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Experimental determination of net protein charge and A_tot and K_a of nonvolatile buffers in human plasma

¹Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1; and ²Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61802

Submitted 31 January 2003 ; accepted in final form 26 March 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
The mechanism for an acid-base disturbance can be determined by using the strong ion approach, which requires species-specific values for the total concentration of plasma nonvolatile buffers (A_tot) and the effective dissociation constant for plasma weak acids (K_a). The aim of this study was to experimentally determine A_tot and K_a values for human plasma by using in vitro CO₂ tonometry. Plasma PCO₂ was systematically varied from 25 to 145 Torr at 37°C, thereby altering plasma pH over the physiological range of 6.90–7.55, and plasma pH, PCO₂, and concentrations of quantitatively important strong ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^-, lactate) and buffer ions (total protein, albumin, phosphate) were measured. Strong ion difference was estimated, and nonlinear regression was used to calculate A_tot and K_a from the measured pH and PCO₂ and estimated strong ion difference; the A_tot and K_a values were then validated by using a published data set (Figge J, Rossing TH, and Fencl V, J Lab Clin Med 117: 453–467, 1991). The values (mean ± SD) were as follows: A_tot = 17.2 ± 3.5 mmol/l (equivalent to 0.224 mmol/g of protein or 0.378 mmol/g of albumin); K_a = 0.80 ± 0.60 x 10^-7; negative log of K_a = 7.10. Mean estimates were obtained for strong ion difference (37 meq/l) and net protein charge (13+.0 meq/l). The experimentally determined values for A_tot, K_a, and net protein charge should facilitate the diagnosis and treatment of acid-base disturbances in critically ill humans.

plasma pH; strong ion difference; anion gap; metabolic acidosis

Plasma proteins provide the major contribution to A_tot, and, therefore, plasma protein concentration independently affects acid-base balance. The role of plasma protein concentration in acid-base balance is well recognized in human and veterinary medicine, with hypoproteinemia and hyperproteinemia causing alkalemia and acidemia, respectively (5, 11, 20, 29). The most widely used method to assign a value for A_tot in human plasma has been calculation from the plasma protein concentration by using the estimate of net protein charge obtained by Van Slyke and colleagues (39) in 1928. To obtain this estimate, human plasma proteins were assumed to have the same alkali titration curve as horse serum proteins (40); this assumption provided an estimate for the net protein charge of human plasma ([total protein] = 70 g/l, where brackets denote concentration) of 16.9 meq/l. In a 1964 study, Van Leewen (38) estimated that the net protein charge in human plasma was 12.6 meq/l, a value that was similar to the estimate of 12.0 meq/l obtained by Figge and colleagues (10) in 1992. Many investigators assumed that these estimated values for net protein charge (16.9, 12.6, or 12.0 meq/l) were equivalent to the value for A_tot; however, this is an erroneous assumption, because the value for A_tot must always be greater than that of net protein charge, and because A_tot is expressed in different units (mmol/l) than net protein charge (meq/l) (6).

Species-specific values for A_tot and K_a should be experimentally determined when using the strong ion approach to describe acid-base equilibria (4, 35, 36). Values for A_tot (14.9 or 15.0 mmol/l) and K_a (2.1 or 2.2 x 10^-7) have been experimentally determined for equine plasma (4, 35), but different values have been experimentally determined for cattle plasma (A_tot = 25.0 mmol/l; K_a = 0.9 x 10^-7; Ref. 7) and cat plasma (A_tot = 27.4 mmol/l; K_a = 1.0 x 10^-7; Ref. 21). For human plasma, only theoretical estimates for A_tot (24.1 mmol/l) and K_a (1.1 x 10^-7) are available (6). Interestingly, these theoretical A_tot and K_a estimates predicted that net protein charge in human plasma was 15.3 meq/l, which was similar to the estimate of Van Slyke et al. (16.9 meq/l) (39), but greater than that of Van Leeuwen (12.6 meq/l) (38) and Figge et al. (12.0 meq/l) (11). The purpose of this study was, therefore, to experimentally determine A_tot and K_a values for human plasma and, from this information, calculate net protein charge. We accomplished our objectives by performing in vitro CO₂ tonometry of plasma from eight healthy humans. We also validated the experimentally determined values for A_tot and K_a using published data (11) and compared the predictive accuracy of these values with theoretical estimates (6) or derived estimates (10, 32).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
Blood and plasma collection. Twenty milliliters of venous blood were collected into lithium-heparin tubes from the antecubital veins of eight healthy humans (26–46 yr old; 4 men, 4 women). Lithium-heparin tubes were used instead of sodium-heparin tubes for blood collection, because the measured sodium component of plasma collected into sodium-heparin can be increased by up to 2 meq/l, increasing the measured SID (44). In addition, lithium-heparin dissociates in plasma into a strong cation (lithium) and strong anion (sulfite), with no change in actual SID or measured SID, because plasma lithium and heparin concentrations are not routinely measured.

