Direct reading glucose electrodes detect the molality of glucose in plasma and whole blood.

It is the activity that determines the direction of chemical processes, transport, etc. and thus provides the clinically more relevant information. Direct reading glucose electrodes consume glucose at a rate proportional to the glucose activity in the sample. The activity equals the molality (mmol glucose per kg water), so results from direct reading glucose electrodes must differ from the conventionally measured glucose concentration. This was observed in 159 whole blood samples which gave higher results from a direct reading glucose electrode than by our conventional method (y = 1.21x - 0.37 mmol/l). However, adjustment for the different water concentration due to salt, plasma proteins, and hemoglobin occupying space, gave results equal to the concentrations (y = 1.00x - 0.28 mmol/l, r = 0.997). Furthermore, results for samples with constant glucose concentration and varying albumin concentration correlated with the albumin concentration (r = 0.989), but not after adjustment for water concentration (r = 0.037, n.s.).

Relation between ionised calcium and pH in infants with acute acid-base disturbances.

Plasma ionised calcium, [Ca2+], and pH were negatively correlated in 21 infants with disturbed acid-base homeostasis. The slopes of the individual regression lines indicated a mean decrease in [Ca2+] of 42% per pH increase. (Mean delta log[Ca2+]/delta pH = -0.184, SEM = 0.050, p less than 0.001). The value was not significantly different from the relation between [Ca2+] and pH in vitro, when whole blood or plasma was equilibrated with CO2-air mixtures. pH and [Ca2+] must be measured and considered together to allow a proper interpretation of the result.

Donnan effect or protein interference in ionised calcium measurements?

The observation that the ionised calcium concentration is higher if measured in the original serum sample than in an ultrafiltrate is examined by means of a theoretical model. It is concluded that this difference is a predictable consequence of the Donnan equilibrium and is not a result of protein interference with the calcium-specific electrode.

Serum ionized calcium, parathyroid hormone and phosphate in uremic patients during and between hemodialysis.

The serum concentrations of actual ionized calcium (at actual pH), adjusted ionized calcium (at pH 7.4), pH, intact parathyroid hormone (PTH) and phosphate were studied in ten patients during and between two hemodialysis sessions using a dialysate containing 1.66 mmol/l of calcium. Actual ionized calcium and adjusted ionized calcium increased during hemodialysis from 1.19 to 1.38 and 1.43 mmol/l, respectively (mean values) and returned to predialysis values within five and nine hours postdialysis. Serum PTH decreased from 165 ng/l to 55 ng/l (median values) during hemodialysis but two-hour postdialysis the level did not differ significantly from the predialysis level. Serum phosphate decreased from 2.05 mmol/l to 1.11 mmol/l during hemodialysis, and increased slowly towards the predialysis level. The level of pH increased from 7.40 to 7.47 during hemodialysis and reached predialysis level after nine hours. In a multivariate analysis including actual and adjusted ionized calcium, pH, phosphate and PTH, only actual or adjusted ionized calcium was associated with the level of PTH. We conclude that the effect of dialysate calcium on the levels of ionized calcium and PTH is of very short duration postdialysis.

Ionized calcium and cyclic AMP in plasma and urine. Biochemical evaluation in calcium metabolic disease.

Measurement of ionized calcium and cAMP in plasma and urine are used as sensitive parameters for the evaluation of calcium disorders. Ionized calcium is accepted as the biologically active form of calcium in the extracellular fluid, while urine cAMP provides an in vivo receptor assay for the biologically active parathyroid hormone. When urine is included as part of the calcium metabolic investigation it usually requires 24 h urine collection with a variety of different laboratory tests. Ionized calcium and cAMP are described in the literature in terms of several derived quantities, nomenclatures, and units which are rather unsystematic. The author developed reliable techniques and proposed systematic names and symbols and reference values for these quantities. Due to the lack of guidelines for the collection of urines in calcium metabolic evaluation, the author presented a simplified protocol (4 h standardized urine collection). In clinical investigation plasma and urine cAMP have been used to differentiate idiopathic hypoparathyroidism from pseudohypoparathyroidism (PsHP) based on the results of i.v. injection of parathyroid hormone (PTH). Nephrogenous cAMP has also been used for the detection of primary and secondary hyperparathyroidism with a high nosographic sensitivity (90%) (Broadus). The author showed that measurement of cAMP after i.v. PTH was a reliable and sensitive test to establish the diagnosis of PsHP, and that the urinary cAMP was useful for the diagnosis of secondary hyperparathyroidism in patients with jejunoileal bypass, but could not confirm the high nosographic sensitivity for the diagnosis of primary hyperparathyroidism. Further data are needed for proper conclusion. Although pursued vigorously the research into idiopathic stone formation using different protocols has not prevented stone recurrence nor indicated where further progress might be made. For the evaluation of recurrent calcium disease, the author proposed a simplified 4 h standardized urine collection with plasma albumin, urinary pH, standardized excretion rate of calcium, plasma phosphate glomerular filtration rate, and nephrogenous cAMP as the most important parameters. In this way the author obtained a sensitivity of 93% and specificity of 95.6% for the diagnosis of recurrent stone former. The test may therefore be of value for predicting the risk of recurrent stone formation in the single stone former.

