 0.002671/0.997 = 0.002679 in 0;;.


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338 PLASMA PROTEIN FROM SPECIFIC GRAVITY

From observations of hdair and Adair (US), it appears that proteins, unlike most crystalloid solutes, may dissolve without measurable shrink- age in volume, the volume of the solution formed equaling the volume of the solvent plus the volume of the dry protein. In the case of edestin and hemoglobin the apparent specific volumes of both proteins were found t’o be 0.744 f 0.002 in aqueous solution, and the specific volume (l/(density)) in the dry state was also found to be 0.744 f 0.002, from density values of 1.345 f 0.003. The apparent behavior of the proteins in dissolving without volume change is consistent with the linear form of the equation, P = a(G - b).

From the a of 365 of Equation 1, one calculates for the pathological plasmas of Table II a VP of 0.724, smaller than the VP of 0.730 calculated for normal plasma. Pedersen (19) found for human albumin a VP of 0.736, for r-globulin, 0.718. It consequently appears possible that changes in the ratio of y-globulin to albumin could cause changes in VP of the order required to cause the average difference of 2 per cent in PN between t,he normal and pathological plasmas of the same gravity in our series.

For a protein, fetuin, from the plasma of a new-born calf, Pedersen (19) found a VP of 0.70, suggesting that, the presence of abnormal proteins, as well as changes in the distribution of normal ones, may influence V, and hence the a value of the equation P = a(G - b).

Fig. 2 shows no correlation between the A:G ratio, determined by the Howe method, and the P,-- PN difference. Present data do not suffice to show whether correlation would become evident if the y-globulin fraction was determined separately, and if the influence of other factors, such as error in calculating protein as 6.25 X N in pathological plasma, were eliminated.

Factors Determining Constant b in Equation P = a(G - b)-The value 1.0070 of the constant b approximates the gravity of a solution of the plasma crystalloids. This was demonstrated by an experiment in which normal plasma was filtered under pressure through cellophane. Filtrat,es, free of protein, showed by micro pycnometer 0:: values approximating 1.0070 within the limit of error, ~1~0.0003, of the measurement.

Per unit concentration, the increment in gravity caused by the organic crystalloids is of t,he order of the increment caused by protein (0.0027), while the mineral constituents cause from 2 to 2.5 times as great increment,s. Per gm. per 100 ml., the increments caused by the chief plasma crystalloida are as follows: urea 0.0029, glucose 0.0039, NaCl 0.0071, NaHC03 0.0062, NC1 0.0064, CaClz 0.0087. The total increment in gravity due to the crystalloid solutes of normal plasma may be approximately estimated by assuming that of the total mineral salts, indicated by a total base of 154 m.eq. per liter, 105 m.eq. have the effect of NaCl and the remainder the


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ARCHIBALD, AND EDER

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effect of NaHC03, and that the effects of the nitrogenous crystalIoids approximate that calculated by assuming that all the non-protein nitrogen is urea. The respective gravity increments caused by the solutes thus calculated are NaCl 0.0036, other mineral salts 0.0025, glucose (100 mg. per 100 ml.) 0.0004, non-protein nitrogenous solutes (30 mg. of non-protein N per 100 ml.) 0.0002, total increment 0.0067.

The lipides, as non-protein constituents, could affect the value of b if they altered significantly the gravity of the non-protein phase of the plasma. From the fact that fats in general are lighter than water, one might expect their presence in plasma to lower the gravity of the non-protein phase, and

to.6 - .

. +0.4 - .

l * l .

. .

+0.2 - . * .

3 .

’ +o, . . Auerage pathologzcel plasma

2 . .

0.. . l * .

-0.2- . l

. l * * .

. . -0.4- .

. .

-0.6’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

Albumin:gIobulinratio

FIG. 2. Lack of correlation between the albumin-globulin ratio and the P, - PN difference in nephrotic plasmas.

to cause markedly negative errors in gravity-calculated proteins when the plasma lipides are high. However, Popjak and McCarthy (20) found that the mean apparent partial specific volume of the lipides in plasmas ex- amined by them was 0.996, indicating a value of 1.004 for the density, Di6, of the lipide phase; this corresponds to a D i! of 1.0070, identical with the b value, assumed in Table IV as the 0:: of the non-protein phase of plasma. In part, the apparent high density of the lipide phase, compared with ordinary fat, is attributable to the fact that about 40 per cent of the mixed plasma lipides ordinarily consists of cholesterol, which has a density of 1.067. It is possible also that the loose combination of the lipides with proteins, which makes varying amounts of lipides invisible in plasma, may


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decrease the apparent specific volume of the lipides. The plasma lipides, at least up to the maximal concentrations (about 3 gm. per 100 ml.) studied by Popjak and McCarthy (20), appear to have little effect on the relation of plasma gravity to protein concentration.

