Review of Calcium Methodologies** · of bilirubin and hemoglobin in visual titra tions could be...

A n nals o f C lin ic a l a n d L a b o r a t o r y Science, Vol. 5, No. 3 Copyright © 1975, Institute for Clinical Science

Review o f Calcium Methodologies**

BENNIE ZAK, Ph.D.,* EMANUEL EPSTEIN, PH.D.,f AND EUGENE S. BAGINSKI, Ph .D 4

Departments of Pathology, *Wayne State University School of Medicine and Detroit General Hospital,

Detroit, MI 48201 f William Beaumont Hospital, Royal Oak, MI 48072

| Si. Joseph Mercy Hospital, Pontiac, Ml 48053

ABSTRACT

A review of calcium methodologies for serum has been described. The analytical systems developed over the past century have been classified as to type beginning with gravimetry and extending to isotope dilution-mass spectrometry by covering all of the commonly used technics which have evolved during that period. Screening and referee procedures are discussed along with comparative sensitivities encountered between atomic absorption spectrophotometry and molecular absorption spectrophotometry. A procedure involving a simple direct reaction for serum caloium using cresolphthalein complexone is recommended in which high blanks are minimized by repressing the ionization of the color reagent on lowering the dielectric constant characteristics of the mixture with dimethylsulfoxide. Reaction characteristics, errors which can be encountered, normal ranges and an interpretative resume are included in its discussion.

In tro du ctio nIt is difficult to look at almost any area

of metabolism without encountering calcium. It is involved in the clotting mechanism,138’152 enzyme reactions,63’68’160’169 sodium and potassium transport,42 bone formation,55 bone resorption,66 membrane function,148 with vitamins,82’85 with hormones98’168 and it is intricately interwoven in phosphate metabolism.177 Just as calcium is inseparably enveloped in the maintenance of normal body functions, so does it also act as a warning signal through high and low concentrations in a variety

* * Supported in part by the Detroit General Hospital Research Corporation.

of pathological circumstances.105’109’129’141’ 149,173,187 j n conjunction with other constituents such as in its relationship to phosphate,81 vitamin A,85-111 parathyroid hormone,9 magnesium,25 potassium,42’93 calcitonin37 and chloride,122 as examples, its diagnostic implications are widened. It is also important to consider posture and venous occlusion on the values obtained for serum calcium since they have been shown to affect calcium values.38’75

Calcium is widely distributed throughout the body and most body fluids. The body contains roughly 1,100 g on the average, with very little, less than one gram, circulating in the plasma. Most of it is in

1 9 6 ZAK, E P ST E IN AND BAGINSKI

the skeletal structure and some of the rest fairly well distributed in all body fluids. It is excreted in both feces and urine with the daily loss estimated at approximately0.5 g. Since this loss must be replaced and because not all of the calcium ingested is finally absorbed,33’73 the amounts required daily are somewhat in excess of the amounts lost.89 Several reasons for some of the absorbed calcium being excreted back into the intestine have been described.73 In conditions such as pregnancy, more calcium is needed than would be required for the same individual in the non pregnant state. Another recent finding of significant physiological interest is that astronauts in the weightless state lose considerable amounts of calcium.17 Calcium is also known to have a role in sickle cell anemia,170 an important genetic disease which has been studied in terms of calcium and red blood cell membrane metabolism.

All analytical procedures which merit sustaining clinical biochemical interest follow an evolutionary pathway. Calcium, certainly one of the most important essential metals of animal physiology, has been subjected to a continuous development of analytical schemes. The essentiality of the knowledge required for understanding its many faceted physiological activities has resulted in unceasing research over a period of many years. Simply listing what is known or of interest in its physiology and biochemistry would entail a vast and considerable effort. Calcium is, therefore, biologically important and naturally ubiquitous; analytically, it has been somewhat recalcitrant. From a clinical chemist’s point of view, all of the myriad methods proposed for its determination now seem to be narrowing down to molecular absorption (M AS) or atomic absorption spectrophotometry (AAS).

Early in the historical pathway of calcium methodology, gravimetry and titrimetry were encountered as main analytical

techniques for the determination of calcium in biological fluids.71’110 A great impetus was then given to the development of calcium methodology when ethylene diamine tetraaeetic acid (E D TA ) and then other complexones began to be used for the titrimetric determination of metals in conjunction with colorimetric complexing indicators used to determine the endpoints of those titrations. Direct titrimetry then evolved to the use of these indicators as color reagents for the spectrophotometric determination of several metals. In between visual titrimetry and colorimetry, a hybrid analytical system developed, marrying the two techniques and resulting in the process of the spectrophotometric titration. In this case, a photometer was substituted for the eye to detect the change in color of the calcium-color reagent complex over that of the free color reagent, since most of the indicators used were reagents which existed in two color forms, one for the metal- free state and one for the metal-bound state. Since fluorescence is inherently a more sensitive process than colorimetry, fluorimetric indicators were also developed and with these materials, visual titrations, instrumental titrations and direct fluorimetric methods were also developed. Other physico-chemical instrumental techniques were then developed where the foremost among these at the present time has been AAS.

C lassification

D ir e c t G r a v im e t r y

The quantitative determination of serum calcium began with gravimetry. As early as 1871, Pribham precipitated calcium directly from serum with ammonium oxalate and then weighed the washed precipitate.128 Such determinations by means of gravimetry in a clinical laboratory seem of little more than historical interest at the present time even though the precipitation step itself is still in relatively common use. There

REV IEW O F CA LCIU M M ETHODOLOGIES 197

was some extension of gravimetric techniques into contemporary analysis, for derivatives of calcium isolated in the gravimetric mode have sometimes been used in a purification or isolation scheme leading to a final determination by analytical instrumentation such as absorption or emission photometers,117 emission spectrographs,155 polarographs,16 and others.45’171 As further evidence that this is still an important analytical pathway, some relatively recent examples of the use of this precipitation device for purification have also been described.142’159

D e r iv a t iv e T i t r im e t r y

Gravimetry continued as an analytical technique until Kramer and Tisdall reported a then advanced system in which the washed calcium oxalate was dissolved with acid, and the oxalate moiety was titrated at an unstated warm temperature with standardized potassium permanganate to a self indicated endpoint.94 Tisdall then modified the procedure by describing in somewhat greater detail the process of precipitating and washing the calcium oxalate precipitate.164 From that point on, the procedure for titrating the acid dissolved calcium oxalate at a still undefined warm temperature was similar to the one previously described. The main difference then was the use of two washings instead of three and decanting rather than syphoning off the supernatant fluid. Clark and Collip reported that very slight variations in the Cramer and Tisdall procedure caused serious error.26 They therefore modified Tis- dall’s modification and included the range of temperature required (70 to 75 °) to titrate the oxalate successfully. Except for the control of temperature, the main difference in technique was in the description of the way the oxalate precipitate in the tube was to be washed and drained. This same procedure is presently used but some other modifications have appeared along the way.

For example, cerium has been substituted for permanganate as a titrant with heat, or by overstepping and back titrating with iron or by direct titration without heat because of a solvent change which increased the oxidation potential.71 These modifications led to colorimetric processes in which one overstepped with a fixed amount of eerie ion and then measured the color of residual cerium after oxidation was completed.183 A similar colorimetric procedure for measuring residual permanganate has also been described.44

The permanganate titration could have been carried out by adding a small amount of manganous sulfate to the dissolving sulfuric acid followed by a room temperature titration with potassium permanganate.92 Since this is a self catalyzed reaction in which the formation of some manganous ion is necessary before the reaction can proceed, either the heating step to form some manganous ion to catalyze the reaction or the addition of the manganous ion is necessary.

D ir e c t T it r im e t r y

The development of metallochromic indicators has been recently reviewed by Diehl40 and the natures of the reactions of several of the indicators for calcium have been described. In addition, a wealth of other literature in review or book form pertaining to mechanisms, physical properties and applications of these indicators are available.39’109’112’150’181 The action of metallochromic indicators has been shown to be somewhat complicated in that they are capable of acting in a dual role. One role for each is that of a metal complexer while the other role is that of an acid-base indicator.137 In addition, there have been reports of side reactions by buffer ingredients as further complications to “alter the conditional constants of the complexes.”1’145

The early titrant and perhaps the best known one was ED TA which was used in

1 9 8 ZAK, E P ST E IN AND BAGINSK1

conjunction with a variety of indicators such as murexide,56 eriochrome black T,40'101 cresolphthalein complexone5’137 and others.53’54’ eo,79,9i,i4T J3ecause 0f ^ subjective nature of the visual method, this led to the substitution of the spectrophotometer for the human eye in the titration of serum calcium. Also, some of the problem encountered with interfering colors such as those of bilirubin and hemoglobin in visual titrations could be minimized by a judicious choice of wavelength in spectrophotometric titrations. In the totally visual circumstance, colors and color changes were difficult to determine when a second indicator color and perhaps a third interference color was introduced. But even when interfering colors were not present, the two color indicator with one color owing to the metal complex and the other owing to the free form of the indicator was sometimes difficult to use, especially when there was a relatively small concentration of calcium to be determined. If the calcium content was relatively high, the sample relatively large and the titrant relatively strong as in a water hardness titration,41 it was relatively easy to detect visually a color change because a small volume of strong titrant transformed the system abruptly from one color form to the other. However, in considering a biological fluid such as serum, where the amount of calcium was but a small fraction of the amount titrated in water hardness determinations, and the concentration of the titrant was relatively much weaker, then the change from one color form to another visually similar one was more gradual than abrupt and difficulty was often encountered in perceiving this change. Therefore, spectrophotometric titrations were considered for the determination of calcium because such a development would enable the analyst to minimize sample size while at the same time eliminating the basic problem of trying to visualize when one color changed completely

to another similar one. A popular metallochromic indicator for calcium has been eriochrome black T (E B T ); however, it reacted poorly with calcuim. Since magnesium can undergo a better indicator change than calcium with such an indicator compound, it was then included into the procedure as a constituent of the titrant. When magnesium as the Mg-EDTA complex is used in such a calcium titration with EDTA as the titrant along with EBT as the indicator, the EBT is called an indirect metal indicator.123 In this case, EDTA forms a more stable complex with calcium than with magnesium, and magnesium is released to form a more stable complex with EBT than does calcium because the calcium-EBT complex has too weak a binding constant to give a satisfactory titration. Therefore, EDTA first binds calcium; magnesium is thereby released to react with EBT and this latter complex provides the titrated metal for the endpoint reaction as described by the equations which follow:

Ca++ + EDTA + Mg EDTA + EBT—^ C a ED TA + Mg EBT

As the titrant mixture is added to calcium ions, an exchange takes place to form Mg EBT. When no more calcium ion is present, it is the Mg EBT which is finally titrated as follows:

Mg EBT + EDTA — > Mg EDTA + EBT (wine) (blue)

This actually results in a much sharper change either visually or spectrophotomet- rically than could have been possible had magnesium been excluded from the titrant. This is graphically illustrated in figure 1 where it can be seen that the spectrophotometric titration was superior when varying concentrations of magnesium were present ( curves B -E ) than when calcium and magnesium standards are titrated with this mixed reagent. The titration of calcium is shown in figure 2 for a range of 1.25 to 5.0

REVIEW O F CA LCIU M METHODOLOGIES 1 9 9

F i g u r e 1. Effect of varying magnesium concentration of EDTA titrant in calcium titrations. A through E contain 0.0, 2.0, 7.5, 10.0 and 30.0 meg per ml of magnesium. (Reproduced with the permission of Analytical Chemistry.190)

mEq per liter at 660 nm, the peak maximum of EBT. In the beginning of the titration, the exchange between calcium and magnesium is taking place and this is indicated by the descending limb. When Mg-EBT is subsequently titrated to liberate strongly absorbing EBT, the sharply ascending portion of the curve appears, followed by a leveling off when the magnesium which had exchanged with the calcium was used up. In figure 3 is demonstrated the same titrimetric phenomenon for magnesium alone for 1.05 to 4.20 mEq per liter without any exchange process for calcium. The end-point for either metal is graphed where the tangents to the ascending and levelled off portions of the curve intersect as shown in figure 3. Curves inverse to those depicted here and showing a less sensitive absorbance change would have been obtained if the metal-bound color at 540 nm had been chosen instead of the free form. The importance of this hybrid system lies in the fact that it can be the basis for the next evolutionary step in analysis using

F i g u b e 2. Titrimetric curves for calcium at 660 nm. A through D contain 1.25, 3.75 and 5.00 mEq per 1 of calcium. (Reproduced with permission of Analytical Chemistry.190 )

the same indicators, the spectrophotometric determination of calcium.

In the case of the one color indicator such as cresolphthalein complexone,5 even the macro-titration was considered to be somewhat difficult, because the ionic form

Milliliters of Titrant

F i g u b e 3. Titrimetric curves for magnesium at 660 nm. A through D contain 1.05, 2.10, 3.15 and 4.20 mEq per liter of magnesium. (Reproduced with permission of Analytical Chemistry.190)


pH

F i g u r e 4. Variation of color of cresolphthalein complexone with calcium and hydrogen ion concentration. (Reproduced with the permission of Interscience Publishers.137)

of the indicator, some of which was present at the pH of the titration and whose concentration was variable with pH ,56 presented the identical spectrum as the calcium complex. In such a circumstance, the titration color change could be difficult to visualize because of the fairly deep residual color of the ionized form of the indicator.181 This ionization could be reduced to some extent by lowering the pH,115 or it could be repressed by incorporating a low dielectric solvent into the mixture.191 It is clearly shown by a pCa vs pH plot in figure 4 that this one color indicator works best across the range of pH 10.5 to 11 when used in the all aqueous manner described for it.13T It also indicates just as clearly that the indicator itself can be strongly colored in an alkaline medium.

D e r iv a t iv e A n a l y s is

Many of the techniques devoted to calcium determination were of the derivative

analysis type usually one in which a precipitate of calcium was formed followed by weighing,27 or by a determination of the resolubilized precipitating species containing calcium either by titrimetry28’52’74’158’164 or colorimetry,48’49’156 though in some cases other physico-chemical techniques were employed.15’24’69’114’186 One other kind of derivative treatment involved an oxalate precipitate which was then destroyed by heat or an oxidizing acid. The residual calcium was then titrated acidimetrically in a borate medium.156 More recently, the residual calcium was treated with a color reagent and spectrophotometry replaced the acidimetric step.159 Many colorimetric reagents have been used in derivative determinations of calcium. These include chloranilic acid,48’49’80’!87 napthalhydroxamic acid,13’165’185 oxalate-trihydroxy benzoic acid,166 2-chloro-5-cyano-3,6 dihydroxyben- zoquinone,134 oxalate-cerium,71 oxalate-per- manganate44 and others.3’76’118’153

The principles mainly involved in these determinations have been either to solubilize a colored derivative of calcium and then measure the colored anion or to react the colorless anion of the precipitate with a colored oxidant where the latter lost some of its color on being reduced by the anion during the reaction process. The residual color then became the measure of the amount of calcium precipitated. Heteropoly blue reactions of molybdate with precipitated calcium phosphate followed by reduction of the complex have also been successfully applied.117 One other device used to increase reaction sensitivity obtained with a derivative was developed for some of the already colored precipitating agents such as chloranilic acid. Here a metal, iron, was added to the precipitate dissolving solution and it reacted with and enhanced the color obtained from the reactant and thereby acted as a sensitivity device in the procedure.157

REVIEW OF C A LC IU M M ETHODOLOGIES 2 0 1

M o l e c u l a r A b s o r p t io n S p e c t r o p h o t o m e t r y

The tediousness of the spectrophotometric titrations along with the subjective character of its visual counterpart in perceiving a color change undoubtedly led to the spectrophotometric use of indicator- metal complexes as measures of the calcium to be determined.

The source of color reagents presently used for the colorimetric determination of calcium came from the early titrimetric determinations of Schwarzenbach125’150 and others.5’22’30’62’97’100’127’130 Although the number of reagents used to determine calcium by color is extensive, the most consistently used other than in derivative precipitation analysis have been sodium alizarin sulfonate (SA S ),31 methylthymolblue (M T B ),18 cresolphthalein complexone (C P C ),32’146 glyoxal bishydroxyanil (G H A ),107’112 murexide (M )135 and several others which have seen somewhat lesser use.57’65’S8’97’103> 127,128,134 p erhaps the most popular reagent of the present in manual and automated procedures is CPC because of its initial success on the Auto Analyzer.86

The primary source of interference with calcium color reactions has been magnesium which also reacts to some extent with the same complexones.180 In order to obviate the problem, procedures have been described in which calcium was isolated in order to remove it from magnesium,159 magnesium was added to the standards at a fixed concentration in order to minimize its effect81 or a chelating agent to tie up magnesium was included in the reagent to eliminate the interference entirely.32’146 The latter approach has been the simplest and the most effective and, in fact, has made colorimetric procedures possible which are as accurate as and more sensitive than atomic absorption when considered from a signal generated per solution concentration point of view. Other trace metals such as copper, iron, and zinc which might inter

fere with the reaction of complexones can be effectively removed from the reaction by the inclusion of cyanide into the reagent system.

A t o m i c A b s o r p t io n S p e c t r o p h o t o m e t r y

The most significant advance of the last two decades in calcium methodology ( and in metal analysis in general) has been the development of AAS. The principles of its operation have already been described so many times in reviews106’119’184 and books,36’ 136,154 that it would be superfluous to go over them again. Excellent values for calcium are obtained in AAS merely by high dilution of the sample with an acid lanthanum solution. This eliminates the need to remove the proteins or interfering phosphate anions, a major problem in the determination. Methods in which proteins are eliminated by precipitating them out or dialyzing the calcium away from them are also used,90’105 but on a routine basis they seem to offer no substantial advantages. In fact, in the case of dialysis in automation, it has been demonstrated that there is some disadvantage in that the amount of calcium dialyzed from an all water system is lower than that obtained with a proteinaceous system.105’106’119 Since this phenomenon complicates calibration by making protein standards mandatory, it would seem that dialysis could be avoided in favor of simple dilution with appropriate lanthanum solutions perhaps containing some organic solvent or surfactant to aid in accelerating aspiration rates and thereby increasing the signal obtained from the same concentration of calcium in the diluted solution.94’140 Some other aspects of AAS can be described which are also quite interesting.

Robinson points out that the Beer-Lam- bert law cannot be applied directly in AAS because the number of unexcited, absorbing atoms is not known and this is undoubtedly true.139 However, from a practical point of view, one can easily deter


mine the amount of signal generated for a given concentration (solution concentration) of a metal under a given set of analytical conditions. Therefore, one should be able to base an absorptivity calculation on the concentration of calcium in the solution under analysis. If reagent and instrument conditions can be changed later so that more ground state atoms are available to absorb light, the practical “apparent” absorptivity could be reevaluated." By this means, one may very well be able to carry out comparative sensitivity studies and to decide on comparative sensitivity characteristics, a subject which has been somewhat controversial in the past.47’154 This does not attempt to detract from any concept of sensitivity which concerns itself with detectability, the ability to detect a given signal above background noise.