One milliliter of venous blood was immediately analyzed to characterize the normal values, and plasma was harvested from the remaining 19 ml by centrifugation that was completed within 30 min of collection. Plasma was frozen at -70°C and stored for up to 2 mo before being thawed at room temperature immediately before CO₂ tonometry was performed. The University of Guelph ethics committee approved this study.

CO₂ tonometry of plasma. Plasma samples were tonometered (IL 235, Instrumentation Laboratory, Lexington, MA) for 20 min at 37°C over a PCO₂ range of 25–145 Torr by using a gas mixture containing 20% CO₂ and 80% normal air. This produced a pH range of 6.90–7.55.

Blood and plasma analyses. The fresh blood sample and all tonometered plasma samples were analyzed in duplicate on a Statprofile 9+ (NOVA Biomedical, Canada, Mississauga, Ontario) for blood-plasma gas analysis (pH, PCO₂) and determination of [Na⁺], [K⁺], [Ca²⁺], [Cl^-], and [lactate^-]. Table 1 provides a summary of the variability and measurement methodology for each variable. An untonometered plasma sample was analyzed in duplicate (Dacos multianalyzer, Coulter Electronics, Hialeah, FL) to determine strong cation (Mg²⁺) and nonvolatile buffer ion (total protein, albumin, and inorganic phosphate) concentrations.

Calculation of SID. Strong cation (Na⁺ + K⁺ + Ca²⁺ + Mg²⁺) and strong anion (Cl^- + lactate) concentrations were assumed to be constant during CO₂ tonometry and an ionic equivalency assigned to those variables (Mg²⁺, lactate) not measured by using ion-selective potentiometry. Accurate measurements of SID are difficult to obtain in plasma (6, 31, 42) because of cumulative measurement error, presence of unknown strong anions (12, 15), and differences in equipment and methodology (18, 28); SID was, therefore, initially estimated by using three methods: SID₃ = {([Na⁺] + [K⁺]) - [Cl^-]}; SID₄ = {([Na⁺] + [K⁺]) - ([Cl^-] + [lactate])}; and SID₆ = {([Na⁺] + [K⁺] + [Ca²⁺] + [Mg²⁺]) - ([Cl^-] + [lactate])}. A constant value for SID₃, SID₄, and SID₆ was assigned by using the mean value for all CO₂ tonometered samples from each subject. This minimized the effect of measurement variability in strong ion concentrations. A constant value for SID is one of the assumptions of the strong ion approach; SID is invariant over the physiological range of pH, because strong ions are fully dissociated at physiological pH (4, 6, 33). Because SID₃, SID₄, and SID₆ represented constant values, the terms were expressed as [SID₃]_constant, [SID₄]_constant, and [SID₆]_constant, respectively.