+1.3 x

+I.2

+1.1 L-

+1.ok +0.9-

+0.8 - nz

i ::,‘- .

4 2 +0.5-

c +0.4-

2 g +0.3.-

3 3 +0.2= g h +0.1- .a

$j fO.O-

P+* -O.l-

-0.2-

-0.3 -

-0.4-

x

BaD,” x XoDO

-x@o 0 x Mean x for @dogid plarnu with NPN < 100

000% x x

?P xx

00 00 Mean for normal plasma

q3* K

x)0 o Misc. hospital cases. x x

x Cases of Bright’s Disease. x

0 x

Non-protein nitrogen, mg./lOO ml.

FIG. 3. Effect of high plasma non-protein nitrogen concentrations on the P, - PN differences in pathological plasmas.

In Table II a large proportion of the subjects in the group with Bright’s disease were lipemic, but this group, like the miscellaneous pathological group in the same table, showed Pa values that, if calculated by the equation for normal plasma, averaged 2 per cent above the nitrogen-calculated proteins (see Figs. 2 and 3).


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In exceptional plasma, with lipemia so gross that a large layer of fat could be removed by centrifugation, an increase in plasma gravity as the result of centrifugation has been observed of an order to indicate that the material removed had a 0:: less than 1.007; but even in such cases, some of which are included in Fig. 2, the effect of the Iipides on P, did not lower the latter by more than 0.2 gm. per liter.

Sources of Error in Calculation of Weights of Proteins As 6.25 X N in Pathological Plasma

Part of the deviation of P, from P, noted in Table II is probably due t,o error, not in PG, but in PN calculated as 6.25 X N. The factor, 6.25, is based on an assumed constant protein nitrogen content of 16.0 per cent,, found in analyses of fat- and ash-free total proteins of normal human plasma (13). The different fractions of normal human plasma, analyzed by Dumas combustion by Brand, Iiassell, and Saidel (21), had the following percentages of nitrogen: albumin 15.95, /3-globulin 14.84, y-globulin 16.03, fibrinogen 16.90. It is evident that either abnormalities in the proportions in which the different normal fractions are present in the total plasma protein mixture, or the presence of abnormal proteins, may significantly aher the percentage of nitrogen in the mixture, and the accuracy of the calculation of gm. of protein as 6.25 X gm. of N. Elimination of variat,ion in protein nitrogen content as a source of error in chemically determined proteins could be accomplished by determining the proteins gravimetrically (22), as has been done by Lloyd et al. (12) in cases of famine edema, or by wet carbon combustion of the lipide-free proteins by the method of Hoag- land and Fischer (23).

A relatively small plus error is introduced into PN by the nitrogen content of the phosphatides. The aqueous protein precipitants ordinarily used, such as trichloroacetic and tungstic acid solutions, form protein precip- itates which carry down with them all of the lipides of plasma. Hence in the usual procedures for determining protein nitrogen, including that of the present papers, the nitrogen of the phosphatides is included. The approximate effect may be estimated as follows: The phosphatides comprise about 25 per cent of the total lipides. The nitrogen content of the phosphatides is about 2 per cent; hence the nitrogen content of the total lipides is about 0.5 per cent. An increase in plasma lipides of 3 gm. per 100 ml. would increase the apparent PN by about 0.1 gm. per 100 ml., and make the gravity-calculat’ed proteins appear by comparison to be that much too low. The error in PN caused by lipide nitrogen is negligible (about 0.02 gm. per 100 ml.) in normal plasma, but may become significant in gross lipemia. Such an effect could be a factor in some of the relatively few


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cases in Figs. 2 and 3 in which Pa calculated by the equation for normal plasma falls below PM.

Magnitude of Intrinsic Error in Calculation of Proteins from Gravity in Normal and Pathological Plasmas

By intrinsic error is meant error in excess of that due to error in measurement of the specific gravity; it is the error due to variation of a and b in the equation P = a(G - b) from their assumed values. To estimate the magnitude of the maximal intrinsic error from results in Tables II and III, we subtract from the observed maximal P,- P, difference the sum of the estimated effects of maximal errors in the gravity and the Kjeldahl methods used, and obtain a result which is presumably larger than the true intrinsic error of PO (because error in PN due to variation from the factor 6.25 is ignored), but is the best estimate that can be made at present of the intrinsic error.