Another area of interest with AAS (and other spectrophotometry) lies in the use of the scale expander and its effect on sensitivity. Somewhat frequently of late one can encounter described analytical conditions in which it is apparently believed that absorbance variation in spectrophotometric measurements are not only dependent on the Beer-Lambert considerations of concentration and light path as a function of wavelength but also as scale expansion. Somehow the “expansion” of absorbance is made possible by a simple turn of the scale expander knob.19-43 This is surprising and unexpected if the scale expander concept misleads to both its meaning as well as its value. Apparently it is believed by some that scale expansion increases the absorbance rather than that it enhances readibil- ity of the scale.19’43 There would be no confusion in terms if sensitivity could be defined in terms of signal generated per unit concentration as just described rather than by magnification of an unchanging absorbance number, since slope steepness in calibration determines procedural sensitivity. Readability or detectability are a

kind of measure of sensitivity, but absorptivity still seems a most reasonable comparative measure for absorption systems even if it can only be determined in some apparent manner.179 Therefore, although some authors suggest that scale expansion is a means of expanding sensitivity,83’182’184 it might be more appropriate if they were to redefine sensitivity with a meaning related to a concentration phenomenon, or else to state that scale expansion is a means of expanding readability, for the latter may have different meanings for different individuals.

Should one then agree to the concept that true sensitivity is an absorptivity function and that practical absorptivity should be based on the calcium concentration in solution, one might also have to agree that AAS using the flame system is not more sensitive for calcium than MAS using a chelating color reagent because sensitivity is therefore defined by the change in absorbance per unit concentration per centimeter of light path (A /C /cm ). If AAS is a reproducible system as it apparently is, then for any given set of analytical conditions, it should have a reproducible absorptivity.58 Even if one allows the unit light path for atomic absorption to be 10 cm and uses those absorbance values for comparison to MAS, AAS for calcium at least as presently published is still at a lower order of sensitivity. In other words, if AAS could be reported as is MAS in terms of molar absorptivity, then some sort of system sensitivity comparison of a practical nature would allow one to determine this relationship to one’s own satisfaction. If one were to achieve an absorbance of 0.1 for 2 meg per ml concentration of calcium using a 10 cm light path in AAS (as the authors do), the molar absorptivity would be 200 liters per mole centimeter. If one were to achieve an absorbance of 0.60 for 1 meg per ml concentration of calcium using a 1 cm light path in MAS (as the authors

REV IEW O F C A LC IU M METHODOLOGIES 2 0 3

also do), the molar absorptivity would be 24,000 liters per mole centimeter. The ratio of sensitivity here would therefore yield a factor of 120 in favor of MAS. But, even if one were to consider that 10 cm in AAS should be the unit light path, then MAS would still be 12 times as sensitive from a practical point of view.

Another problem in understanding sensitivity is that concentration in the measured solution is not always considered. For example, a spectrophotometric reaction of fixed molar absorptivity has an inherent sensitivity related to the reaction. However, it is possible to increase absorbance readings obtained for any given sample size by concentration of the color. This is often described as being a more sensitive procedure or reaction.23 Descriptions for obtaining increased sensitivity by such means are certainly moot for both AAS as well as MAS. For even though true absorptivity may not be easy to determine, it is present, and the theoretical limit of sensitivity for a given system can be based on it. Making a solution more concentrated in absorbing atoms or chromophores is a way of getting more signal without changing inherent sensitivity. Several excellent articles have described the concepts of sensitivity and its relationship to precision in AAS l31>132>133

F l a m e S p e c t r o p h o t o m e t r y

Unlike AAS for calcium, it is difficult to get a flame emission picture in which interference from other cations or from certain anions is not manifested. This has been rather unfortunate because of the ease with which a flame measurement is made.14 The use of an internal standard such as lithium in the emission mode will not solve the problem because the calcium signal is depressed by phosphate whereas the lithium signal is not.161 Phosphate is a negative interference whereas at the same time the same sample contains positively interfering

cations such as sodium. In addition, if one wanted to use an internal standard, it would be necessary for the instrument to be able to measure both metals simultaneously, which is possible for some but not all flame emission systems. However, if simultaneous measurements could be made, it would be worthwhile then to use an internal standard other than lithium which would undergo the same relative enhancements and inhibitions as does calcium. If separation of calcium or removal of the interferences were resorted to, then the ease of measurement would be lost even as the accuracy might be increased. Nevertheless, a number of reports have indicated that calcium in serum can be measured by flame emission by the use of a hot flame such as acetylene-oxygen and a secondary serum standard which would provide an average flame background somewhat similar to that of the measured sample in terms of enhancement or inhibition.

Not much has been done in the clinical laboratory with the use of standard additions in flame photometry. This method can be effective in those circumstances in which the system in which the measurement is made has a relative influence on the constituent being measured. By putting the standard inside the sample, presumably both portions of metal, from standard and from sample, would be similarly influenced. Therefore if the emitted light from the metal of the sample were enhanced or inhibited in a totally relative way, then so would that of the standard added to the sample. Even though the amount of light emitted from both calcium components is different than that of a pure standard, as described, as long as the change is relative, the calculation could be accurate, probably more accurate than if the standard and the sample were determined separately. In a way, the use of a secondary serum sample performs the same function, the difference being that if each sample exerts a different

2 0 4 ZA K, E P ST E IN AND BAGINSKI

influence on the emissivity of calcium, then it would be better practice to put the standard in each sample than it would be to have a separate and single serum standard for all samples.

Several reports have been made on the successful use of the flame for the determination of serum calcium in which different analytical devices for achieving accuracy were described. One of these used total destruction to remove proteins, followed by solubilization to the original serum volume and measurement against standards in radiation buffer solutions which approximated normal serum.84 In another system calcium was determined by correction for the flame background by using a serum standard which approximated the radiation conditions of the sample.71’110 Still another procedure was developed163 in which automatic background subtraction was attained by the use of a specially designed photometer based on Margoshes and Vallee instrumentation108 for eliminating the effect of cation interference. In addition, the incorporation of EDTA into their radiation buffer reagents eliminated anion interference. The overall result was an interference free flame photometric system which automatically subtracted the enhancing background effect of cations such as sodium and potassium and eliminated the inhibiting effect of anions such as phosphate or sulfate.

In spite of all of the work described for calcium determination by flame emission, there is comparatively little of this kind of analysis practiced at the present time in clinical laboratories, even though there is at least one commercial system advertising for this purpose.

A u t o m a t io n

Any discussion of automation for the determination of calcium should be primarily concerned with continuous flow systems86 because discrete sample analyzers are presumably mimicking manual procedures by

carrying out direct serum determinations. Problems could arise for the latter systems if, in automating, some modifications were made which altered the accuracy of the procedure. When calcium was determined on the DuPont ACA using an MTB procedure modified from the description of Gindler,61 the presence of magnesium caused serious interferences even though a complexing agent for magnesium, 8-hy- droxyquionoline sulfate (8-HQ), was included in the procedure. This procedure has recently been “recalled” and replaced by a CPC procedure in which the effect of magnesium appears to have been removed.

All discrete sample analyzers determine calcium directly in raw serum usually by employing either CPC or SAS as the color reagent and 8-HQ to bind magnesium. Jaundice and hemolysis can be tolerated but turbidity owing to lipids creates a light scattering problem in measurement.

Another real problem is encountered in the dialysis phase of continuous flow systems where, owing to variable rates of diffusion for calcium, differences in dialysis rates were obtained because the standard was aqueous and the sample proteinaceous. In such a circumstance, all values obtained were high and this effect was minimized by including a protein, albumin, in the standards. The problem was described as being caused by a Donnan effect.105 Another means of obviating thé problem was to incorporate polyvinyl pyrolidone into the standards, which made both samples and standards dialyze their calcium at the same rate. These investigators claimed that the proteinaceous-aqueous diffusion difference was not due to a Donnan effect at all.4 In the case of the multiphasic continuous flow system, the problem of differing dialysis has always been minimal even though the solution to the problem was inadvertent, because the standard, by necessity, had to be a secondary serum standard with determined values for all constituents measured.

REVIEW OF CA LCIU M METHODOLOGIES 2 0 5

Aside from the use of dialysis, the chemistry of all automation systems are identical with those of manual methods. The advantages of automation are precision, speed and a saving in labor. The newer Auto Analyzers, AA-II for example, can determine calcium at higher rates of analysis than the older systems of AA-1 because of presumably better washout characteristics and a more favorable sample to reagent ratio which approaches steady state characteristics. However, one novel approach to automated acceleration for the old AA-1 system has been described.10 In this circumstance, sampling time was decreased so that 100 samples per hour could be handled instead of 50 samples per hour. The method proposed increasing the size of the sample and all volumes pumped by the reagent lines by a factor such as two. If the sampling time was then halved, then the ratio of sample volume to reagent volume would be the same as at the slower sampling time. Delay time needed for color reaction could be increased by lengthening the delay coils by whatever volume factor could provide the needed time.

F l u o r e s c e n c e

Fluorimetric reagents for the determination of calcium have also seen extensive use.78’87’100’113’175 Most commonly, the indicator has been calcein and both manual and automated procedures have been developed for this determination.2’12’50’51’72’116 The handling of the sample has been virtually identical to that of the colorimetric approaches, so fluorescence offers the same simplicity and a much greater sensitivity. However, even though it is more sensitive, fewer laboratories seem to favor it than do those using colorimetry or AAS. Some factors have been reagent stability, bile pigment and lipid interference, and the problem of adsorption of calcium to glass.112

A bioluminescent compound, aequorin,78 has been suggested for the determination

of calcium in serum, but thus far it has not seen widespread use even though it is a sensitive reactant for calcium.