In preliminary data analysis, strong ion (Na⁺, K⁺, Ca²⁺, and Cl^-) concentrations measured by ion-selective electrodes were regressed against pH. Neither [K⁺] or [Cl^-] varied with pH; however, [Na⁺] varied inversely with pH for six of the eight plasma samples (mean linear regression equation: [Na⁺] = -3.98 pH + 173.5), and [Ca²⁺] varied inversely with pH for all eight plasma samples (mean linear regression equation: [Ca²⁺] = -1.29 pH + 11.9), where [Na⁺] and [Ca²⁺] were in meq/l. Over the pH range in this study (6.90–7.55), this corresponds to a mean change in [Na⁺] and [Ca²⁺] of 2.6 and 0.8 meq/l, respectively. A possible reason for the observed pH dependence of measured [Na⁺] and [Ca²⁺] was poor selectivity of the sodium and calcium electrodes to H⁺. The potentiometric selectivity coefficient defines the ability of an ion-selective electrode to distinguish the primary ion from other ions in the same solution; the smaller the value for the selectivity coefficient, the less susceptible is the electrode to changes in the concentration of the interfering ion (3). Although reported selectivity coefficients of the Ca²⁺, Na⁺, and K⁺ electrodes for H⁺ are 0.16–16, 0.2–3.2, and <0.0001, respectively (1), these selectivity coefficient values were not high enough to explain the pH dependency of [Na⁺] and [Ca²⁺] during CO₂ tonometry over a pH range of 6.90–7.55.

The most likely reason for the pH dependency of measured [Na⁺] and [Ca²⁺] was salt-type binding of sodium and calcium to plasma protein; as pH decreases, the net protein charge decreases (becomes less negative), thereby "releasing" electrostatically bound sodium and calcium and increasing plasma [Na⁺] and [Ca²⁺] when measured by ion-selective potentiometry (3, 19, 23). As we observed, the magnitude of this effect was more pronounced for Na⁺ than Ca²⁺, because 36 mmol of sodium and 0.8 mmol of calcium are electrostatically bound to plasma proteins for each liter of human plasma (3). To account for the effect of pH on electrostatically bound sodium and calcium, we calculated the SID for each tonometered plasma sample from the measured values for [Na⁺], [K⁺], [Ca²⁺], [Mg²⁺], [Cl^-], and [lactate] and termed this value [SID₆]_variable, because the value varied with pH during CO₂ tonometry.

Calculation of A_tot and K_a. Measured values for pH and PCO₂, calculated values for [SID₃]_constant, [SID₄]_constant, [SID₆]_constant, and [SID₆]_variable, the six-factor simplified strong ion electroneutrality equation (4)

(1)

and the Marquardt nonlinear regression procedure (13, 30) were used to solve simultaneously for A_tot and K_a, where [A^-] in Eq. 1 is the net charge of plasma nonvolatile buffers. To facilitate accurate calculation of values for A_tot and K_a, Eq. 1 was expressed in the following form

(2)

by applying known values for the solubility of CO₂ in plasma (S; 0.0307 mmol · l^-1 · mmHg^-1) (2) and the negative logarithm of the apparent equilibrium dissociation constant (pK'₁; 6.120 at [NaCl] = 0.16 mmol/l; interpolated from Table II, Ref. 14). With the use of the value of 6.120 for pK'₁ calculated actual plasma [HCO₃] (mmol/l) at 37°C (25); similarly, the four methods used to calculate SID provided a value in terms of concentration. This means that Eq. 2 estimated a value for A_tot in terms of concentration (mmol/l). The form of the simplified strong ion electroneutrality equation used in Eq. 2 was selected because it provided the narrowest confidence intervals for the estimated values of A_tot and K_a. Initial estimates for A_tot of 5–30 mmol/l in increments of 5 mmol/l and initial estimates for K_a of 0.1–3.0 x 10^-7 in increments of 0.1 x 10^-7 were used for the nonlinear regression procedure.

Because the true value for SID was unknown, a fifth nonlinear regression procedure was performed to simultaneously estimate values for A_tot, K_a, and SID (called [SID]_estimated), with initial estimates for [SID]_estimated of 30–45 meq/l in increments of 5 meq/l.

