A RAPID METHOD FOR THE ESTIMATION OF CAERULOPLASMIN LEVELS IN HUMAN SERUM , THE ESTABLISHMENT OF THE CLINICAL NORM AND THE STUDIES OF THE CAERULOPLASMIN LEVELS IN VARIOUS CONDITIONS

A rapid method was developed for the estimation of caemloplasmin levels in human subjects and the reliability of the method established by carrying our optimal condition and routine condition variance tests. The caemloplasmin levels in 94 Sri Lankans were investigated and the clinical norm established. Theserum caeruloplasmin levels lie between the range 1.19 2.78/hnoles/l (18-42 mfi) with the mean of 1.98 pnoles/l (29.95 mg%) and the standard deviation of 0.38 ~ o l e s l l (5.72 mg%). The caemloplasmin levels were also investigated in persons suffering . from liver diseases and in pregnant mothers. The values were raised in 9ral hepatitis, and in pregnancy but decreased in Wilson's disease and alcoholic cirrhosis.


Introduction :
Copper is found in serum as metaloproteins.About 96% of the-plasma copper is firmly bound to the a 2globulin and known as caeruloplasmin.Most of the non-caeruloplasmin bound copper, is attached to the semm albumin.It is believed that copper performs catalytic functions in the living organism.Copper influences erythropoiesis and its deficiency impairs absorption and transport of iron and decreases haemoglobin synthesis.
Caemloplasmin is estimated by several methods,192*3*495*6 but the best and the most convenient method is to measure the intensity of the purple coloured complex formed by the oxidation of para phenyl diarnine (PPD) to give quinone types of compounds.This complex has a characteristic absorbance at 525 nm or 530 nrn.Henry e t a15 recorded the optical density at 10 minutes and 40 minutes incubation at 3 7 O ~, after the addition of semm.The difference between the two figures was a measure of the -

*Depafiment of Biochemistry, General Hospital, ~u n c n e ~d a ,
Sn' Lanka.
serum oxidase activity and normal sera produced a different optical density of 0.28 to 0.57 at 530 nm.They used an empirical figure of 5000 to convert , the optical density at 530 nm per minute at 25OC, t o mg caemloplasmin per 100 ml of serum.Conversion factor in this case became 5000 x Tf x 1/30 1 where Tf is the temperature correction.ouch in^ however, recorded the optical density at 525 nm after 1 5 minutes incubation at 3 7 O ~ and reported the activity as difference in optical density or mg of caemloplasmin per 100 ml, by means of the formula 150 x OD525 1.7 where ODS,, is optical density at 525 nm.The conversion t o international units was calculated as follows -mg% x 10 x 1000 = .151,000 is the molecular weight of 151,000 caeruloplasmin.This procedure c a n n o t b e adopted as a routine method in most Sri Lankan laboratories because of the non-availability of visible spectrophotometers.It was decided t o estimate the colour intensity both colorimetrically and spectrophotometrically in order to correlate the findings between the two readings.
In the recent past, there have been very many requests for the estimation of caeruloplasmin levels in patients suffering from Wilson's disease.But no data is available on caeruloplasmin levels in Sri Lankans.This and the non-availability of a rapid method, prompted modification of the existing method using a calorimeter which could then be used in most provincial laboratories.

Reagents
Paraphenylene diamine dihydrochloride powder obtained from BDH Chemicals Ltd, was brown in colour because of its auto-oxidative properties.It was therefore recrystallized using ethanol and water.After recrystalisation it was a colourless compound having a melting point of 140'~.It is necessary to store this compound in an amber coloured bottle in a dessicator to prevent oxidation.
'Sodium mide (a white powder) was also obtained from BDH Chemicals Ltd.
The buffer used for the assay was a glacial acetic acid sodium,acetate buffer of molarity 0.55 M and pH 5.2.(20 ml glacial acetic acid and 98.3g anhydrous sodium acetate dissolved in 1 litre).The pH was checked by a pH meter, PYE UNICAM Model 291 mk 2.
To establish clinical norms, the blood samples from normal donors were obtained from the Blood ~& k , General Hospital, Colombo.All samples were screened by the Blood Bank for haemoglobin, Wasserman reaction, total proteins and for malarial parasites.

Pathological specimens
Samples of blood received by the Biochemistry Department of the Medical Research Institute from several institutions in the country for routine assay for hepatitis, cirrhosis, Wilson's disease and other abnormalities, were used t o estimate the caeruloplasmin levels.Samples of the blood from women in various stages of pregnancy were obtained from the Castle Street Hospital for Women, Borella.

Quality Control Serum
The pooled serum used for the estimation of optimal condition variance (OCV) and routine condition variance (RCV) was from samples of serum received for routine biochemical estimations.This serum was dispensed in 1 ml portions, in sterile bottles and stored at -20°c.

Assay of caeruloplasmin activity
One ml of buffered substrate (0.1% w/v paraphenylene diamine dihydrochloride in acetate buffer, freshly prepared) was pipetted into eachof t y o tubes, test and blank, covered with liquid paraffin, and pre-incubated for 5 min to attain 3 7 O ~, using a water bath.To the test, 0.1 ml of serum was added and incubated for exactly 1 5 minutes more.Then 5 ml of azide solution (0.2% w/v) was added both t o the blank and t o the test t o inhibit the enzymic reaction and 0.1 ml of serum added t o the control and the purple colour was measured using Klett Summerson calorimeter with filter No.54 (green), and Corning -253 spectrophotometer at 525 nm against the blank as zero.The relationship between the colorimetric and spectrometric readings is shown in Figure 1.It was interesting t o note that the colorimetric readings were found to be 500 times that of the spectrometric readings (525 nm).This was so in almost all the readings.Therefore the empirical formula of ouch in^ becomes ( 0 .3 ' ~ colorimetric reading) -1.7 mg of caeruloplasmin per 100 ml of serum.