I o n ic a n d U l t r a f i l t e r a b l e C a l c i u m

Ultrafilterable calcium and ionic calcium are measureable in several kinds of analytical systems. Calcium not bound to protein is of two kinds, either ionic or bound to anions such as phosphate, citrate and carbonate; they are important enough to merit special analytical consideration.6’106’ ii9,i43 Continuous flow automation,144 po- tentiometry using ion specific electrodes,121 ultrafiltration,46 and others64’172 are techniques which have been used to determine ionic or ultrafilterable calcium. The best documented system of the present for the important determination of ionic calcium is undoubtedly potentiometric using a specific ion electrode which is both sensitive and reproducible. Although some effort has been expended in attempting to determine ionic calcium by colorimetric procedures either manual67 or automated,144 they have not yet seen popular acceptance.130 Several excellent presentations on the subject of the modem measurement of ionic calcium have appeared which give excellent coverage to both the methodology and the meaning of its results.95’96

M is c e l l a n e o u s I n s t r u m e n t a l T e c h n iq u e s

There are a variety of other instrumental techniques aside from those previously described which play little or no role in the present clinical laboratory systems for calcium determinations. The reasons may be any one of several such as expense of equipment, skills required, lack of speed or just that they do not provide enough additional accuracy to merit using them in the face of the negative characteristics for the routine laboratory just enumerated. Such systems which are here but still used relatively infrequently include neutron activa


tion analysis,69 isotope dilution,114 coulometric titrations,24 atomic fluorescence,34’ 186 potentiometric titrations with a calcium electrode,102’176 emission spectography,155 polarography,77 X-ray fluorescence,120 chromatography35 and mass spectrometry.188 There are undoubtedly other instrumental techniques or variations aside from these mentioned, but they are of no current consequence for the clinical laboratory.

S c r e e n in g P r o c e d u r e

The inclusion of calcium in screening procedures has been tested and found to be extremely useful.70’162’174 That such procedures for the determination of calcium work well is in accord with the experiences of many automation users. Problems with dialysis are known4’105’106’119 and can be solved in a general way by incorporating protein into the standards, mathematically correcting for the protein effect in an average way or by carrying out direct analysis. It has previously been pointed out that ideal screening procedures should be quite accurate.189 If they were not, there would be a need to repeat a rather large number of questionable results. However, continuous flow screening using an SMA- 12-60 has proved that a system can be provided in which the screen is as good as AAS for both screening and backup modes of analysis.59 Discrete sampling robots with access to the same basic CPC procedures should have similar screening capabilities with the possible exception of the specimen which remains turbid after treatment with reagents.

R e f e r e e P r o c e d u r e

The Clark-Collip modification of the Cramer-Tisdall technique had been accepted as a yardstick procedure prior to the publication of a “referee” system using AAS. Its ability to serve as a referee procedure is based on the assumption that the final reading step measures only calcium,

with a signal which is uninhibited and unenhanced, that is no relative error occurs, and also in which no absolute negative or positive interferences are possible. The validity of this claim is based on a comparison to a primary referee procedure involving an isotope dilution-mass spectrometric method (ID-M S) to determine all values.19’20’114 In addition, the material used to standardize the AAS system had to be of very high purity, and NBS-SRM calcium carbonate (SRM 915) was used in all o f the studies. Serums compared had their calcium content predetermined by the ID-MS method which obviously had to be considered as an absolute measurement entirely without error. Once it could be demonstrated that the blind values obtained by ID-MS were consonant with AAS, then it could be determined that the procedure was accurate. The primary thrust of the description was not really the development of an AAS procedure but rather it was a meticulously detailed description on the preparation of uncontaminated solutions and glassware and their handling in all the steps of the procedure without incurring contamination in the process. Approximately one week was the time suggested in order to perform the method, so it was obviously not meant to be a routine procedure. All of the many rigid specifications of the referee procedure from maintaining purity of water, all reagents and all glassware to the precise operation of a very precisely operating atomic absorption spectrophotometer “performed by workers well acquainted with the techniques of analytical chemistry” had to be established as iron clad principles in order for the “overall accuracy of the method to be achieved.” Obviously, this is an extremely limited operation, not presently available to many if not most of all clinical chemists. It has not been established in the field that this is the very precise procedure it is described to be.19 In fact, there is some

REVIEW O F C A LC IU M METHODOLOGIES 2 0 7

evidence that others have not found this an easy procedure to duplicate.124

F u t u b e o f C a l c i u m M e t h o d o l o g y

It is difficult to predict at this point in time what the future will hold for the determination of a metal such as calcium. There will undoubtedly be physico-chemical systems in which calcium and perhaps several other metals will be measured rapidly, accurately, precisely, and directly in biological fluids by an automatic sample handling system using very small quantities of materials. However, before that comes about, there is no question but what the immediate future will still lie with manual and automated spectrophotometric measurements either with MAS or with AAS procedures. Both seem to fill the present need well and it is difficult to believe that they will not still continue to do so for several years to come.

D irect C reso lph th alein Com plexone P rocedure for Seru m C alc iu m

P r i n c i p l e

Calcium reacts with cresolphthalein complexone in an alkaline dimtethyl sulfoxide solution to form an intensely absorbing chromophoric complex. High blanks are minimized to a certain extent by the lowered dielectric constant characteristics of the final solution mixture which serves to repress the ionization of the color reagent, a primary source of the blank reading.

R e a g e n t s

Cresolphthalein complexone color reagent. Forty mg of the color reagent are weighed into a calcium-free beaker containing 1 ml of concentrated HC1 in which the CPC is dissolved by swirling. The solution is transferred to a 1 L volumetric flask by washing it over with 1 dl of C.P. dimethyl sulfoxide. Eight-hydroxy quinoline (2.5 g) is added with mixing to effect so

lution. This is then diluted to one 1 with calcium free distilled H20 and stored in a polyethylene bottle.

Diethylamine buffer solution. Forty ml of diethylamine are added to an aqueous solution containing 0.5 g of KCN. The mixture is diluted to one 1 with H20 and stored in a polyethylene bottle.

Ethylene bis ( oxyethylenenitrilo) tetraacetic acid ( EG TA ). A distilled H20 solution of 0.5 percent is prepared. Its purpose is for use in preparing blanks for turbid specimens.

Stock standard (1 g of calcium per liter). A stock solution is prepared by dissolving2.5 g of C aC 0 3 in calcium free H20 using a minimum amount of concentrated HC1. The solution is diluted to one 1 with moreh 2o .

Working standards. The stock calcium solution is diluted to prepare 50 to 200 mg per 1 working standards using calcium free water.

P r o c e d u r e

Twenty /¿I of serum are pipeted into a 4 ml Auto Analyzer cup containing 1 ml of CPC solution. One ml of the DEA buffer solution is added and the solution is mixed. Similar volumes of the 100 mg per 1 of standard and a control are carried through the same process along with a blank prepared from the reagents alone. No H20 is necessary to substitute for the serum volume in the reagent blank because the volume change is too small to change the blank absorbance by a measurable amount. Alternatively, the two reagents can be pipeted into each tube before the sample is added. This tube can then be used to zero the instrument. On the addition of sample, the same tube can be used to determine the concentration of the sample. Standards would be prepared in the same manner. Although this is a means of accounting for contamination in the tube in which the reaction takes place, premixing of these rea


gents gives a less stable solution if the reagents are mixed together and allowed to stand for long periods. As described, the reagents seem to work well with no stability problems. Another serious problem using the internal self-blanking system described, is that an aspirating cuvet system cannot be used because two solutions, the blank and the sample (or standard), must be measured independently in the same cuvet. If there were a small contamination in the vessel, it would then be preblanked or self blanked,178 before the addition of the sample or standard to be measured.

C o r r e c t io n f o r T u r b id it y

If the 100:1 ratio of reagent to sample still results in light scatter when handling a noticeably turbid specimen, it is possible to salvage the determination without preparing a protein free filtrate (P F F ) or resorting to total destruction of the sample. Routinely, it is only necessary to add 20 ¡j1 of EGTA solution to samples and the reagent blank and then to redetermine the absorbance of the cuvets against the treated blank. The absorbance remaining after the calcium complex color is thus discharged is the residual turbidity absorbance that needs to be subtracted to correct the total absorbance previously obtained with the turbid sample (obtain all absorbance at 575 nm).

C a l c u l a t io n

The reaction of CPC with calcium in this procedure obeys Beer’s law up to 200 mg per 1. Therefore, calculations can be made routinely by the use of a single standard. Since the reagents are stable enough to yield consistent absorbance values, a calibration curve can be prepared using all of the 50 to 200 mg per 1 standards. The calibration curve is used to ensure linearity for a reagent set, while the single standard and control are used routinely.

D is c u s s io n

A direct reaction for serum calcium was chosen over the alternatives of PFF formation or total destruction for several reasons. Direct reaction lessened the chances of contamination because of fewer reagents and minimal treatment of the sample, differences were not observed which might be caused by any natural contaminants of serum when the direct procedure was compared to a total destruction procedure and dialysis became unnecessary if continuous flow automation was used.10’191 The latter procedure therefore avoided the problem of differing dialysis between standard and sample,105 and made accelerated sampling10’191 and handling by a discrete sample analyzer a more likely probability. The most serious problem encountered using the direct reaction involved the occasional turbid specimen which exhibited light scattering even at high dilution in an alkaline medium. However, the routine solution of this problem as described under procedure was not difficult. Ideally, the turbidity problem should be eliminated without resorting to a correction by a process of elimination of the light scatter by some reagent modification. Such a reagent has been described briefly in which surfactants have been added to both reagents of the present procedure so that turbidity owing to lipemia is not encountered.104 If this modification of the present procedure works, then even this occasionally occurring problem should be eliminated.

The high absorbance of the reagent blank is caused by the ionization of the CPC reagent in alkaline solution.181 This is shown in figure 1 where the variation in color of both CPC and its calcium complex as a function of pH and pCa are depicted.137 In this all aqueous situation, the best choice of pH is between 10.5 to 11.0. However, when dimethylsulffoxide is incor- ported into the reagents, the ionization of the CPC is repressed and the blank absorb

REVIEW O F CA LCIU M M ETHODOLOGIES 2 0 9

ance decreased. This also makes it possible to elevate the pH somewhat in order to achieve a higher absorbance with the calcium complex. Since both the complex and the CPC itself have the same spectrum, and since blank absorbance cannot be totally avoided by the repression phenomenon which may also interfere with complex formation if it is not controlled, the compromise conditions described for final alkalinity and dimethylsulfoxide concentration were chosen.