What is "ionized calcium"?

"Ionized calcium", in clinical chemistry, designates the free calcium ions which are present in the biological fluids as hydrated ions: Ca2+, 12 H2O. The calcium ion selective electrode measures differences in activity, however, values are reported not as the activity, but as the activity divided by the activity coefficient (gamma) of Ca2+ in "normal plasma". The latter is assumed to be equal to the activity coefficient (gamma) in a calibration solution of CaCl2 + NaCl with an ionic strength of 0,16 mol X kg-1. We have estimated the activity coefficient in this calibration solution to be gamma Ca2+ = 0,3040 (molal basis) or gamma Ca2+ = 0,3048 (substance concentration basis). The derivation of gamma Ca2+ is based on a convention for derivation of the activity coefficient of the chloride ion. We have used the Debye-Hückel equation extended by the Stokes-Robinson hydration theory plus the Bates-Staples-Robinson convention that the hydration number for Cl- is zero. International consensus is needed concerning the convention to be used for deriving the activity coefficient of Ca2+ in the calibration solutions and concerning the most appropriate value for gamma Ca2+ in normal plasma.

Ionized calcium during dialysis and ultrafiltration.

The changes in ionized calcium during dialysis and ultrafiltration with albumin containing solutions were highly dependent on the total electrolyte concentration. Protein interference on the calcium electrode could not explain the observation, which was, however, consistent with the Donnan theory. The uneven distribution of sodium ion also agreed with a Donnan membrane potential.

Elimination of the erythrocyte effect on the liquid junction potential in potentiometric measurements on whole blood using mixed salt bridge solutions.

An effect of erythrocytes on the liquid junction potential of the junction "concentrated KCl whole blood" has previously been described, the effect being a positive bias of +1,4 mV with a high erythrocyte volume fraction (0.75). We have studied the erythrocyte effect with different concentrated salt solutions, and found a similar positive bias with KBr, KJ, KNO3, K2SO4, but a negative bias with NaCl, NaNO3, LiCl and CaCl2. The erythrocyte bias may be eliminated by mixing salts with opposite effect, e.g. KCl + NaCl (1.0 + 7.0 mol/l), KNO3 + NaNO3 (1.0 + 3.0 mol/l) KNO3 + Li-acetate (0.15 + 4.0 mol/l). The use of a mixed salt bridge solution may be advantageous for eliminating the erythrocyte bias when measuring ionic activity (e.g. Na+, K+, Ca2+) directly in whole blood.

Nomograms for calculating the concentration of ionized calcium of human blood plasma from total calcium, total protein and/or albumin, and pH.

Two nomograms are described. One is a Cartesian nomogram similar to the original McLean-Hastings nomogram but supplemented by an albumin scale and a pH scale to allow calculations in case of an abnormal albumin/globulin ratio or an abnormal pH. The other is an alignment nomogram. The underlying arithmetic equations, which are slightly different for the two nomograms are described. Both refer to 37 degrees C. Calculated and measured ionized calcium are compared for 24 specimens and it is concluded that the calculated value may be subject to a considerable bias amounting to 0.2 mmol/l in the worst cases. Nomograms size A4 and BASIC program are available on request.

Variability of the calcium ion activity with pH in stone-forming and non-stone-forming urine.

In recurrent renal stone-formers (N = 20) and matched healthy adults (N = 20), the actual activity of ionized calcium (alpha Ca2+) and pH were determined in whole urine with an ion-selective electrode. No significant difference was found for the actual median activity of ionized calcium, however the actual median pH was significantly higher in stone-formers compared to healthy adults (pH = 5.57 vs. pH = 5.24; p < 0.005). The relationship between Ca2+ activity and pH was studied in each collected urine by titration with HCl/NaOH. In all urines the Ca2+ activity decreased with increasing pH in a typical bifasic manner. All curves showed a characteristic "breaking point" at a similar median pH in the stone-formers and in the healthy adults (pH = 6.81 vs. pH = 6.77) (NS). However the slope of the curves in the stone-formers and healthy adults changed from a median value of delta lg alpha Ca2+/delta pH of -0.139 and -0.173 (NS) respectively, to a highly significant difference of -1.326 and -1.053 (p < 0.0001) between the groups, indicating increased binding/precipitation of Ca2+ in stone-formers than in healthy adults supporting the theory of the lack of inhibitors in stone-formers. The strong relationship between the activity of ionized calcium and pH, combined with a higher actual pH and a higher decrease of ionized calcium with pH in stone-formers than in healthy adults, indicates hydrogen ion as a major factor in stone-formation. The close relationship between Ca2+ activity and pH indicates the need for simultaneous measurements of the pH in order to interpret data for the Ca2+ activity. In order to preserve a low urinary pH, where Ca2+ is predominantly in a free ionic state, our results suggest that treatment with acidifying salts could be a logical choice in order to prevent stone-formation.

