Plasma bicarbonate assays-time for a new look?

Personal View Ann cu«Biochem 1993; 30: 233-237

Plasma bicarbonate assays-time for a new look?

A L Miller

From the Department of Chemical Pathology, The University College and Middlesex School of Medicine, London WIP6DB, UK

Additional key phrases: acid base imbalance;

electrolytes; total carbon dioxide; blood gas analysis

The traditional 'electrolyte profile' still provided by many laboratories consists of the two principal cations of the plasma, sodium and potassium, the two principal anions, chloride and bicarbonate, together with some indicator of glomerular filtra- tion rate, such as urea or creatinine, or both. As will be emphasized shortly, bicarbonate itself cannot readily be measured, but a clinically useful approximation can be obtained by measuring the total CO₂ content of the plasma.

This grouping of assays has its origins in the history of the clinical laboratory. Measurement of the anions long antedates the development of flame-emission spectrophotometry! in the early 1950s, which made measurements of sodium and potassium practical procedures for the hospital laboratory, capable of providing valuable clinical information with an acceptable turn-round time.

The cation measurements were added to those of the anions already provided to give the profile.

This focused attention on the electrical neutrality of the extracellular fluid, leading to the reporting of ion concentrations in milliequivalents rather than mass units and the development of the familiar bar-diagrams, in which columns of equal length represent cation and anion concentrations, and to the concept of the 'anion gap' which will be further discussed later. Increasing under- standing of the pathophysiology of the body fluids, based on the earlier work of Gamble.i-' led to a rapid rise in demand for electrolyte assays. This was met by automation, mainly using Technicon AutoAnalyser equipment, culmi- nating in the development of dedicated multi- channel analysers, such as the SMA 6/60,

This article was commissioned by the Clinical Laboratory Investigations Subcommittee of the Scientific Committee of the Association of Clinical Biochemists.

specifically designed to meet this particular work-load.

Initially, the measurement of 'bicarbonate' had particular importance, since it was the only prac- tical means of assessing acid-base status. Despite the fact that rapid and reliable measurements of blood pH andPco2 , which on their own permit full assessment of acid-base disturbances, are now readily available the plasma bicarbonate is still seen by many as providing valuable clinical information.

MEASUREMENT OF PLASMA BICARBONATE

At this point, it may be helpful to review the methods available, and to remind ourselves that none of them is a true measure of the bicarbonate concentration in plasma. There was no confusion in the minds of those who originally described these methods. They were meticulous in their terminology. The time-honoured gasometric methods pioneered by Van Slyke" were correctly described as measuring the total CO₂content of the plasma, recognizing that the CO₂evolved on acidification of plasma, though principally derived from bicarbonate, came also from CO₂in simple physical solution as well as from carbamino- compounds and carbonic acid. AutoAnalyser methods, where the CO₂ liberated by acidification of plasma in a closed system diffused through into a reagent stream to produce a pH change reflected in an indicator colour change, also measured total CO_{2 ,} Electrometric measure- ment similarly measures total CO_2,which diffuses through a silicone-rubber membrane to produce a change in H+ ion activity in a buffer solution which is monitored by a pH electrode.' Even in the case of contemporary enzymatic assays utilizing the enzyme phosphoenolpyruvate carboxylase (EC 4.1.1.31) any impression of specificity is spurious, since substrates other than bicarbonate (including gaseous C00 can be utilized in the reaction" and, in any case, removal of bicarbonate from the reaction mixture leads to shifts in 233

existing equilibria. Since all these methods give a rough approximation to the actual bicarbonate in most situations, careless use of words has allowed us to slip into calling them bicarbonate assays.

Moreover, whilst in the past great care was taken in collection of the blood sample to ensure that CO₂was not lost to theair before analysis,"

such precautions have now been largely aban- doned. Samples are often received at room temperature, in partly-filled tubes, and with an unknown interval having elapsed between collec- tion and separation of the plasma. The plasma samples are often loaded on to analysers in open cups and left for a variable period before analysis, again carried outinreaction vesselsopen to the air. Thus, there is opportunity for an unknown loss of CO₂ from the plasma, a process which continues as further CO₂is formed by the breakdown of bicarbonate, and accounts for the well-known fall in measured plasma total CO₂if adequate precautions are not taken to prevent such losses." These factors introduce a variable and unknown error into the results we furnish to clinical colleagues-an error to which both we and they appear in- different!