A sixth nonlinear regression procedure was performed to calculate A_tot and K_a. This was done to compare A_tot and K_a values obtained by using the six-factor simplified strong ion model (described previously) with Stewart's eight-factor strong ion model (36). Although we have shown algebraically (4) and graphically (6) that Stewart's eight-factor strong ion model contains two redundant factors [apparent equilibrium dissociation constant for the ion product of water () and apparent equilibrium dissociation constant for ], it was of interest to determine whether the eight-factor model provided more accurate estimates for A_tot and K_a. Measured values for pH and PCO₂, calculated values for [H⁺](= 10^-pH), either [SID]_variable or [SID]_estimated, the carbonate ion concentration ([]), and the hydroxyl ion concentration ([OH^-]), Stewart's eight-factor strong ion electroneutrality equation (36)

(3)

and the Marquardt nonlinear regression procedure (30) were used to solve simultaneously for A_tot and K_a. To facilitate the nonlinear regression procedure, Eq. 3 was expressed in the following form

(4)

Where pK'₃ is the negative logarithm (10.22) of the apparent equilibrium dissociation constant for HCO₃ (K'₃ = 6 x 10^-11) and pK'_w is the negative logarithm (13.36) of the ion product of water (K'_w = 4.4 x 10^-14). The form of the strong ion electroneutrality equation used in Eq. 4 was selected because it provided the narrowest confidence intervals for the estimated values of A_tot and K_a when pH was changed by CO₂ tonometry.

R² values were calculated for the seven fitted nonlinear regression models by using the values obtained during CO₂ tonometry of each human plasma sample: the six-factor simplified strong ion model and five different methods ([SID₃]_constant, [SID₄]_constant, [SID₆]_constant, [SID₆]_variable, [SID]_estimated) for estimating SID, and the eight-factor strong ion model and two different methods ([SID₆]_variable, [SID]_estimated) to estimate SID. The calculated A_tot values were indexed to the total protein (A_{tot tp}) and albumin (A_tot-alb) concentration, and mean ± SD values for A_tot, A_{tot tp}, A_{tot alb}, and K_a were determined. A P value <0.05 was considered significant.

Comparison and validation of calculated A_tot and K_a values. Serum electrolyte concentrations, PCO₂, protein concentrations, and pH values were extracted from a published data set of human serum filtrands with experimentally induced changes in PCO₂, SID (expressed as concentration), and [total protein] (Ref. 11, Table 2). These data were used to compare and validate the mean A_{tot tp}, A_{tot alb}, and K_a values obtained by using the six-factor simplified strong ion model and five different methods for assigning a value to SID, [SID₃]_constant, [SID₄]_constant, [SID₆]_constant, [SID₆]_variable, and [SID]_estimated, or the eight-factor strong ion model using [SID₆]_variable and [SID]_estimated. In addition, the calculated A_{tot tp} and K_a values were compared with previous estimates [A_{tot tp} = 0.344 mmol/g of total protein, K_a = 1.05 x 10^-7 (6); A_{tot tp} = 0.340 mmol/g of total protein, K_a = 0.56 x 10^-7 (32); A_{tot tp} = 0.334 mmol/g of total protein, K_a = 0.42 x 10^-7 (10, 11)]. Calculated A_{tot alb} values were also compared with previous A_tot estimates derived from the albumin concentration [A_{tot alb} = 0.572 mmol/g of albumin (6); A_{tot alb} = 0.553 mmol/g of albumin (32); A_{tot alb} = 0.545 mmol/g of albumin (10, 11)].

Serum pH was calculated by using the six-factor simplified strong ion equation (3) in the following form

(5)

the measured variables PCO₂, SID = ([Na⁺] + [K⁺] + [Ca²⁺] + [Mg²⁺] - [Cl^-] - 1.5), and [total protein]; known values for S (0.0307 mmol · l^-1 · mmHg^-1) (2) and pK'₁ (6.120) (14, 25); and the calculated values for A_{tot tp}, A_{tot alb}, and K_a. The subtraction of 1.5 from the SID value represented the estimated charge on the unmeasured strong anion sulfate in the serum filtrand (11). The calculated pH was then compared with the measured pH by using linear regression analysis, and the R², coefficient, and intercept values were used for comparison with the line of identity (slope = 1; intercept = 0).

Calculation of net protein charge. The [A^-] (in meq/l) at physiological pH (7.40) was calculated from the experimentally determined values for A_tot and negative log of acidic dissociation constant (pK_a) (8) as

(6)

The value for [A^-] calculated in Eq. 6 represents the net negative charge of nonvolatile plasma buffers (albumin, globulin, phosphate); the value for [A^-] is, therefore, pH dependent. Protein and phosphate also have a pH-independent negative charge that acts as a strong anion charge (41). In albumin and globulin, this is due to carboxyl, phenolic, and guanidium groups (37), and on phosphate it is due to the moiety.