As ma,ximal experimental errors in Tables II and III we will assume ~0.0001 for gravities measured by pycnometer, ~0.0006 for gravities measured by the copper sulfate method (2), and ~0.5 per cent of the nitrogen determined by Kjeldahl analysis (13). The gravity errors of the two methods correspond to errors of ~0.04 and f0.22 gm. respectively of protein per 100 ml. of plasma. The nitrogen error, for a plasma of mean normal protein content, corresponds to ~~0.04 gm. of protein per 100 ml. The combined maximal experimental errors of Pa and PN thus estimated are ho.08 gm. of protein per 100 ml. of plasma when gravities are measured by pycnometer and ho.26 gm. when gravities are measured by the copper sulfate method. Errors in P, that may be due to deviations from the factor 6.25 are not included in the estimates.

Sormal Plasma-In the three groups of observations in Table III, the maximal deviations of P, from PN, calculated as twice the standard deviations, are rt0.19, f0.21, and ~0.27 gm. per 100 ml., compared with estimated maximal experimental errors of f0.08, f0.26, and f0.26 gm. per 100 ml., respectively. The observed standard deviat.ion X 2 exceeds the estimated experimental errors by 0.11 gm. per 100 ml. in the first group, falls short by 0.05 in the second, exceeds by 0.01 in the third.

It appears probable that the intrinsic error in the calculation of prot,ein concentrations in normal human plasma from gravities by Equation 2 does not exceed 0.1 gm. per 100 ml. It also appears that deviations of the protein nitrogen content from 16.0 per cent in normal plasma are small; otherwise error in P, from such variation would have caused the deviation of P, from P, to exceed more definitely the deviation predictable from the errors in gravity measurement and Kjeldahl analysis.

Pathological Plamnas-In the pathological plasmas of Table II, the maxi-


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ma1 deviations of Pa from PN, calculated as twice the standard deviations, are 0.44 and 0.52 gm. per 100 ml. in the two groups. These definitely exceed t’he 0.26 gm. difference estimated as the maximum attributable to errors in the Kjeldahl nitrogens and in the gravities determined by the copper sulfate method. The intrinsic error of gravity-estimated proteins in pathological plasmas of the types covered by the cases in Table II would accordingly be estimated at about 0.2 gm. per 100 ml., if the P, values could be assumed to be without error from use of the factor 6.25. Since there is reason to assume that part of the deviation of P, from PN in pathological plasmas may be due to error in the factor 6.25, it appears probable that the maximal intrinsic error of gravity-calculated proteins in pathological plasmas of the type in Table II (without non-protein N over 100 mg. or very high blood sugar) is something less than 0.2 gm. per 100 ml.

More extensive data, with all gravities precise to f0.0001 unit, and with chemical determinations of the proteins by a method not dependent on an assumed constant nitrogen content, would provide more satisfactory statis- tics for estimation of the intrinsic error of PO. The above estimates, however, appear to provide a fair approximation of the accuracy with which proteins can be calculated in human plasma from gravities measured without significant error.

Probable Causes of Greater Deviations of Gravity-Calculated from Nitrogen- Calculated Proteins in Pathological Than in Normal Plasmas--In cases of gross glucemia, or such high blood non-protein nitrogen concentrations as are exemplified in Table V, abnormally high crystalloid concentrations can cause gravity-calculated nroteins to be too high.

If cases with such gross crystalloid increases (Table II) are excluded, however, pathological plasmas show a variability in the relation of gravity- calculated to nitrogen-calculated proteins that appears to be due to variations in either the nitrogen contents or specific volumes of the proteins themselves. The fact that, for a given specific gravity, the average plasma protein content was 2 per cent less in the pathological plasmas of Table II than in the normal plasmas of Table I could be due either to lower nitrogen content (15.7 versus 16.0 per cent) or lower specific volume (0.724 versus 0.730) of the proteins of the pathological group.

Some of the results in the literature appear to indicate also that, in conditions of malnut.rition, abnormalities in t,he plasma proteins similarly affect the Pa - PN difference. Hoch and Marrack (8) observed that, in plasmas from a group of subjects suffering from malnutrition, the nitrogen- calculated protein content averaged 0.54 gm. per 100 ml. lower than in normal plasmas of the same specific gravity, the abnormality being in the same direction observed in the pathological plasmas of Table II. Because


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there is no reason to suspect the presence of such gross increases of crystalloid solutes (e.g. 400 mg. of glucose or 500 mg. of urea per 100 ml. above normal) as would be necessary to cause the observed effect in Hoch and Marrack’s subjects, it appears that the observed l’, - PM difference may have been due to abnormality in the proteins. The abnormal relation between PN and PG in the malnourished group could be caused either by a decrease in specific volume of the proteins from the 0.730 of normal plasma proteins to 0.706 in the malnourished group, or by a decrease in nitrogen content from the 16 per cent in normal proteins to 14.8 per cent in the malnourished group, or by smaller shifts in both values.