Quantitatively, the difference between measured 'bicarbonate' (strictly the 'total CO_{2 ' )} and the true value may not be great, and it may be of little clinical relevance. The largest component of the total CO₂other than bicarbonate is the CO₂in simple physical solution which, at the partial pressure existinginnormal subjects, is only about

1. 3 mmol/L. The difference, however, needs to be recognized.

An alternative approach is to derive the actual bicarbonate concentration by application of the Henderson-Hasselbalch equation to data obtained by 'blood-gas' analysis. The validity of such calculations has been repeatedly questioned, mainly on the grounds that a constant pK for carbonic acid cannot be assumed. It has been suggested by Flear et al.⁹that variation in pK could lead to errors in the calculated bicarbonate of ±60070. However, such variation requires extreme shiftsinpK-from 5·84 to 6· 30-around the value of6'1 usually appliedinthe Henderson- Hasselbalch equation, and these must be unusual in clinical practice. Earlier, Flear and his colleagues'?had set out cogently the case against the use of the calculated bicarbonate, document- ing the theoretical considerations involved.

CLINICAL USEFULNESS OF PLASMA BICARBONATE

Given that there is no wholly satisfactory method for either the measurement or the calculation of the plasma bicarbonate, it is worth attempting to establish the clinical usefulness of the test. Such an evaluation is particularly timely since the continu- ous-flow systems and electrometric methods, which used relatively cheap methods, are now being replaced by discrete analysers using the costly PEP- carboxylase method. In our laboratory the reagent costs of bicarbonate assays soared to £27 000 pa.

TABLE 1. Changes inH+. Peo₂and CO2content in acid base disturbances Blood gases

H+ _Peoz

Respiratory acidosis Increased Increased

Respiratory alkalosis Decreased Decreased Metabolic acidosis Increased Decreased

Metabolic alkalosis Decreased '?Increased (compensatory)

Combined respiratory/ Increased Increased metabolic acidosis

Fully compensated Normal Increased or decreased disturbance

Total COzcontent (bicarbonate) Increased (may be a useful

indicator of developing acidosis inchronic lung disease) Decreased (could assist in recogni-

tion of over breathing) Decreased (useful indicator of

developing acidosis in renal disease and monitoring progress of known metabolic acidosis) Increased (useful indicator of

alkalosis in vomiting, K+

depletion etc.) Decreased (indicating the

metabolic component: non contributory without_Peoz) Increased or decreased (non-

contributory)

The limitations of 'bicarbonate' assays in the assessment of acid-base pathology are obvious and familiar (Table 1). The bicarbonate concentration in the plasma is the resultant of its rate of formation from available cO₂ and its utilization as buffer anion; it follows that plasma bicarbonate is reduced in both metabolic acidosis and in respiratory alkalosis, a problem which caused initial confusion over the nature of the acid-base disturbance in the early stages of salicylate poisoning. Blood pH and Pco₂ measurements allow full assessment of both the primary and secondary components of any acid- base disturbance.

Itis important here to distinguish between pH and [H+]. What is measured is the pH, the measure of hydrogen ion activity, but when considering the clinical significance of changes it is easier to think in terms of [H + ] , the hydrogen ion concentration. The reasons for this are simple and familiar. pH is a logarithmic scale so that a fall of O·3 in the pH represents a doubling of [H+]; the use of [H+1makes changes more readily comprehensible. Moreover, [H+] bears a linear relationship toPco₂so that a change in Pco₂ should lead to a predictable change in [H +], deviation from this predicted change indicating a non-respiratory component to the disturbance. Thus, we measure pH but think in terms of [H+].

Returning to the diagnosis of acid-base dis- orders, the [H+] indicates the presence of acidosis or alkalosis, and consideration of the Pco₂in the context of that [H+] usually clearly indicates whether any change inPco₂is primary (as in a respiratory disturbance, where the direction of change would explain the observed change in [H + ] ) or secondary (as in metabolic disturbances, where the change inPco₂would tend to minimize the change in blood [H+]).