The pH-dependent component of net phosphate charge ([phosphate^-]; in meq/l) at physiological pH (7.40) was calculated as

(7)

The net protein charge (in meq/l) at physiological pH (7.40) was calculated as [A^-] - [phosphate^-]. This calculates the pH-dependent component of protein charge and not the total protein charge.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
Blood and plasma analyses. The values for venous blood from eight humans are presented in Table 2. A total of 157 CO₂ tonometered plasma samples were analyzed, representing 16–23 tonometered samples from each human. Representative PCO₂ and pH values obtained during CO₂ tonometry of plasma from two humans are shown in Fig. 1.

View larger version (10K): [in this window] [in a new window]   Fig. 1. Representative data from 2 human plasma samples during CO2 tonometry.

Calculation of SID. For normal plasma, mean values for [SID₃] (40.9 meq/l), [SID₄] (40.0 meq/l), and [SID₆] (43.0 meq/l) were obtained (Table 2). Different mean values for [SID₃]_constant (43.8 meq/l) (Tables 3, 4, 5), [SID₄]_constant (41.9 meq/l), [SID₆]_constant (46.0 meq/l) (Tables 3, 4, 5), and [SID]_estimated (37.1 meq/l) were obtained during tonometry (Tables 6 and 7). The mean range for [SID₆]_variable during tonometry was 7.5 meq/l (Tables 8 and 9).

Calculation of A_tot and K_a. The R² value for all nonlinear regression models was >0.98, indicating excellent fit to the data. The calculated values for A_tot and K_a depended markedly on the value assigned to SID (Tables 3, 4, 5, 6, 7, 8, 9; Fig. 2).

View larger version (15K): [in this window] [in a new window]   Fig. 2. Plot of calculated total weak acid concentration value (indexed to total protein) (Atottp) against estimated value for strong ion difference (SID). Values are means ± 1 SD. Note that the calculated value for Atottp depends on the estimated value for SID.

When pH = 7.40 and PCO₂ = 40 Torr, the actual [HCO₃] = 23.4 mmol/l (calculated from the Henderson-Hasselbalch equation when S = 0.0307 mmol · l^-1 · mmHg^-1 and pK'₁ = 6.120). Accordingly, the true SID of human plasma at pH = 7.40 can be calculated by using the simplified strong ion electroneutrality equation (Eq. 2) so that SID = 23.4 + 11.5 = 34.9 meq/l. The calculated value for true SID was within the 95% confidence interval (29–45 meq/l) estimated by using nonlinear regression (Table 6). Applying the measured values for the mean venous blood values in this study Table 2; pH = 7.37, PCO₂ = 51 Torr, A_tot = 17.2 mmol/l (calculated from a [total protein] of 76.9 g/l), K_a = 0.80 x 10^-7, S = 0.0307 mmol · l^-1 · mmHg^-1, pK'₁ = 6.120} to Eq. 5 predicted that SID = 39 meq/l, which was also within the 95% confidence interval (29–45 meq/l) for the calculated SID value.

Comparison and validation of calculated A_tot and K_a values. With the use of a data set containing 72 serum filtrands from two humans and calculating A_tot from the [total protein], the highest R² value (0.967) was obtained from the A_tot and K_a estimates obtained with [SID]_estimated in the six-factor strong ion model and A_tot indexed to [total protein] (Table 10). The most accurate values were, therefore, A_tot = 17.2 ± 3.5 mmol/l (equivalent to 0.224 mmol/g of protein), K_a = 0.80 ± 0.60 x 10^-7, and pK_a = 7.10. These estimated values for A_tot and K_a were only one of four pairs of values where the fitted regression line was the same as the line of identity (slope = 1; intercept = 0); the values for A_tot and K_a obtained by Siggaard-Andersen et al. in 1977 (32) and Constable in 2001 (6) also fitted the line of identity, but had lower R² values. In addition, Stewart's eight-factor strong ion model with [SID]_estimated (Table 10) was very close to the six-factor strong ion model (R² = 0.965). The A_tot and K_a values derived by Figge et al. in 1992 (10) from their data set (11) did not fit the line of identity.