Relation between pH and ionized calcium in vitro and in vivo in man.

We investigated the in vitro and in vivo relation between pH and ionized calcium during acute respiratory pH changes. The in vitro slope delta 1gcCa2+/delta pH determined in 20 healthy volunteers after equilibration at two different pCO2 values was -0.22 +/- 0.04 (mean +/- 2 SD) for whole blood and -0.24 +/- 0.04 (mean +/- SD) for serum. The in vivo slope delta 1gcCa2+/delta pH during acute respiratory changes in 10 healthy volunteers was -0.17 +/- 0.04 (mean +/- 2 SD) for capillary blood. A mathematical formula which describes the relation between pH and ionized calcium is presented.

Serum-ionised magnesium in patients with acute myocardial infarction. Relation to cardiac arrhythmias, left ventricular function and mortality.

Measurement of serum-ionised magnesium has recently become available, and we report the first study of the relation of this parameter to arrhythmias, left ventricular function and mortality in patients with an acute myocardial infarction. Serum-ionised magnesium was determined in 217 consecutive patients admitted to hospital with an enzyme confirmed AMI. 70 healthy subjects acted as a control group. The main study parameters were occurrence of arrhythmias, left ventricular function estimated by echocardiography, and mortality after 10 months for the AMI patients. AMI patients had significantly lower serum-ionised magnesium compared to healthy controls but the level of serum-ionised magnesium in the acute phase of a MI was neither related to arrhythmias, left ventricular function nor mortality.

[Osteoporosis-profile--a bad examination?]. / Osteoporoseprofil--en dårlig undersøgelse?

A so-called osteoporosis-profile has become increasingly popular among Danish doctors. Future bone loss in peri- and postmenopausal women is estimated by means of an algorithm comprising serum osteocalcin and alkaline phosphatases as markers of bone formation and urinary calcium and hydroxyproline excretions, corrected for creatinine, as markers of bone resorption. The osteoporosis-profile, performed by Medi-Lab, was tested in six females aged 46-56 years. Three different diets were used on three successive week-ends, each ending with a collagen-free supper and an overnight fast. Individual changes in estimated bone loss of up to 200-300% were found, demonstrating that the test was useless in a single person.

[Ionized calcium, total calcium and albumin corrected calcium in the serum in 1213 patients with suspected calcium metabolic diseases. A prospective multicenter study]. / Ioniseret calcium, totalcalcium og albumin-korrigeret calcium i serum hos 1.213 patienter mistaenkt for calciummetabolisk sygdom. En prospektiv multicenterundersøgelse.

The correlation between serum ionized calcium, serum total calcium and albumin corrected total calcium was investigated in a prospective multicentre investigation of 1,213 patients with suspected calcium metabolic disease. 31.0% of the patients were misclassified when serum total calcium was measured instead of serum ionized calcium. The diagnostic discrepancy between the two methods decreased with the calculation of albumin corrected total calcium or calculated ionized calcium (17.9%). On justing for the analytical error connected with the measurement of ionized calcium, 11.2% of the patients were still misclassified. It is not possible precisely to predict serum ionized calcium from the measurement of serum total calcium and we recommend measurement of serum ionized calcium in patients believed to have calcium metabolic disease.

The calcium ion activity and the standardized excretion rate of calcium in urine of healthy adults.

A standardized protocol is described for the study of the calcium excretion in urine. After 12 h of fasting, urine is collected during 4 h with a water load of 10 ml per kg body weight. Urine is also collected during the following 20 h period on the habitual water and calcium intake. Reference values for 48 healthy adults are given as 0.25 and 0.75 quantiles. The measured activity of calcium ions (Ca2+) in urine is 0.09-0.27 mmol/kg for the 4 h period, 0.34 to 0.52 mmol/kg for the 20 h period; pH values are 5.61-6.43 (4 h) and 5.46 to 6.04 (20 h). The concentrations of total calcium are 0.67-2.05 mmol/l (4 h) and 3.16 to 4.94 mmol/l (20 h). The value for the excretion rate of calcium (standardized to a creatinine clearance of 100 ml/min) is 1.30-3.24 mumol/min for the 4 h period and 3.06-4.88 mumol/min for the 20 h period, with no significant difference between the results for men and women. The relationship between the Ca2+ activity and pH was studied in urine titrated with HC1 or NaOH. In all urine the Ca2+ activity falls with increasing pH in a typical biphasic manner. This indicates the need for simultaneous measurement of the pH in order to interpret data for the Ca2+ activity in urine.