TABLE V

Cases with Non-Protein Nitrogen Greater Than 100 Error of PC

Gby cuS(,, method

PG = 365 x PN = 6.25 x Non- rotein estimated by

Case No. (G - 1.0070) protein N k assuming excess non-protein

(a) @I (6) (cl N*isnure --

,gm. per 100 ml. gm. gcr 100 ml *mm. per 100 ml gm. 9cr 100 Pd. pn. per 100 fnl.

1 1.0280 7.67 6.63 +1.04 0.272 f0.55 2 1.0242 6.28 5.58 +0.70 0.249 +0.50 3 1.0256 6.79 6.48 +0.31 0.275 +0.55 4 1.0249 6.53 6.17 +0.36 0.185 +0.35

* Excess non-protein nitrogen is taken as the amount above 0.030 gm. per 100 ml. The increment of G per gm. of urea per 100 ml. is taken as 0.0029 (see p. 338), and hence as 0.00621 per gm. of urea N per 100 ml. The effect of “excess non-protein N” on G is hence estimated as a G increment of 0.00621 X “excess non-protein N,” and the error in PO caused by non-protein N is taken as 365(0.00621 X “excess non- protein N”) = 2.27 X “excess non-protein N.”

A similar difference from normal subjects (9) in subjects with famine edema (12) was noted by Lloyd et al. (see Table I). Lloyd et al. compared the gravities of the plasmas of their famine group with protein concentrations determined, both gravimetrically, as described by Robinson and Hogden (22), and by Kjeldahl, and concluded that both factors, low protein specific volume and low protein nitrogen content, contributed to the posi- tive value of the P, - P, difference. Gravimetrically determined pro-

tein concentrations averaged 0.35 gm. per 100 ml. lower in plasma of the same gravity in the famine-stricken than in the normal group (compare data of Lloyd et al. for 1945 and 1948, Table I); this difference, being independent of nitrogen values, may be attributable to smaller specific volume (greater density) of the proteins in the starved group. If this is the case, the equation P = a(G - b) would require, for starved subject,s, an a value still smaller than that of Equation 1 (Table IV).


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ARCHIBALD, AND EDER

345

.4pparent Partial Specific Volume of Proteins in Normal Human Plasma’

The data of Table III permit calculation by Equation 5 of the partial specific volume of the proteins in normal plasma from t’he a and b values of the equation P = a(G - b). As pointed out, the a and b values may be varied considerably without a statistically significant change in the accuracy of the equation for calculating P. However, it appears that the correct value for a in normal plasma, with G measured by pycnometer, is within the range 373 f 15, with the corresponding b in the range 1.0070 f 0.0009. The corresponding VP range calculated by Equation 5 from these a and b values is 0.730 f 0.010. This is not out of line with Peder- sen’s (19) values of 0.736 for human albumin and 0.718 for r-globulin, which together constitute about 70 per cent of normal human plasma protein, or with Popjak and McCarthy’s (20) value of 0.729 for rabbit proteins. In earlier (1928) work on the proteins of horse serum, Svedberg and Sjiigren (24) found higher and nearly identical values, 0.748 for the albumin fraction and 0.745 for the total globulins; however, they determined their protein concentrations by weighing the heat-coagulated proteins, which may have contained some occluded fat.

Range of Normal Plasma Protein Concentration-The data of Table III indicate 7.4 f 0.6 gm. per 100 ml. as the normal range. The data were obtained by macro-Kjeldahl analyses performed with precautions for accuracy (13), and presumably indicate the approximate range for plasma drawn from the blood of normal healthy young adults in the standing or sitting position. As a number of authors have shown, however (cf. Lange (25)), change from the upright to the horizontal position causes a decrease in plasma protein concentration, which is measurable in a few minutes and approaches a plateau in about an hour. It is due to dilution of t.he blood by interstitial fluid. Lange found the average protein concentrations in plasma of blood drawn from subjects lying down to be 0.55 gm. per 100 ml. lower than in plasma drawn while the subjects were standing. For subjects in bed, therefore, a range of about 6.8 f 0.6 gm. per 100 ml. may be normal.

SUMMARY

From comparison of plasma specific gravities with protein concentrations calculated from accurate Kjeldahl analyses as N X 6.25 the following equations have been derived: from thirty-seven normal plasmas, P = 373 (G - 1.0070); from 91 pathological plasmas without gross elevation of blood urea or sugar, P = 365 (G - 1.0070). P indicates the gm. of protein per 100 ml. of plasma, G the specific gravity of the plasma, the density of water at’ the same temperature being taken as unity.