With the use of the same data set and calculating A_tot from the albumin concentration, the highest R² value (0.960) was obtained from the A_tot and K_a value obtained with [SID]_estimated, followed very closely by Stewart's eight-factor model with [SID]_estimated (R² = 0.959) (Table 11). The A_tot value was equivalent to 0.378 mmol/g of albumin. As before, the estimated values for A_tot and K_a obtained with [SID]_estimated was one of four, where the fitted regression line was the same as the line of identity; the values for A_tot and K_a obtained by Siggaard-Andersen in 1977 (32) and Constable in 2001 (6) also fitted the line of identity, but had lower R² values. The A_tot and K_a values derived by Figge et al. in 1992 (10) from their data set (11) did not fit the line of identity.

Calculation of net protein charge. The [A^-] at physiological pH (7.40) for each tonometered human plasma sample was calculated from the experimentally determined values for A_tot and pK_a obtained by using [SID]_estimated: [A^-] = A_tot/[1 + 10^{( pKa-pH)}] = 10.8 ± 4.0 meq/l (Table 6). This estimate for [A^-] reflected the charge assigned to the pH-dependent components of albumin, globulin, and phosphate; because the pH-dependent component of phosphate charge at physiological pH (7.4) was 1.0 meq/l (calculated by using Eq. 7 and data in Table 2), the mean net protein charge in the eight human plasma samples attributed to nonvolatile buffer ions was 9.8 ± 4.0 meq/l = 0.215 meq/g albumin ([albumin] = 45.5 g/l) or 0.127 meq/g total protein ([total protein] = 76.9 g/l).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
In this study, we experimentally determined and validated values for A_tot (17.2 mmol/l) and K_a (0.8 x 10^-7) of human plasma. We also found that the values for A_tot and K_a depended markedly on the estimated value for SID. Determining the true value for SID remains the major difficulty in applying the strong ion approach to acid-base disturbances.

This appears to be the first study to use nonlinear regression to simultaneously estimate values for A_tot, K_a, and SID; the values obtained for A_tot and K_a at the same time as SID provided the most accurate prediction of pH from known values for PCO₂, [Na⁺], [K⁺], [Ca²⁺], [Mg²⁺], [Cl^-], [lactate], and [total protein] in filtrands of human plasma. This suggests that the true mean SID of the eight plasma samples was 37 meq/l. This value was lower than that estimated as [SID₃] ([Na⁺] + [K⁺] - [Cl^-] = 41 meq/l), [SID₄] ([Na⁺] + [K⁺] - [Cl^-] - [lactate] = 40 meq/l), and [SID₆] ([Na⁺] + [K⁺] + [Ca²⁺] + [Mg²⁺] - [Cl^-] - [lactate] = 43 meq/l); less than the estimated value of 40–42 meq/l (15, 16, 17, 33); but similar to the value calculated by Watson in 1999 (41) (38 meq/l). Clearly, unidentified strong anions are present in human plasma. These unidentified strong anions include sulfate, D-lactate, nonesterified fatty acids, and ketoacids; however, concentrations of these anions are too low to explain all of the unmeasured anion charge.