Actual ionized calcium and pH in blood collected in capillary or evacuated tubes.

Results are presented for the concentration of actual ionized calcium (cCa2+), the actual pH and ionized calcium at pH = 7.4 [cCa2+(7.4)] measured in capillary whole blood, venous serum from vacuum tubes (Venoject) and serum equilibrated to pCO2 approximately equal to 5.3 kPa from 57 renal patients. The mean value for cCa2+ was higher for venous serum than for capillary blood (1.29 mmol/l versus 1.26 mmol/l), corresponding to the difference in pH (in venous serum). This difference could be eliminated by separating the serum at 4 degrees C rather than room temperature. The cCa2+ (7.4) was the same for capillary blood, venous serum (Venoject) and equilibrated serum from blood collected in plain glass tubes, Venous serum could be stored at 4 degrees C for 4 h without significant change in cCa2+ and pH, and for 1 week at 4 degrees C for the measurement of cCa2+(7.4). In patients with acid-base disturbance cCa2+ is preferable to the measurement of cCa2+(7.4).

Six direct radioimmunoassays of estradiol evaluated.

We evaluated the analytical performance of six commercial direct radioimmunoassays of 17 beta-estradiol, those from Radioassay Systems Laboratories, IRE Medgenix, Biotecx Laboratories, Farmos Spectria, International CIS, and Diagnostic Products Corp. The mean value for estradiol (E2) and the within- and between-run CVs were determined for serum pools and control materials, measured in seven to 10 runs with each method. Mean values for E2 in pooled sera deviated by 75% to 350% from the means measured with our routinely used extraction method. Between-run CVs ranged from 4% to 14% for the direct assays as compared with 10% for the extraction assay. We also investigated, for two of the direct assays, the effect of extraction with diethyl ether before radioimmunoassay, with respect to improvement in the agreement with our extraction method. All of the assays were easy to perform and results were obtained within 4-5 h, but we conclude that matrix effects may be expected in direct assays of estradiol.

Increased activity of ionised calcium in gall bladder bile in gall stone disease.

The actual activity of ionised calcium (Ca2+) in gall bladder bile determined with an ion-selective electrode was significantly higher in patients with gall stone disease (n = 15) than in patients without gall stones (n = 10) (0.43 mmol/kg v 0.31 mmol/kg; p < 0.05). No change in the Ca2+ activity in any of the gall bladder bile samples was observed during equilibration with CO2. During titration with HCl/NaOH, however, the Ca2+ activity fell with increasing pH in a biphasic manner, with the breaking point occurring at a significantly lower median pH in patients with gall stones than in patients without (pH 7.1 v 8.2; p < 0.0001). The combination of a higher activity of calcium in bile and precipitation of bile salts taking place at a lower pH in patients with gall stone disease than in patients without gall stones suggests a major role for calcium and pH in the pathogenesis of gall stones. Strict anaerobic sampling is not necessary for the measurements of Ca2+ in gall bladder bile, because the Ca2+ was not significantly affected by the changes in pCO2. The metabolic studies suggest, however, that simultaneous measurements of the activity of Ca2+ and pH is important in order to interpret data for the calcium activity in gall bladder bile.

Performance characteristics of creatine kinase-MB isoenzyme measured with an immunoenzymometric and an immunoinhibition assay in acute myocardial infarction with and without thrombolytic therapy.

In a time study we compared the analytical and clinical performance of the Tandem Icon QSR CK-MB enzyme-immunoassay (Hybritech) (creatine kinase-MB) and a creatine kinase-MB immunoinhibition method (Boehringer Mannheim GmbH) (creatine kinase-B). Two hundred and ninety-nine serum samples from 38 patients suspected of acute myocardial infarction were collected at regular intervals during 48 hours. Twenty-nine patients were diagnosed as having acute myocardial infarction, of whom 19 received thrombolytic therapy. Although highly correlated, the large scatter around the regression line at low values indicated a different clinical performance of the two methods. We evaluated and compared test performance at different decision levels by means of frequency distributions and predictive values of positive and negative results. For early diagnosis of acute myocardial infarction (4 hours after onset of pain) the Hybritech creatine kinase-MB method gives acceptable predictive values. In thrombolytic treated acute myocardial infarction patients, the peak creatine kinase-MB and creatine kinase-B concentrations were reached after 13.0 h and 13.6 h after the onset of pain, compared with 19.8 h and 17.8 h for patients without thrombolytic therapy.