Quality control
Before the introduction of any method of assay for routine use the WHO has recommended1 ' that optimal condition variance (OCV) should be determined by analysing 20 replicate speciments.The results of such analysis of the OCV, given in Figure 2, show that the variation did not exceed + 2 SD (standard deviation) of the mean.In this case the value of the SD was 0.099 p moles/l (1.49 mg%).OCV was calculated by the equation S D / ~ x 100 where SD is standard deviation and iT is the arithmetic mean.It gave a value of 4.76%.OCV varies from estimation t o estimation and the values of enzymes such as amylase, SGPT and Lactate dehydrogenase are 6, 7 & 8 respectively.'Therefore, the value 4.76% obtained by the present method seems satisfactory.
After OCV was established for the method, the routine condition variance (RCV) was also established over a period of 20 days, using the same sample under identical conditions.During RCV estimation, at least two random serum samples were included in each batch of assay.The RCV, shown in Figure 3, was 8.11% which seems satisfactory.

Clinical Norms
The clinical norm was established on 8 5 males and 9 females between the ages of 20 and 60.The frequency of distribution of the caeruloplasmin levels in Sri Lanka is shown in the form of a histogram in Figure 4.The standard deviation was 0.379 p moles/l (5.72 mg?40) and arithmetic mean was 1.98 p moles,'l (29.95 mg%) of serum.The lowest value was 1.20 p moles/l (18.1 mg%) and the highest value 2.74 p moles/l (41.5 mg%) of serum.The normal range was from 1.19 t o 2.78 p moles/l (18 to 42 mg%) of serum.
Statistical normality of the sampling was established by plotting the results on a normal probability paper, as s h ~w n in Figure 5.The resulting straight line confirmed the sampling as satisfactory and the results as

Gaerulopla-smin Levels '
Upper S c a l e -JJ moles/l Lower S c a l eme statistically acceptable.
The results (Table 1 ) show most values t o be between 1.66 and 2.32 p molesll (25 and 35 meo) which i s 2 that the raised caeruloplasmin levels in pregnancy reflect an adaptive increase in caeruloplasmin to meet the increasing demand for fetal haemoglobin synthesis.The caeruloplasmin levels of the pathological specimens (Table 3) show that in viral hepatitis the caeruloplasmin levels are increased.However, in alcoholic cirrhosis where there is degeneration of the liver cells, the value is low.The increase in the caemloplasmin levels in viral hepatitis and the other liver diseases is probably caused by the leak of the enzyme from liver cells due to necrosis.The present study included two cases of Wilson's disease for caeruloplasmin assay, one being 55% of the lowest limit of normality and the, other 28% (Table 3).
Total protein was determined by the Biuret m e t h ~d , ~ albumin by the bromocresol green binding m e t h ~d , ~ bilirubin by treatment with diazotised sulphanilic acid,9 serum glutamate oxalocetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) by the 2,4 dinitro phenyl hydrazine method1 O and alkaline phosphatase by the antipyrine method.ll

3. Results and Discussion 3 . 1
Correlation between colorimetry and spectrophotometry

Figure 1 . 4 Analytical
Figure 1.Relationship between colorimetric .andspectrophotometric readings Caeruloplasmin was assayed as described under 2.5.The intensity of the colour was measured using Klett Summerson calorimeter with filter No.54 and spectrometric readings recorded on a Corning-253 spectrometer against the blank at zero.

Figure 2 .DateFigure 3 .Figure 4 .
Figure 2. Optimal condition variance for the assay of serum caeruloplasmin Caeruloplasmin was assayed as described under 2.5.Tests were carried out on pooled serum on 20 replicate specimens at the same time under optimal conditions.Colorimetric readings were recorded on a Klett Summerson using filter No. 54.SD = Standard deviation.

Figure 5 .
Figure 5. Normal probability distribution Caeruloplasmin was determined'as described under.2.5.%P loo was calculated where n is the sample size (94) and r is the numbk 1s ascending order of the values.The graph was plotted on a normal probability paper.2r -1 x 100 figures on the vertical axis and cae~loplasmin levels on the X 2 n axls.

Table 1 . Caeruloplasmin pattern in normal Sri Lankans Caeploplasmin p moles 11 mg% No. of cases Males
moles/l (20 to 50 mg%) of serum.The values established in the present study -1.19 to 2.78 p moles/l (18 t o 42 mg%o) of serum -closely resemble the reported values suggesting that ethnic factors have no impact on caeruloplasmin levels in adults.Pathological specimens It is known that in certain diseases and during pregnancy, the caeruloplasmin content varies considerably from normal levels.'Caeruloplasmin levels in pregnancy and in viral hepatitis were found t o be elevated and the present findings are in close agreement with the previously published results.The caeruloplasmin levels of pregnant women, given in Table 2, show values in the range of 2.65 p moles/l (40 mg%) and above.The zirithmetic mean of these cases is 2.93 * 0.349 p moles/l (44.27 ?5.27 mg%) of serum whereas the normal values was shown t o be 1.98 ?0.378 p moles/l (29.95 ?5.72 mg%) of serum.These findings are in agreement with those reported earlier.' The pregnancy cases studied had caeruloplasmin levels above the normal mean + 2 SD 2.74 p moll1 (41.39 mg%).It is suggested