The procedure described the use of a constant blank whose absorbance is subtracted from the sample and standards. This requires some explanation because of the complexity of the reaction.29 The true blank is regressive and therefore variable and it owes its regressive character to the fact that some of the CPC is removed by reaction with the calcium of the standard or sample. Subtracting the full amount of the reagent blank color causes those specimens which have the most calcium to have the lowest residual blank color remaining. Therefore, the constant blank subtracts the least color from the lowest concentrations, the most from the highest concentrations and thereby causes a relative error in the calibration curve. Since such a relative error results in no change in the linearity of a calibration curve, its only effect is to cause a small decrease in the slope of the curve. This means that the apparent molar absorptivity is slightly lower than the true molar absorptivity, but is without real effect on the calibration characteristics of the system.29

S o u r c e s o f E r r o r

The primary source of error to consider in a microprocedure involving a 20 ¡A size sample containing approximately 2 meg of calcium, a ubiquitous metal, has to be contamination. As little as 0.1 meg of contamination in glassware would cause a positive error of 5 percent. If the sample size was

1 ml and it contained a corresponding 100 meg of calcium, the same error would be an almost unmeasurable 0.1 percent. Certainly, the latter error would have to be considered insignificant.

Some compounds such as EDTA or acting like EDTA may be present to tie up a portion of the serum calcium and make it unavailable for reaction with CPC because of a much more favorable binding constant. In this instance, the described procedure would give a lower value than the true total concentration. However, while the EDTA was present in serum and until the EDTA was spilled, that calcium would not be available for its normal functions. A truer total value for calcium concentration would be obtained by atomic absorption, but both values might be needed if both kinds of information were useful to the diagnostician. Obviously then, anticoagulants such as oxalate or EDTA should not be used although the presence of either would be signaled by the lack of a peak absorbance reading.

The other interferences one should consider are jaundice, hemolysis and turbidity owing to lipids. Jaundice and moderate hemolysis are obviated by wavelength, but turbidity creates a critical condition which must be taken care of by a choice of PFF formation, wet ashing or, preferably, sample blanking using EGTA to eliminate the color of the calcium complex. A recent report of the use of a combination of surfactants and dimethyl sulfoxide to eliminate the turbidity problem is still being evaluated. For the present, it can only be described as a promising approach.

N o r m a l R a n g e s

The normal range obtained with this procedure is 4.20 to 5.25 m Eq per 1 with a mean of 4.75 mEq per I.

R e s u m é o f C l i n ic a l I n t e r p r e t a t io n s

Some conditions which can result in lowered serum calcium values are acute


hyperphosphatemia, hypoparathyroidism, maternal tetany, nephritis, nephrosis, osteomalacia, osteoporosis, neonatal tetany, pancreatitis, pseudohypoparathyroidism, pseudo-pseudohypoparathyroidism, rickets, starvation, steatorrhea and surgical hypoparathyroidism. Conditions which can result in elevated serum values include acute bone atrophy, bone neoplasm ( metastatic), disuse atrophy, hyperparathyroidism (primary and secondary), hyperthyroidism, hypervitaminosis, hypoadrenalism, hypo- phosphatasia, idiopathic hypercalcemia, milk-alkali syndrome, multiple myeloma and sarcoidosis.2’151

R eferences

1. Aikens, D. A., Schmuckler, G., Sawdek, F. S., and Reilley , C. N.: Increased selectivity in chelometric titrations through endpoint location by linear extrapolation. Copper as photometric indicator. Anal. Chem. 33: 1664-1671, 1961.

2. Alexander, R. L. J r .: Evaluation of an automatic calcium titrator. Clin. Chem. 27:1171- 1175, 1971.

3. Alonso, A., T umilasci, O. R., and N ikonov, J. M.: Improvement of a direct colorimetric method for calcium determination. Clin. Chem. Acta 27:549-551, 1971.

4. Amador, E. and Neeley , W. E.: Automated serum calcium analysis corrected for dialysis effects. Amer. J. Clin. Path. 58:707—717,1972.

5. Anderegg, G., F lashka, H., Sallm an , R., and Schwarzenbach, G.: Metallindikitoren VII. Ein auf erdkaliionen ansprechendes Phtalein und seine analytische Verwendung. Helv. Chim. Acta 37:113-120, 1954.

6. Arras, M. J.: Measuring ionized calcium. Postgrad. Med. 45:57-60, 1969.

7. A rv a n , D. A .: Observations on an automated method for determination of serum and urine calcium. Amer. J. Clin. Path. 45:358-360, 1966.

8. Ashton, A. A.: The use of tetracycline as a fluorescent indicator in the compleximetric microdetermination of group II cations. Anal. Chim. Acta 35:543-545, 1966.

9. Auerbach, G. D., Potts, J. T. Jr., and Chase, L. R.: Polypeptide hormones and calcium metabolism. Ann. Intern. Med. 70: 1243-1265, 1969.

10. B aginski, E. S., Marie, S. S., Clark, W. L., Salancy, J. A., and Zak, B .: Accelerated automated microdetermination of serum calcium. Microchem. J. 27:293-301, 1972.

11. B a g i n s k i , E. S., M a r i e , S. S., C l a r k , W. L., a n d Z a k , B.: Direct microdetermination of serum calcium. Clin. Chim. Acta 46:49-54, 1973.

12. B a n d r o w s k i , J. F. a n d B e n s o n , C. L.: Investigation of the use of calcein in the ultramicro fluorimetric determination of calcium. Clin. Chem. 28:1411-1414, 1972.

13. B e c k , G. a n d B e r l i , W.: Nephelometrische Bestimmung von Calcium und Magnesium mit Natriumnapthalhydroxamat. Mikrochim. Acta, pp. 24-29, 1957.

14. B e l k e , J. a n d D i e r k e s m a n n , A.: A flame photometric method for the determination of sodium, potassium and calcium in biological fluids. Arch. f. exper. Path. u. Pharmakol. 205:629-646, 1948.

15. B r e y e r , B. a n d M c P h i l l i p s , J.: The indirect polarographic determination of calcium by chloranilic acid. Analyst 78:666-669, 1953.

16. B r e y e r , B. a n d M c P h i l l i p s, J.: An indirect polarographic determination of calcium. Nature 272:257, 1953.

17. B r o d z i n s k i , R. L., R a n c i t e l l i , L. A., H a l l e r , W. A., a n d D e w e y , L. S.: Calcium, potassium and iron loss by Apollo VII, VIII, IX, X, and XI Astronauts. Aerospace Med. 42:621-626, 1971.

18. B u d e s i n s k y , B.: Xylenol orange and methylthymol blue as chromogenic reagent chelates. Analytical Chemistry; A Collection of Monographs, volume 1. Flaschka, H. A. and Barnard, A. J., eds. New York, Marcel Dekker, Inc., pp. 15-47, 1967.

19. C a l i , J. P., B o w e r s , G. N. Jr., a n d Y o u n g ,D. S.: A referee method for the determination of total calcium in serum. Clin. Chem. 29:1208-1213, 1973.

20. C a l i , J. P., M a n d e l , J., M o o r e , L., a n d Y o u n g , D. S.: Standard Reference Materials: A referee method for the determination of calcium in serum. NBS Special Publication 260-36, U.S. Department of Commerce, 1972.

21. C a n t a r o w , A. a n d T r u m p e r , M.: Calcium and inorganic phosphate metabolism. Clinical Biochemistry, Philadelphia, W. B. Saunders Company, pp. 228-239, 1962.

22. C a t l e d g e , G. a n d B igg s, H. G.: A new indicator for the chelometric measurement of calcium in serum and urine. Clin Chem. 22: 521-526, 1965.

23. C h r i s t i a n , G. D.: Medicine, trace elements, and atomic absorption spectroscopy. Anal. Chem. 42.-24A-40A, 1969.

24. C h r i s t i a n , G. D., K n o b l o c h , E. C., a n d P u r d y , W. C.: Coulometric generation of ethylene glycol bis-(B-aminoethyl ether )- N,N-tetraacetic acid. Titration of calcium in the presence of magnesium. Anal. Chem. 37: 292-294, 1965.

25. C h u t k o w , J. G.: Lability of skeletal muscle magnesium in vivo. A study in red and white muscle. Mayo Clin. Proc. 49:448-454, 1974.

REVIEW O F CA LCIU M M ETHODOLOGIES 2 1 1

26. Clark, E. P. and Collip , J. B.: A study on the Tisdall method for the determination of blood serum calcium with a suggested modification. J. Biol. Chem. 63:461-464, 1925.

27. Clark, G. W.: The microdetermination of calcium in whole blood, plasma and serum by direct precipitation. J. Biol. Chem. 49: 487-517, 1921.

28. Clarke, G. W.: Effect of hypodermic and oral administration of calcium salts on the calcium content of rabbit blood. J. Biol. Chem. 43:89-95, 1920.

29. Clark, W. L., B aginski, E. S., Marie, S. S., and Zak, B.: Spectrophotometric study of a direct determination of serum calcium. Mi- crochem. J. (in press).

30. Close, R. A. and West , T. S.: A new selective metallochromic reagent for the detection and chelatometric determination of calcium. Talanta 5:221-230, 1960.

31. Connerty, H. V. and Briggs, A. R.: Determination of serum calcium by means of sodium alizarin sulfonate. Clin. Chem. 11:716- 728, 1965.

32. Connerty, H. V. and Briggs, A. R.: Determination of serum calcium by means of orthocresolphthalein complexone. Amer. J. Clin. Path. 45:290-296, 1966.

33. Cramer, C. F.: Aspects of intestinal absorption of calcium, phosphorous and magnesium. Methods and progress. Meth. Achievm. Exp. Path. 6:172-192, 1972.