We believe that unaccounted protein and phosphate charge are responsible for most of the unmeasured strong anion charge in human plasma. Protein and phosphate charge have two components: a fixed charge that functions as a strong anion [the net difference in charge between carboxyl, epsilon-amino, phenolic, and guanidium groups in proteins (41) and in phosphate], and a variable charge that functions as a nonvolatile buffer ion (due mainly to imidazole groups in protein and and in phosphate) (Fig. 3). To quantify the pH-independent and -dependent components of protein and phosphate charge, the concentrations of protein and phosphate would need to be varied. When the effect of variations in protein concentration on pH was investigated in human plasma, the net anionic charge assigned to albumin at pH = 7.40 has been 0.25 meq/g (9), 0.268 meq/g (24), 0.27 meq/g (41), 0.33 meq/g (20), or 0.408 meq/g (29). All five estimates for net anionic charge for albumin were higher than that obtained in this study (0.215 meq/g albumin at pH = 7.40); however, our estimate reflects only the pH-dependent (nonvolatile buffer ion) charge of albumin and not the strong ion charge. Accordingly, the negative charge on albumin should be compartmentalized into a pH-independent strong ion charge (0.305 - 0.215 = 0.090 meq/g albumin, where 0.305 is the mean of the 5 estimates for the net anion charge of albumin and 0.215 is our estimate for the nonvolatile buffer ion charge of protein, assuming only albumin contributes to protein charge), a pH-dependent nonvolatile buffer ion charge, which can be calculated from the known values for pH, phosphate concentration, and the negative logarithm to the base 10 of the dissociation constant (1.58 x 10^-7) of , and experimentally determined values for A_tot and K_a. The formula for calculating net protein charge (in meq/l) from the albumin concentration (in g/l) and the phosphate concentration (in mmol/l) is, therefore

(8)

where protein charge is indexed to the albumin concentration, and the pH-dependent component of phosphate charge is subtracted from the assigned nonvolatile buffer ion charge. At normal values for pH (7.40), [albumin] (41 g/l), and [phosphate] (1.2 mmol/l), Eq. 8 calculates pH-independent protein charge = 3.7 meq/l, pH-dependent nonvolatile buffer ion charge = 10.3 meq/l, and pH-dependent phosphate charge = 1.0 meq/l; the net protein charge of human plasma = 3.7 + 10.3 - 1.0 = 13.0 meq/l. This value for net protein charge was similar to that obtained by Van Leewen in 1964 (38) (12.6 meq/l) and Figge et al. in 1992 (10) (12.0 meq/l).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Schematic of the effects of 3 independent variables on pH. Note that albumin, globulin, and phosphate contribute to both SID and total weak acid concentration (Atot). NEFAs, nonesterified fatty acids.

A similar approach can be applied to calculating net protein charge from the [total protein]. The net anionic charge assigned to total protein in human plasma has been 0.179 meq/g (38), 0.243 meq/g (29), or 0.26 meq/g (20). These three estimates for net total protein charge were higher than that obtained in this study (0.127 meq/g total protein at pH = 7.40); however, as discussed previously, our estimate reflects only the pH-dependent component and not the pH-independent (strong anion) component. Of these three estimates, the value obtained by Van Leewen (0.179 meq/g) (38) appears to be the most accurate, as it was developed from a net protein charge of 12.6 meq/l. Accordingly, protein charge can be compartmentalized into a pH-independent strong ion charge (0.179 - 0.127 = 0.052 meq/g total protein, where 0.179 is Van Leewen's estimate and 0.127 is our estimate for the nonvolatile buffer ion charge of plasma proteins) and a pH-dependent buffer ion charge, which can be calculated as described previously for albumin (Eq. 8). The formula for calculating net protein charge (in meq/l) from the [total protein] (in g/l) and the [phosphate] (in mmol/l) is, therefore

(9)

where protein charge is indexed to the [total protein] and the pH-dependent component of phosphate charge is subtracted from the assigned nonvolatile buffer ion charge. At normal values for pH (7.40), [total protein] (70 g/l), and [phosphate] (1.2 mmol/l), Eq. 9 calculates the net protein charge of human plasma = 3.6 + 10.4 - 1.0 = 13.0 meq/l. This value for net protein charge was identical to that obtained from the albumin concentration alone.

Which method should we use clinically to calculate net protein charge? The R² values from linear regression of calculated pH against measured pH (Tables 10 and 11) indicated that expressing A_tot in terms of [total protein] provided the most accurate fit to the data.

