A rapid complexometric method for the estimation of calcium in bone, dentine and enamel

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610

A rapid complexometric

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method for the estimation of calcium

in bone, dentine and enamel In recent years many complexometric biological developed

materials

have been described.

to determine

the Ca content

procedures Almost

for the determination

all of these procedures

of serum, milk,

of Ca in have been

urine and faecesLM5. None

appears to be suitable for a rapid, direct determination of Ca in trichloracetic acid (TC4) extracts of bone, dentine or enamel. The present method fulfils thisrequirement and is an adaptation of the technique described by MOKI~ by which estimations of Cn may be carried out upon TCA extracts of serum, muscle and kidney. The procedure described by 8ffORI,however, is unsuitable for estimations of Ca in TCA extracts of the hard tissues since the precipitation of some of the (13 at the high PH, necessitated by the indicator,

results

in premature

and impermanent

end-points.

This difficulty

been overcome in the present method by the addition of citrate prior to titration. similar procedure was adopted by BXXIU ct al. 7 who determined the Ca content ashed samples of mineralised tissue, but the end-point droxy-r-(2-hydroxy-4-sulpho-I-naphthyla~o)-3~~apht~~oic that

of calcein

(fluorescein-bismcthylene-imino-diacetic

has A in

of their indicator, Calred (2-hyacid) is not as definite as acid),

used in the

present

method.

Most of the reagents described are included here for completeness

below are the same as those used by Mom6 but and clarity.

Reagents Potassium hydroxide : 3 X. Citrate : I g citric acid dissolved in IOO ml water and the solution adjusted to PH 7 by the addition of NaOH. Indicator: 4 mg calcein dissolved in TOO ml 0.25 N KOH.

This solution

will keep for 1-2 weeks if stored in a

refrigerator. C.DTA stock solution: 45 g CDT‘4 (I, z-diaminocyclohexane - N,N,N’,N’tetracetic acid) dissolved and made up to I 1 with water. CDTA working solution: CDT.4 stock solution diluted IOO times. TCA solution: 20 g TCA per IOO ml aqueous solution. Standard Ca solution: 0.1001 g CaCO, dried at IIO', is transferred to a roe-ml graduated flask, dissolved by the addition of 5 ml X HCl and diluted to IOO ml. r ml of this solution contains 20 pequi\F. fn.

Prepavatim

of TCA extracts

Approximately 40 mg dry, defatted bone, dentine or enamel are transferred to large test tubes and extracted overnight by IO ml of 20% TCA solution. After extraction, the sides of the tube are washed down with 5 ml water and the precipitated protein spun down. hliquots of the supernatant (0.5 ml in the case of bone and dentine, and 0.4 ml for enamel) are pipetted into large tubes in which the titrations are carried out.

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3 ml citrate

solution

is pipetted

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011

into each aliquot

of the TCA extract

after mixing, by 2 ml indicator and 5 ml KOH. The solution working solution until the green fluorescence of the indicator viewed against

a black background.

It is important

followed,

is titrated with C,DTA has disappeared when

that similar lighting

conditions

are used for all titrations. Prefiaratioll

of standard

A standard

curve

curve is prepared,

The Ca content

as described

of samples is estimated

by

MORI,

using

o-30

by direct comparison

,uequiv. Ca.

with the standard

curve. Refirodmibility

of the method

To obtain an indication

of reproducibility,

extracts

of bone, dentine

and enamel

were titrated by the above procedure. The results are presented in Table I, which includes a comparison between the values obtained by the present technique and those given by the Clark-Collip method as modified by WEIDMANN AND ROGEKS~ The above determinations were carried out by different operators and at different times. The repeatability of the method is clearly very high. The results obtained agree to within less than 17; with those given by the Clark-Collip method, and values

obtained by the present procedure are more consistent than those given by oxalate ~recipit~~tiol~, presumably because of the extreme simplicity of the procedure. Once a TCA extract is prepared, the determination takes about 5 min. The solution of CDTA recommended by MORI and used in the present work may be satisfactoriiy replaced by a solution

of EDTA

(diamino-ethane-tetra-acetic

acid).

In confirmation of the work of MORI it was found that the presence of Mg had little effect on results. A solution containing equivalent amounts of Mg and Ca gave Ca values only about 1% higher than those obtained in the absence of Me;. The citrate added to the TCA extract prevents the precipitation of Ca ion which otherwise occurs due to the high concentration of phosphate present in the hard tissues.

612

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I would like to express my thanks to Dr. S. M. WEIUMANN

for helpful advice and

criticism and to Mr. B. WRAY for technical assistance. The work was carried out with the assistance of a grant from the Medical Research Council of Great Britain. Biological

Research

LTnit,

J. A. WEATHERELL

School oj Dentistry,

Lrwiversity

of Leeds

(Great Britain)

1 H. D. APPLIXO.\T,.\I. \VEST, M. RIANDEL ANU .-\. M. SALA, Clip. C/w%, 5 (1959) 30 6 I>.N. BARON AND J. I_ BELL, J. CZi+z.Pathol., IL (1959) 143. 3 I'.CARTER ANL) J. CLEMEXT-METRAL, Cli~a.Chinz. .4cta, 4 (1959) 357. 4 I<.CARUBELLI, \V. 0. S~XITH ANL) J, I;.HARIMARSTEN, Clip?.Chewz., 5 (1959) 45. 5 H. 1'.MALMSTADT AND T. I'.HAUJIIOANXOU, Cli~z.Chew., 5 (1959) 50. 6 K. bIOR1, .4vch.Biochem. Biophys., 83 (1959) 552. i I<. L’. I
Received

March rgth,

1960

The mucoproteins

of the eye lens

BELLOWS' has suggested that mucoprotein acts as a cement which holds the individual fibers of the bovine eye lens loosely together. Kr~us~~deduced the presence

of mucoproteins in the bovine lens by precipitating the lens proteins with acetic acid and determining the nitrogen distribution of the various layers of the lens. PERMUTT~ confirmed this by a histochemical method in which the lens polysaccharides were stained. We have examined the mucoproteins in the individual human lens and have tried

to find a correlation

between

the mucoprotein

appearance of its transparency. We have used the polarographic

content

method because

of the lens and the clinical

of its great sensitivity.

Accord-

ing to WINZLER~ and KALOUS~ the mucoproteins which pass into the sulphosalicylic acid filtrate are responsible for the polarographic activity of the filtrate. Although in the case of blood serum other polarographically active agents interfere6, it is possible to determine the differences of the mucoprotein content by this method. The intracapsullary extracted lenses were dried, weighed and homogenized in 3 ml of distilled water; I ml of the homogenate was denatured for 45 min with I ml of 0.1 N NaOH. The sample was then deproteinized with I ml of a zoq, sulphosalicylic acid solution and 0.5 ml of the sulphosalicylic acid filtrate was examined polarographically in I ml of the classical ~~KL)ICKA'~' solution. We examined IO clear lenses (extracted within 3 h post mortem) and 12 cataract lenses (after intracapsular extraction of seuile cataracts). The average height of the polarographic wave under experimental conditions was 81 3: 13 mm (calculated for a weight of I g of the normal lens) while the average height of the wave for cataract lenses was 127 & zq mm/g. The difference is significant even for P