34. D agnall, R. M., Kirkbright, G. F., West , T. S., and Wood, R.: Multichannel atomic fluorescence and flame photometric determination of calcium, copper, magnesium, manganese, potassium and zinc in soil extracts. Anal. Chem. 43:1765-1769, 1971.

35. De, A. K. and Bhattacharyya, C. R.: Combined ion exchange—solvent extraction (CIESE) studies of metal ions on ion exchange papers. Anal. Chem. 44:1686-1688,1972.

36. D ean, J. A. and Rains, T. C.: Flame Emission and Atomic Absorption Spectrometry. New York, Marcel Dekker, Inc., 1971.

37. D eftos, L. J., Murray, T. M., Powell, P., Habener, J. F., Singer, F. R., Mayer, G. P., and Potts, J. T. J r .: Calcium, Parathyroid Hormone and the Calcitonins, Talmadge, R. V. and Munson, P. L., eds., Amsterdam, Ex- cerpta Medica, pp. 140-151, 1972.

38. D ent , C. E.: Some problems of hypoparathyroidism. Brit. Med. J. 3:1419-1425, 1962.

39. D ieh l , H.: Calcein, Calmagite and 0,0'-Di- hydroxyazobenzene. Titrimetric, Colorimetric and Fluorimetric Reagents for Calcium and Magnesium. Columbus, OH, G. Frederick Smith Chemical Company, 1964.

40. D ieh l , H.: Development of metallochromic indicators. Anal. Chem. 39.-30A-43A, 1967.

41. D ieh l , H., Goetz, C. A., and Hach, C. C.: The versanate titration for total hardness. J. Amer. Water Works Assoc. 42:40-48, 1950.

42. D u n n , M. J.: Red blood cell calcium and magnesium: Effects upon sodium and potassium trasport and cellular morphology. Bio- chim. Biophys. Acta 352:97-116, 1974.

43. E h r l i c h , E., C o e l h o , R., a n d G u i l l a i n , F.: A simple automated method for the determination of low concentrations of inorganic phosphate. Anal. Biochem. 50:503-508, 1972.

44. E l l i o t t , J. E. a n d P e a r s o n , P. B.: A direct photoelectric method for the determination of serum calcium. J. Lab. Clin. Med. 32:1262- 1266, 1946.

45. F a l e s , F. N.: Evaluation of procedures for urinary calcium. Clin. Chem. 20:549-558,1964.

46. F a r e s e , G., M a g e r , M., a n d B l a t t , W. F.: A membrane ultrafiltration procedure for determining diffusible calcium in serum. Clin. Chem. 16:226-228, 1970.

47. F a s s e l , V. A.: Measuring trace elements. Science 264:819-820, 1969.

48. F e r r o , P. V. a n d H a m , A. B.: A simple spectrophotometric method for the determination of calcium. Amer. J. Clin. Path. 28: 208-217, 1957.

49. F e r r o , P. V. a n d H a m , A. B.: A simple spectrophotometric method for the determination of calcium. II. A semimicro method with reduced precipitation time. Amer. J. Clin. Path. 28:689-693, 1957.

50. F i n g e r h u t , B. a n d M i l l e r , H.: Direct determination of calcium in icteric serum. Clin. Chem. 9:360-364, 1963.

51. F i n g e r h u t , B., P o o c k , A., a n d M i l l e r , H.: Automated fluorimetric method for the determination of calcium. Clin. Chem. 15:870- 878, 1969.

52. F i s k e, C. H. a n d L o g a n , M. A.: The determination of calcium by alkalimetric titration-II. The precipitation of calcium in the presence of magnesium, phosphate, and sulfate with applications to the analysis of urine. J. Biol. Chem. 93:211-226, 1931.

53. F l a s h k a , H. a n d G a n c h o f f , J.: Photometric titrations-III. The consecutive titration of calcium and magnesium. Talanta 8:720-725, 1961.

54. F l a s h k a , H. a n d Sa w y e r , P.: Photometric titrations-VI. The determination of submicro quantities of calcium and magnesium. Talanta 9:249-263, 1962.

55. F o u r m a n , P., R o y e r , P., L e v e l l , M . J., a n d M o r g a n , D. B.: Calcium Metabolism and the Bone, 2nd ed., Oxford and Edinburgh, Black- well Scientific Publications, 1968.

56. F r i e d m a n , H. S. a n d R u b i n , M. A.: Clinical significance of the magnesium-calcium ratio. Clin. Chem. 2:125-133, 1955.

57. F u n a h a s h i , S., Y a m a d a , S., a n d T a n a k a , M.: A kinetic method of determination of calcium in the presence of magnesium. Anal. Chim. Acta 56:371-376, 1971.

58. F u w a , K. a n d V a l l e e , B. L.: The physical basis of analytical atomic absorption spec


trometry. The pertinence of the Beer-Lam- bert law. Anal. Chem. 35:942-946, 1963.

59. Gambino, S. R. and F onseca, I.: Comparison of serum calcium measurements obtained with the SMA 12/60 and by atomic absorption spectrophotometry. Clin. Chem. 27: 1047-1049, 1971.

60. Gilbert, D. L. and MoGann, J.: Titrimetric analysis of calcium and magnesium in muscle. Proc. Soc. Exp. Biol. Med. 97:791-793,1958.

61. G indler, E. M. and King, J. D.: Rapid colorimetric determination of calcium in biological fluids with methylthymol blue. Amer. J. Clin. Path. 58:376-382, 1972.

62. Gran, F. C.: A colorimetric method for the determination of calcium in blood serum. Acta Physiol. Scand. 49:192-197, 1960.

63. Green , N. M. and N eurath, H.: The effects of divalent cations on trypsin. J. Biol. Chem. 204:379-390, 1953.

64. Gupta, G. S.: Plasma ionic calcium determination. Clin. Biochem. 2:31-39, 1968.

65. Gurney, J.: An evaluation of a micromethod for serum calcium determination. Clin. Biochem. 1:85-87, 1967.

66. Hansen, J. W., Gordon, G. S., and Prussin, S. G.: Direct measurement of osteolysis in man. J. Clin. Invest. 52:304-315, 1973.

67. Harnach, F. and Coolidge, T. B.: Determination of ionized calcium in serum with murexide. Anal Biochem. 6:477-485, 1963.

68. Harris, G. L. A., Cove, D. H., and Crawford, N.: Effect of divalent cations and chelating agents on the ATPase activity of platelet contractile protein, “Thrombosthemin.” Biochem. Med. 11:10-25, 1974.

69. H aven, M. C., H aven, G. T., and D unn, A. L.: Simultaneous determination of calcium, copper, manganese, and magnesium in serum by neutron activation analysis. Anal. Chem. 38:141-143, 1966.

70. H eedman, P. and Stenstrom , G.: Clinical findings in patients with hypercalcemia. Acta Med. Scand. 193:167-173, 1973.

71. H enry, R. J.: Clinical Chemistry, Principles and Technics. New York, Hoeber Medical Division, Harper & Row, pp. 356-378, 1964.

72. H ill , J. B.: Automated fluorimetric method for determination of serum calcium. Clin. Chem. 22:122-130, 1965.

73. H offman , W. S.: The Biochemistry of Clinical Medicine. 3rd ed., Chicago, Year Book Medical Publishers, Inc., pp. 501-503, 1964.

74. H usdan, H. and Rapoport, A.: Estimation of calcium, magnesium, and phosphorus in diet and stool. Clin. Chem. 25:669-679,1969.

75. Husdan, H. and Rapoport, A., and L ocke,S.: Influence of posture an the serum concentration of calcium. Metab. 22:787-797,1973.

76. Ingman, F. and Ringbom , A.: Spectrophoto- metric determination of small amounts of

magnesium and calcium using calmagite. Mi- crochem. J. 20:545-553, 1966.

77. Ir v i n g , E. A. a n d W a t t s , P. S.: Estimation of calcium and magnesium in blood serum by the cathode-ray polarograph. Biochem. J. 79:429-432, 1961.

78. Iz u t s u , K. T. a n d F e l t o n , S. P.: Plasma calcium assay with use of the jellyfish protein, aequorin, as a reagent. Clin. Chem. 28:77- 79, 1972.

79. Ja c k s o n , S. H. a n d B r o w n , F.: Simultaneous determination of calcium and magnesium of serum by a single chelometric titration. Clin. Chem. 20:159-169, 1964.

80. Jo h a n s s o n , A.: Choice of indicators in photometric titrations. Anal. Chim. Acta 62:285- 296, 1972.

81. Jo r d a n , G. W.: Serum calcium and phosphorus abnormalities in leukemia. Amer. J. Med. 42:381-390, 1966.

82. Jo w s e y , J. a n d R i g g s, B. L.: Bone changes in a patient with hypervitaminosis A. J. Clin. Endocr. 28:1833-1835, 1968.

83. K a h n , H. L. a n d Sl a v i n , W.: An atomic absorption spectrophotometer. Appl. Optics 2: 931-936, 1953.

84. K a p u s c i n s k i , V., Moss, N., Z a k , B., a n d B o y l e , A. J.: Quantitative determination of calcium and magnesium in human serum by flame spectrophotometry. Amer. J. Clin. Path. 22:687-691, 1952.

85. K a t z , C. M. a n d T z o g o u r n i s , M.: Chronic adult hypervitaminosis A with hypercalcemia. Metabolism 22:1171-1176, 1972.

86. K e s s l e r , G. a n d W o l f m a n , M.: An automated procedure for simultaneous determination of calcium and phosphorous. Clin. Chem. 10:686-703, 1964.

87. K i n g , J. S. a n d B u c h a n a n , R.: Urinary calcium determination. Three newer methods compared with the Clark-Collip procedure. Clin. Chem. 25:31-34, 1969.