However, albumin is the most important buffer in plasma (73% of total buffering), with globulins contributing 22% of total buffering and phosphate 5% of total buffering (32). It is widely believed that the net protein charge is more accurately calculated from albumin (9, 20, 24, 29, 41) than total protein (20, 29, 38), principally because of individual variations in the albumin-to-globulin ratio. However, the widespread use of ion-selective electrodes has lowered the reference range of the anion gap {[Na⁺] - ([Cl^-] + [])} for human plasma from 8–16 meq/l (9, 24) to 3–11 meq/l (43), indicating that the unmeasured anions exceed the unmeasured cations by 3–11 meq/l. By attributing charges to quantitatively important anions, protein (13.0 meq/l when calculated from albumin or total protein in humans with normal albumin-to-globulin ratios), phosphate (2.2 meq/l), lactate (0.9 meq/l), and quantitatively important cations, potassium (4.2 meq/l), calcium (2.3 meq/l), and magnesium (1.6 meq/l), the net charge that can be attributed to the quantitatively important components of the anion gap is 8.0 meq/l. Because this estimate lies within the range of the normal anion gap (3–11 meq/l) for human plasma, net protein charge can be calculated from either albumin concentration or protein concentration.

Because of difficulties in obtaining the true value for SID, the major clinical utility in using the strong ion approach in critically ill patients is to calculate the strong ion gap to detect and quantify the unmeasured strong cation or anion concentration (7, 8). Based on the previous discussion and the results of this study, the two most accurate equations for calculating strong ion gap (in meq/l) in human plasma are

(10)

and

(11)

where anion gap (in meq/l) = [Na⁺] - ([Cl^-] + []), and [albumin] and [total protein] are in g/l. Equations 10 and 11 assume that the unmeasured strong cation concentration (K⁺, Ca²⁺, Mg²⁺) equals the unmeasured strong anion concentration (lactate, sulfate, nonesterified fatty acids, ketoacids, pH-independent phosphate charge, and other strong anions); the strong ion charge of albumin or protein is included in Eq. 10 or 11. At normal values of pH (7.40), [albumin] = 41 g/l, and anion gap = 7 meq/l, Eq. 10 calculates the strong ion gap -0.4 meq/l. At normal values of pH (7.40), [total protein] = 70 g/l, and anion gap = 7 meq/l, Eq. 11 calculates the strong ion gap -0.2 meq/l. For the venous blood samples from the eight humans in this study, pH (7.37), [albumin] = 45.5 g/l, anion gap = 6.5 meq/l, Eq. 10 calculates the strong ion gap 0.6 meq/l, and with the use of [total protein] = 76.9 g/l, Eq. 11 calculates the strong ion gap 0.7 meq/l. These calculations suggest that the strong ion gap equations (Eqs. 10 and 11) provide a useful method for detecting the presence and quantifying the magnitude of unmeasured anions in the plasma of critically ill human patients; presently the unmeasured anions are suspected to be predominantly associated with uremia (22, 26).

Finally, which value for SID should be used in the strong ion approach? Because the mean actual SID (37 meq/l) for the eight plasma samples was estimated to be 4 meq/l less than [SID₃] = ([Na⁺] + [K⁺]) - [Cl^-] = 41 meq/l (Table 2), 3 meq/l less than [SID₄] = ([Na⁺] + [K⁺]) - ([Cl^-] + [lactate]) = 40 meq/l, and 6 meq/l less than [SID₆] = ([Na⁺] + [K⁺] + [Ca²⁺] + [Mg²⁺]) - ([Cl^-] + [lactate]) = 43 meq/l, and because mean plasma [K⁺] = 4 meq/l, we suggest the following equations for calculating actual SID from measured SID

(12)

(13)

(14)

(15)

Obviously, Eqs. 12–15 assume that the unmeasured anions equal the unmeasured cations and that plasma albumin and total protein concentrations are normal.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
This study was supported by EP Taylor Equine trust fund, by Natural Sciences and Engineering Research Council of Canada, and by Ontario Ministry of Agriculture, Food and Rural Affairs-Equine Program.

	ACKNOWLEDGMENTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
The technical expertise of Dr. Susanne Misiaszek, Bonnie Lambert, and Dr. Nevil Sukra is greatly appreciated.

	FOOTNOTES

 

Address for reprint requests and other correspondence: H. R. Staempfli, Dept. of Clinical Studies, Ontario Veterinary College, Univ. of Guelph, Guelph, Ontario, Canada N1G 2W1 (E-mail: hstaempf{at}uoguelph.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 

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