Interpretation is made even simpler and more precise by the several excellent acid-base diagrams which have been published, such as that of Flenley!' which allow the observed changes to be assessed visually. Only in two situations is there any unresolved problem. In the patient with a severe combined disturbance involving both respiratory and metabolic components, thePco₂ reflects only the over-all respiratory component.

And in the patient with a fully compensated disturbance, with a normal [H+], one is left to decide on other grounds whether an observed change inPco₂represents the primary event in a respiratory disturbance, with renal compensa- tion, or respiratory compensation of a metabolic

Plasma bicarbonate assays 235 disturbance. In the first of these two situations the bicarbonate provides help; for example, in the common clinical context of combined respiratory- metabolic acidosis, the raised Pco₂indicates the respiratory element whilst a low bicarbonate indicates the depletion of blood buffer in response to the metabolic acidosis. Itis true that the same conclusion can be reached from consideration of the [H+] and reo, alone, since the linear relationship between these two parameters will be lost, but it is perhaps easier to recognize the 'mixed' nature of the disturbance if a 'bicarbonate' is available. In the case of the fully compensated disturbance, the bicarbonate adds nothing to the other measurements, and interpreta- tion rests mainly on the likely disturbance in the prevailing clinical situation.

Blood gas measurements are now readily avail- able and current clinical practice ensures that these are made in any acute situation requiring full acid-base assessment. Moreover, the required instrumentation is often available in clinical areas such as intensive care units, special care baby units and accident and emergencydepartments, allowing very rapid provision of results in response to rapidly changing clinicalsituations. There is clearly little place for the laboratory measurement of total CO₂ in the context of an acute acid-base disturbance.

If this measurement has any place, it is in relation to the assessment of more chronic disturbances. But most total CO2measurements presently carried out are done as part of an electrolyte profile, mainly on surgical patients (pre- or post-operatively) in whom no acid-base abnormality is either expected or found. Clinical audit was needed to establish both the size and the nature of the valid requirement, followed by consideration of how a reduced workload could be most appropriately met. We therefore decided in 1990to discontinue 'bicarbonate' assays as part

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Ann Clin Biochem1993: 30

of the 'electrolyte profile', though the measure- ment remained freely available to clinicians throughout the 24 h by specific request. The effect on workload was dramatic, as is shown in Fig. I, falling from nearly 10 000 assays per month to fewer than1000within 2 months, a levelof activity which has been maintained.Itwas thus clear that there was little clinical demand for the assay, and it became pertinent to examine the source of the residuum of requests received.Itwas found that 89010 of the demand now comes from the Post- graduate Nephrology Hospitals located within our District, with two major University Hospitals, the other hospitals within the District and general practitioners together contributing only 11%-or fewer than five samples a day. Itwould seem that the perceived clinical need for 'bicarbonate' assays relates mainly to the management of renal disease and, in particular, of chronic renal failure.

This is not surprising since renal failure is probably the best example of a condition in which there may be a chronic metabolic acidosis requiring treatment. A simple measurement of total CO_{2 ,} carried out on a venous blood sample together with other relevant assays will provide evidence of developing metabolic acidosis and some assessment of its severity. Due to the nature of the hospitals served by our laboratory service, we see a larger number of patients with such conditions than the average District General Hospital and we may need to make special provision to meet this demand. The work coming from other sources would certainly not justify maintaining a dedicated channel on a major analyser, employing an expensive chemistry.

CALCULATED PLASMA BICARBONATE

How, then, can a limited, but valid, demand be met? One possibility might be to use the laboratory blood-gas analyser to provide a derived bicarbonate. Despite the limitations dis- cussed above, this parameter has an established place in clinical practice. The conventional use of arterial blood for blood-gas analysis relates to measurement ofPo_2; arterio-venous differences in pH andPeo₂are small and usually relatively constant, the pH being 0,03-0,04 units lower in venous blood and thePeo₂about 1 kPa higher, than in arterial blood. As ReIman states, 'For most practical purposes, venous blood can be used interchangeably with arterial blood if one is interested only in the assessment of acid-base

balance'^.12 In this, he reiterates the view that a distinguished panel formulated in 1966.¹³ Interestingly, a paper by Weilet a[.l4 suggests that in at least one situation where differences may occur the use of venous blood may give the more relevant information. They showed that during cardiopulmonary resuscitation, arterial blood may not reflect the marked reduction in mixed venous pH which indicates the degree of acidosis in the tissues.