88. K i n g s l e y , G. R. a n d R o b n e t t , B. A.: New dye method for direct determination of calcium. Amer. J. Clin. Path. 27:1-8, 1957.

89. K i n n e y , V. R., T a u x e , W. N., a n d D e a r - i n g , W. H.: Isotopic tracer studies of intestinal calcium absorption. J. Lab. Clin. Med. 66:187-203, 1965.

90. K l e i n , B., K a u f m a n , J. H., a n d M o r g e n - stern, S.: Determination of serum calcium by automated atomic absorption spectroscopy. Clin. Chem. 23:388-396, 1967.

91. K n a p p e , E. a n d B o c k e l , V.: Die komplexo- metrische Bestimmung des Calciumgehaltes von urin. Hoppe-Seyler’s Z. fur. Physiol. Chem. 312:186-192, 1958.

92. K o l t h o f f , I. M.: Textbook of Quantitative Inorganic Analysis. New York, The Macmillan Company, pp. 564-566, 1964.

93. K o p i t o , E., E l i a n , E., a n d S c h w a c h m a n , H.: Sodium, potassium calcium and magnesium in hair from neonates with cystic fibro

REVIEW O F C A LC IU M METHODOLOGIES 2 1 3

sis and in amniotic fluid from mothers of such children. Pediatrics 49:620-624, 1972.

94. Krameb, B. and T isdall, F. F.: A simple technique for the determination of calcium and magnesium in small amounts of serum. J. Biol. Chem. 47:475-481, 1921.

95. L adenson, J. H. and B owers, G. N. Jr.: Free calcium in serum. 1. Determination with the ion-specific electrode, and factors affecting the results. Clin. Chem. 19:565-574,1973.

96. L adenson, J. H. and Bowers, G. N., Jr.: Free calcium in serum II. Rigor of homeostatic control, correlations with total serum calcium, and review of data on patients with disturbed calcium metabolism. Clin. Chem. 19:575-582, 1973.

97. L amkin , E. G. and William s, M. B.: Spec- trophotometric determination of calcium and magnesium in blood serum with arsenazo and EGTA. Anal. Chem. 37:1029-1031, 1965.

98. L awson, D. E. M., F raser, D. R., Kodicek, E., Morris, H. R., and William s, D. H.: Identification of 1,2,5-dihydroxycalciferol, a new kidney hormone controlling calcium metabolism. Nature 230:228-230, 1971.

99. L emonds, A. J. and McC lella n , B. E.: Correlation of enhancement of atomic absorption sensitivity for selected metal ions with physical properties of organic solvents. Anal. Chem. 45:1455-1460, 1973.

100. L ever, M.: Bis-Aroylhydrazones of a-dike- tones as reagents for colorimetric and fluorimetric determinations of calcium, cadmium and other cations. Anal. Chim. Acta 65:311- 318, 1973.

101. L ewis, L. L. and Melnick , L . M.: Determination of calcium and magnesium with (ethylenedinitrilo) tetraacetic acid. Anal. Chem. 32:38-42, 1960.

102. L ichtenstein , I. E., Coppola, E., and Aikens, D. A.: Selective potentiometric titration of calcium with EGTA using silver ion indicator. Anal. Chem. 44:1681—1683, 1972.

103. L indstrom, F. and Milligan, C. W.: Derivatives of glyoxal bis (2-hydroxyanil) as direct calcium reagents. Anal. Chem. 36:1334- 1338, 1964.

104. L o n g , R. L.: A rapid calcium determination utilizing an unique lipid dissolving system. Clin. Chem. 20:908, 1974.

105. L ott, J. A. and Herman, T. S.: Increased Auto Analyzer dialysis of calcium and magnesium in presence of protein. Clin. Chem. 17:614-621, 1971.

106. L ott, J. A.: Determination of total and ionic serum calcium. CRC Crit. Rev. Analyt. Chem. 3:41-63, 1972.

107. Mager, M. and F arese, G.: Direct photometric analysis of serum calcium with glyoxal bis (2-hydroxyanil). Clin. Chem. 12:234- 242, 1966.

108. Margoshes, M. and Va llee , B. L .: Instrumentation and principles of flame spectro

photometry. Automatic background corrector for multichannel flame spectrometer. Anal. Chem. 28:1066-1069, 1956.

109. M a r t e l l , A. E. a n d C a l v i n , M.: Chemistry of the Metal Chelate compounds. New York, Prentice Hall, Inc., 1952.

110. M a r t i n e k , R. G.: Review of methods for determining calcium in biological materials. J. Amer. Med. Technol. 33:416-449, 1971.

111. M c C l e a n , F. A. a n d B u d y , A. M.: Vitamin A, vitamin D, cartilage, bones and teeth. Vitamins and Hormones 2:51-68, 1963.

112. M e i t e s, S.: Standard Methods of Clinical Chemistry, vol. 6. MacDonald, R. P., ed. New York, Academic Press, pp. 207-214,1970.

113. M i l l i g a n , O. W. a n d L i n d s t r o m , F.: Colorimetric determination of calcium using reagents of the glyoxal bis (2-hydroxyanil) class. Anal. Chem. 44:1822-1829, 1972.

114. M o o r e , L. J. a n d M a c h l a n , L. A.: High accuracy determination of calcium in blood serum by isotope dilution mass spectrometry. Anal. Chem. 44:2291-2296, 1972.

115. M o o r e h e a d , R. W. a n d B igg s, H. G.: The use of 2-amino-2-methyl-l-propanol ( A M P ) as the alkalinizing agent in an improved continuous-flow calcium procedure. Clin. Chem. 20:386, 1974.

116. M o s e r , G. B. a n d G e r a r d e , H. W.: Fluori- metric ultramicrodetermination of calcium in biological materials. Clin. Chem. 15:376-380,1969.

117. M o s h e r , R. E., It a n o , M., B o y l e , A. J., M y e r s , G. B., a n d Is er i, L. T.: The quantitative estimation of calcium in human serum by flame spectrophotometry. Amer. J. Clin. Path. 21:75-80, 1951.

118. M u n r o , A. M. a n d B a s s i r, O.: Methods for determining calcium in biological materials. J. Biol. Appl. Chem. 13:20-28, 1970.

119. N a t e l s o n , S. a n d P e n n i a l , R.: Colorimetric estimation of ultramicro quantities of calcium in human serum as the complex with alizarin. Anal. Chem. 27:434-437, 1955.

120. N a t e l s o n , S., R i c h e l s o n , M. R., Sc h e i d , B.-, a n d B e n d e r , S. L.: X-ray spectroscopy in the clinical laboratory. I. Calcium and potassium. Clin. Chem. 5:519-531, 1959.

121. O r e s k e s , I., H i r s c h , C., D o u g l a s , K. S., a n d K u p f e r , S.: Measurement of ionized calcium in human plasma with a calcium selective electrode. Clin. Chim. Acta 21:303-313,1968.

122. P a l m e r , F. J., N e l s o n , J. C., a n d B o e c h u s ,H.: The chloride-phosphate ratio in hypercalcemia. Ann. Int. Med. 80:200-204, 1974.

123. P e r r i n , D. D.: Organic Complexing Reagents: Structure, Behaviour, and Application to Inorganic Analysis. New York, Interscience Publishers, 1964.

124. P i c k u p , J. P., Ja c k s o n , M. J., P r i c e , E. M., a n d B r o w n , S. S.: Assessment of the refer

2 1 4 ZA K , E P ST E IN AND BAGINSKI

ence method for determination of total calcium in serum. Clin. Chem. (in press).

125. Pollard, F. H. and Martin, J. V.: The spectrophotometric determination of alkaline earth metals with murexide, eriochrome black T and with o-cresolphthalein complexone. Analyst 81 .-348-353, 1956.

126. Pribam , R.: Berichte über die Verhandlungen der Königlich Sächsischen Gesellschaft der Wissenschaften zu Leipzig. Math.-phys. Classe 23:279, 1971.

127. Prokopov, T. S.: Spectrophotometric determination of calcium. Mikrochim. Acta pp. 429-434, 1973.

128. Radin, H. and Gramza, A. L.: Differential spectrophotometric determination of calcium. Clin. Chem. 10:704-720, 1964.

129. Raisz, L. G.: Calcium Metabolism—Recent Advances in Disease-a-Month, Chicago, Year Book Publishers, Inc., December 1972.

130. Raman, A.: The calcium fractions of normal serum. Clin. Biochem. 4:141-146, 1971.

131. Ramirez-Munoz, J.: Chemico-analytical selectivity and sensitivity in atomic absorption flame photometry. Microchem. J. 15:253, 270,1970.

132. Ramirez-Munoz, J., Shifren , N., and Ha ll , A.: Quantitative sensitivity in atomic-absorption spectroscopy. Microchem. J. 11:204-215,1966.

133. Ramirez-Munoz, J. and Ulrich , W. F.: Relationship between sensitivity and precision in atomic-absorption flame photometry. Microchem. J. 15:244-252, 1970.

134. Rehwoldt, R. E., Chasen, B. L., and Li, J. B.: 2-Chloro-5-cyano-3,6-dihydroxybenzo- quinone, a new analytical reagent for the spectrophotometric determination of calcium (II). Anal. Chem. 38:1018-1019, 1966.

135. Reynolds, E. S. and L inde, R. E.: Colorimetric determination with murexide of microgram amounts of calcium in tissues. Anal. Biochem. 5:246—256, 1963.

136. R iet , B. V. and Wynn, J. B.: Potentiometrie determinations of calcium, magnesium and complexing agents in water and biological fluids. Anal. Chem. 41:158-162, 1969.