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As indicated by the difference between the constants, 373 and 365, the pathological plasmas had on the average 0.98 as much nitrogen-calculated protein per 100 ml. as normal plasmas of the same specific gravity. Data from the literature indicate that plasma of subjects suffering from malnutrition shows a similar difference from normal plasma. The difference could be due to either lower specific volume or lower nitrogen content of the proteins of the abnormal plasmas.

It is estimated that, with errors of gravity measurement eliminated, the error in calculating protein concentration from gravity is less than 0.1 gm. per 100 ml. in normal human plasma, and less than 0.2 gm. in pathological plasma without gross abnormalities of crystalloid concentration, such as sugar or urea concentrations above 200 mg. per 100 ml.

The range of nitrogen-calculated protein concentration observed in plasma of blood drawn from healthy men in the upright position was 6.8 to 8.0 gm. per 100 ml., average 7.39.

The apparent specific volume of the proteins in normal human plasma is estimated at 0.730 f 0.010.

BIBLIOGRAPHY

1. Moore, N. S., and Van Slyke, D. D., J. Clin. Invest., 8, 337 (1930). 2. Phillips, R. A., Van Slyke, D. D., Hamilton, P. B., Dole, V. P., Emerson, K.,

Jr., and Archibald, R. M., J. Biol. Chem., 183, 305 (1950). 3. Lowry, 0. H., and Hunter, T. H., J. Biol. Chem., 169,465 (1946). 4. Barbour, H. G., and Hamilton, W. F., J. Biol. Chem., 69,625 (1926). 5. Weech, A. A., Reeves, E. B., and Goettsch, E., .Z. BioZ. Chem., 113,167 (1936). 6. Cole, W. H., Allison, J. B., and Boyden, A. A., Proc. Sot. Exp. BioZ. and Med.,

64, 215 (1943). 7. Hoch, H., and Marrack, J., Brit. Med. J., 2,151 (1945). 8. Hoch, H., and Marrack, J., Brit. Med. J., 2, 876 (1945). 9. Lloyd, B. B., Cheek, E. B., Sinclair, A. M., and Webster, G. R., Biochem. J., 39,

p. xxv (1945). 10. Meyer, F. L., Abbott, W. E., Allison, M., and McKay, C., Arch. Biochem., 12,359

(1947). 11. Dicker, S. E., J. Physiol., 107, 11P (1948). 12. Lloyd, B. B., Sinclair, H. M., and Tweedy, M. C. K., Biochem. J., 43, p. xvi

(1948). 13. Hiller, A., Plazin, J., and Van Slyke, D. D., J. BioZ. Chem., 176, 1401 (1948). 14. Campbell, W. R., and Hanna, M. I., J. BioZ. Chem., 119, 1 (1937). 15. Van Slyke, D. D., J. BioZ. Chem., 71, 235 (1926-27). 16. Kraemer, E. O., in Svedberg, T., and Pedersen, K. O., The ultracentrifuge,

Oxford, 59 (1946). 17. Van Slyke, D. D., Phillips, R. A., Dole, V. P., Hamilton, P. B., Archibald, R. M.,

and Plaein, J. P., J. BioZ. Chem., 183, 349 (1950). 18. Adair, G. S., and Adair, M. E., Proc. Roy. Sot. London, Series B, 120,422 (1936). 19. Pedersen, K. O., Ultracentrifugal studies of serum and serum fractions, Upsala

(1945).


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20. Popjak, G., and McCarthy, E. F., &o&em. J., 40,789 (1946). 21. Brand, E., Kassell, B., and Saidel, L., J. CZin. Inuest., 23,437 (1944). 22. Robinson, H. W., and Hogden, C. G., J. Biol. Chem., 140,853 (1941). 23. Hoagland, C. L., and Fischer, D. J., Proc. Sot. Exp. Biol. and Med., 40, 581

(1939). 24. Svedberg, T., and Sjiigren, B., .I. Am. Chem. Sot., 60,3318 (1928). 25. Lange, H., Acta med. Stand., suppl. 176 (1946).


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Reginald M. Archibald and Howard A. EderPhillips, Paul B. Hamilton, Vincent P. Dole,

Donald D. Van Slyke, Alma Hiller, Robert A.PLASMA SPECIFIC GRAVITY

PROTEIN CONCENTRATION FROM THE ESTIMATION OF PLASMA

1950, 183:331-347.J. Biol. Chem.

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