O'Leary and Langton'! compared calculated plasma bicarbonate values from arterial blood-gas analysis with 'bicarbonate' measurements made either by the Ektachem (potentiometric) method or by the Technicon continuous-flow colorimetric method, using venous blood collected into heparinized Vacutainer tubes (Becton-Dickinson).

They found poor correlation betweenthe calculated and measured values; however, the mean difference between derived and measured concentrations was - 0·89mmol/L(Technicon) and - 1.26 mmollL (Ektachem). This approximates to the expected difference between arterial and venous blood. The imprecision of the methods used, given here as a coefficient of variation, was Ektachem 4·89%

and Technicon 3,28%, compared with 2·87%

for the derived value. Our recent experience suggests a best achievable precision using a PEP- carboxylase method on the American Monitor Perspective analyser of around 4% in studies over a 6 month period(KPiper, personal communica- tion). The derived bicarbonate value would seem to be at least as satisfactory as other measures when all that is required is a simple indicator of metabolic acidosis.

THE ANION GAP

There remains one last question to be addressed:

the issue of the anion gap. The traditional electrolyte profile in which both chloride and 'bicarbonate' are measured allowed the calcula- tion of an anion gap-usually taken as the difference between the plasma sodium concentra- tion and the lesser sum of the chloride and bicarbonate; this difference is, of course normally due mainly to the unmeasured contribution of protein to the total anion concentration. Addition of strong acid other than hydrochloric acid to the ECF will lead to a fall in bicarbonate, used to buffer the added H+ ions, without any increase in chloride, so that the anion gap is increased.

Thus, the presence of an increased anion gap in a patient with acidosis may suggest the possibility

of lactic acidosis or ketoacidosis. This concept has even been developed to allow the calculation of a 'delta gap',16the difference between the observed increase in the anion gap and the observed decrease in the plasma bicarbonate, to permit an indication of 'mixed disorders'. In simple metabolic acidosis the fall in 'bicarbonate' reflects a more-or-less equal rise in the anions derived from the acid, so that the delta-gap is zero; any factor modifying this relationship will lead to the development of a positive, or negative delta-gap. The subject was comprehensively reviewed by Goodkin et al. in 1984}7 The obvious fallibility of the idea, which is still being promoted, was discussed by Di Nubile.P Argu- ment for the retention of bicarbonate assays along these lines can be strongly challenged on the grounds that the calculated anion gap represents a summation of the analytical errors inherent in the measurements used, with a high coefficient of variation and an ill-defined referencerange.'?

Moreover, in the individual patient baseline data against which changes can be assessed are seldom available. But perhaps the most practical considera- tion is that many laboratories in the UK have given up routine measurements of plasma chloride, so that the issue in relation to the anion gap becomes not whether 'bicarbonate' measurements should be continued, but whether chloride measurements should be restored to the electro- lyte profile!

CONCLUSION

There is no satisfactory method for the measure- ment of bicarbonate in plasma, and the methods for the measurement of total CO_{2 ,}as commonly employed, lack both precision and accuracy.

Clinical information provided by so-called bicarbonate measurements may be misleading, and there is evidence that the real clinical need for such assays is small. There is little justification for inclusion of total CO₂ in the standard 'electrolyte profile', since in most patients on whom 'electrolytes' are requested no acid-base disturbance is either expected or found. The true clinical need relates mainly to patients with chronic renal disease. It is possible that this limited requirement might be most easily met by providing a derived value for actual bicarbonate

Plasma bicarbonate assays 237 from mixed venous blood pH and Peo₂ measurements using a blood-gas analyser.

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Accepted for publication 9 November 1992

Ann Clin Biochem 1993: 30