137. R ingbom, A.: Complexation in Analytical Chemistry. New York, Interscience Publishers, pp. 91-93, 1963.

138. Roberts, P. S.: Electrolytes in blood clotting. Hematol. Rev. 2:111-142, 1920.

139. Robinson, J. W.: Atomic Absorption Spectroscopy. New York, Marcel Dekker, Inc.,1966.

140. Robinson, J. W.: Recent advances in atomic absorption spectroscopy. Anal. Chem. 33: 1067-1071, 1961.

141. Robson, J. R. K. and Brooks, G. J.: The distribution of Ca in fingernails from healthy and malnourished children. Clin. Chim. Acta 55:255-257, 1974.

142. Roe, J. H. and Kahn, B. S.: The colorimetric determination of blood calcium. J. Biol. Chem. 81:1-8, 1929.

143. R o s e , G. A.: Determination of the ionized and ultrafilterable calcium of normal human plasma. Clin. Chim. Acta 2:227-236, 1957.

144. R u s h t o n , M. L., Sa m m o n s , H. G., G o s l i n g , P., a n d R o b i n s o n , B. H. B.: Measurement of total, ultrafilterable and ionized serum calcium. Ann. Clin. Biochem. 10:63-71, 1973.

145. Sa d e k , F. S. a n d R e i l l e y , C. N.: Determination of ammonium and potassium ions in mixtures of alkali metals. Use of mercury (Il)-(ethylenedinitrilo) tetraacetic acid. Anal. Chem. 31:494-498, 1959.

146. Sa r k a r , B. C. R. a n d C h a u h a n , U. P. S.: A new method for determining microquantities of calcium in biological materials. Anal. Biochem. 20:155-166, 1967.

147. Sa t o , H. a n d M o m o k i , K.: Successive photometric titration of calcium and magnesium. Anal. Chem. 44:1778-1780, 1972.

148. Sc h a t z m a n n , H. J. a n d R oss i, C. L .: (Ca2+ + Mg2+) -activated membrane ATPases in human red cells and their possible relations to cation transport. Biochim. Biophys. Acta 241: 379-392, 1971.

149. Sc h w a r t z , T. B. a n d H e d g e s , R. N., Jr .: Hypercalcemia and hypocalcemia. Disease- a-Month, Chicago, Year Book Publishers, Inc., December 1960.

150. Sc h w a b z e n b a c h , G.: Complexones and their analytical applications. Analyst 80:713-729, 1955.

151. Se a r c y , R. L.: Diagnostic Biochemistry. New York, McGraw-Hill Book Company, pp. 132- 142, 1969.

152. Se e g e r s , W . H., M c C o y , L., a n d M a r c i n i a k ,E.: Blood clotting enzymology. Three basic reactions. Clin. Chem. 14:97-115, 1968.

153. Se n d r o y , J. Jr.: Photoelectric determination of oxalic acid and calcium and its application to micro- and ultramicroanalysis of serum. J. Biol. Chem. 144:243-258, 1942.

154. Sl a v i n , W.: Atomic Absorption Spectroscopy. New York, Interscience Publishing Co., 1968.

155. Sm i t h , R. G., C r a i g , P., B i r d, E. J., B o y l e , A. J., Iseri, L. T., Ja c o b s o n , S. O., a n d M y e r s , G. B.: Spectrochemical values for sodium, potassium, iron, magnesium, and calcium in normal human plasma. Amer. J. Clin. Path. 20:263-272, 1950.

156. So b e l , A. E. a n d So b e l , B. A.: The determination of calcium in urine. J. Lab. Clin. Med. 26:585-586, 1940.

157. Sp a n d r i o , L.: An improved method for calcium determination in cerum. Clin. Chim. Acta 10:376-377, 1964.

158. Sr e e b n y , L. M., W a n a m a k e r , B., a n d B e c h l e m , D.: The use of eriochrome blueS.E. (mordant blue 13; C.I. 16680) for the determination of calcium in saliva. J. Biochem. 47:764-770, 1960.

159. St e r n , J. a n d L e w i s , W . H. P.: The colorimetric estimation of calcium in serum with o-cresolphthalein complexone. Clin. Chim. Acta 2:576-580, 1957.

REVIEW OF CA LCIU M M ETHODOLOGIES 2 1 5

160. Stoner, R. E., William s, J. B., Connor, T. B., and Brager, S. H.: Inverse relationship between serum calcium concentration and serum lactic dehydrogenase activity. Metabolism 20:464^73, 1971.

161. T eloh, H. A.: Estimation of serum calcium by flame photometry. A.M.A. Arch. Path. 66:474-481, 1958.

162. T hiers, R. E., Bryan, J., and Oglesby, K.: A multichannel continuous-flow analyzer. Clin. Chem. 12:120-143, 1966.

163. T hiers, R. E. a n d H viid.: Interference-free flame photometry of calcium in serum and urine. Clin. Chem. 8:35-45, 1962.

164. T isdall, F. F.: A note on the Kramer-Tisdall method for the determination of calcium in small amounts of serum. J. Biol. Chem. 56: 439-441, 1923.

165. T rinder, P.: A colorimetric micro-determination of calcium in serum. Analyst 85:889- 895, 1960.

166. Tsao, M. U.: Colorimetric determination of serum calcium. J. Biol. Chem. 199:251-257, 1952.

167. T yner, E. H.: Determining small amounts of calcium in plant materials. Anal. Chem. 20: 76-80, 1948.

168. Vaes, G.: Inhibitory actions of calcitonin on resorbing bone explants in culture and on their release of lysozomal hydrolases. J. Dent. Res. 51:362-366, 1972.

169. Vallee , B. L., Stein , E. A., Sumerwell, W. N., and F ischer, E. H.: Metal content of L—amylases of various origins. J. Biol. Chem. 234:2901-2905, 1959.

170. Van E ps, L. W. S., Schouten, H., Slooff, P. A. M., and Van D elden, G. J. A.: Sodium, potassium and calcium in erythrocytes in sickle-cell anemia. Clin. Chim. Acta 33: 475-478, 1971.

171. Van Slyke, D. D. and Carson, P. E.: A simplified technique for determination of small amounts of calcium as oxalate. Clin. Chem. i 0:352-365, 1964.

172. Varghese, Z.: Determination of plasma calcium fractions. Ann. Clin. Biochem. 10:120-124, 1973.

173. Varo, P.: Mineral element balance and coronary heart disease. Intern. J. Vit. Nutr. Res. 44:267-273, 1974.

174. Waldenstrom, J. G.: Systematic serum calcium screening—will it be necessary? Acta Med. Scand. 193:145-146, 1973.

175. Wallach, D. F. H. and Steck, T. L.: Fluorescence techniques in microdetermination of metals in biological materials II. An improved method for direct complexometric titration of calcium in small serum samples. Anal. Biochem. 6:176-180, 1963.

176. Wa llen , B.: Consecutive titration of calcium and magnesium in ethanol-water mixture. Anal. Chem. 46:304-305, 1974.

177. W a s s e r m a n , R. H.: Calcium and phosphorus interactions in nutrition and physiology. Fed. Proc. 19:636-642, 1960.

178. W a t k i n s , R., W e i n e r , L. M., a n d Z a k , B.: Determination of copper, iron, and zinc from a single small sample. Microchem. J. 16:14— 23, 1971.

179. W a t k i n s , R., W e i n e r , L. M., a n d Z a k , B.: Standardization of the ultraviolet spectrophotometric determination of ribonucleic acid. Zeit. Klin. Chim. Klin. Biochim. 10:56-60,1972.

180. W e a t h e r b u r n , M . W., L o g a n , J. E., a n d A l l e n , R. H.: The elevation of calcium values in the automated o-cresolphthalein complexone procedure by high concentrations of magnesium. Clin. Biochem. 2:159-162, 1968.

181. W e l c h e r , F. J.: The Analytical Use of Ethylenediamine Tetraacetic Acid. New York, D. Van Nostrand Company, Inc., 1958.

182. W e s t , T. S.: Atomic analysis in flames. Endeavour 26:44^49, 1967.

183. W e y b r e w , J. A., M a t r o n e , G., a n d B a r l e y ,H. M.: Spectrophotometric determination of serum calcium. Anal. Chem. 20:759-762, 1948.

184. W illis, J. B.: Analysis of biological materials by atomic absorption spectroscopy. Methods of Biochemical Analysis, vol. 11. Glick,D., ed. New York, Interscience Publishers, pp. 1-67, 1963.

185. W i l l s e s, W . C.: A comparative study of the Clark-Collip titrimetric and the colorimetric micromethod of Trinder for the determination of calcium in serum. Amer. J. Med. Technol. 29:121-126, 1963.

186. W i n e f o r d n e r , J. O. a n d V i c k e r s, T. J.: Atomic fluorescence spectrometry as a means of chemical analysis. Anal. Chem. 36:161- 165, 1964.

187. W o t m a n , S., B i g g e r , J. T. Jr ., M a n d e l ,I. D., a n d B a r t e l s t o n e , H. J.: Salivary electrolytes in the detection of digitalis toxicity. New Eng. J. Med. 285:871-876, 1971.

188. Yoe, J. H. a n d K o c h , H. J. Jr.: Trace Analysis. New York, John Wiley & Sons, Inc. 1957.

189. Z a k , B., E p s t e i n , E., a n d B a g i n s k i, E. S.: Review and critique of cholesterol methodology. Ann. Clin. Lab. Sci. 2:101-125, 1972.

190. Z a k , B., H i n d m a n , W . M., a n d B a g i n s k i ,E. S.: Spectrophotometric titration of spinal fluid calcium and magnesium. Anal. Chem. 28:1661-1665, 1956.

191. Z a k , B., Sa l a n c y , J., C l a r k , W . L., M a r i e ,S. S., a n d B a g i n s k i, E. S.: Accelerated un- dialyzed procedure for serum calcium. Advances in Automated Analysis 1:151-157,1973.

Review of Calcium Methodologies** · of bilirubin and hemoglobin in visual titra tions could be...

Documents

Transcript of Review of Calcium Methodologies** · of bilirubin and hemoglobin in visual titra tions could be...