International Journal of Innovative Pharmaceutical ... · PDF filereferenced to Standard preparations containing highly ... foam is present after re-suspension of the magnetic particles,

REVIEW ARTICLE P.Vaishak Nair et.al / IJIPSR / 2 (8), 2014, 1884-1895

Department of Biotechnology ISSN (online) 2347-2154

Available online: www.ijipsr.com August Issue 1884

STATISTICAL ANALYSIS OF RELATIONSHIP BETWEEN BONE

SPECIFIC ALKALINE PHOSPHATASE, TOTAL SERUM

ALKALINE PHOSPHATASE AND VITAMIN D IN HUMAN SERUM

1P.Vaishak Nair*,

2Dr S.F Maleeka Begum,

3Dr Jose Jacob

1,2Department of Biotechnology, Sri Krishna Arts & Science College, Coimbatore, INDIA

3Department of Biochemistry, Amala Institute of Medical Sciences, Thrissur, INDIA

Corresponding Author:

P.Vaishak Nair

Department of Biotechnology,

Sri Krishna Arts and Science College, Kuniamuthur,

Coimbatore, Tamilnadu, INDIA

Email: [email protected]

Phone: +91 8608899787, +91 9656916679

International Journal of Innovative

Pharmaceutical Sciences and Research www.ijipsr.com

Abstract

Biostatistical analysis of serum variables such as Bone Specific Alkaline Phosphatase, Total Serum

Alkaline Phosphatase and Vitamin D was performed with the aim of determining their intra

relationship in human serum. Various techniques were adopted for the analysis procedure such as

Extraction, Segregation, Estimation & Mathematical Computation. Bone Specific Alkaline

Phosphatase and Total Serum Alkaline Phosphatase were extracted and estimated by using Clinical

Immunometric (Immunocapture) & Dry Chemical methods, where as Vitamin D was subjected for

isolation & estimation by using competitive chemiluminescent immunoassay (CLIA) technology.

Further various biostatistical tools (t-Test, Correlation Analysis, ANOVA and Normal Distribution

Methods) were exploited for detailed analytical calculations.

Key words: Bone Specific Alkaline Phosphatase, Total Serum Alkaline Phosphatase, Vitamin D, Clinical Immunometry, Dry Chemistry, Competitive Chemiluminescent Immunoassay (CLIA) , t-Test,

Correlation Analysis, ANOVA, Normal Distribution.




INTRODUCTION

Normal human blood has two prime components- 1.Plasma 2.Serum. In that serum holds a

number of factors such as Enzymes, Antigens, Antibodies, and Hormones. Hence it is known as

the biochemical centre of blood. Among the constituent variables of serum, Bone Specific

Alkaline Phosphatase, Total Serum Alkaline Phosphatase and Vitamin D plays an important role

in bone formation and metabolism. Any infinitesimal deviation from the normal count of these

variables may lead to deformation of bones, which may further lead to various bone associated

disorders. Vitamin D helps to maintain the Calcium and Phosphate in the serum by its action on

intestine, bone, kidney and parathyroid. In the small intestine, Vitamin D stimulates Calcium

absorption, primarily in the duodenum and phosphate absorption by jejunum and ileum. At high

concentration, Vitamin D increases bone resorption by inducing monocytic stem cells in the bone

marrow to differentiate into osteoclasts and by stimulating osteoblasts. Determination of Vitamin

D is useful in the differential diagnosis of hypercalcimia, hypocalcemia or hypercalciuria and for

evaluating Vitamin D status in health and in bone and mineral disorders. (Hanson DA,1992 (1),

Price PA,1987 (2),& Robins SP,1995 (3) ).

Likewise the bone isoenzyme is the marker of bone formation and is found in osteoblast. Bone

alkaline phosphatase is increased in osteoporosis, osteomalacia and rickets, hyperparathyroidism,

renal osteodystrophy and thyrotoxicosis, acromegaly, bone metastasis, glucocorticoid excess,

Paget’s disease and other disorders increased bone formation. Total alkaline phosphatase and

bone alkaline phosphatase provides the highest chemical sensitivity and specificity in diagnosis

and monitoring of Paget’s disease. Although total alkaline phosphatase is most often used, bone

alkaline phosphatase is more sensitive than total alkaline phosphatase in mild Paget’s disease.

Osteocalcin is relatively insensitive and less useful than bone alkaline phosphatase in Paget’s

disease. In severe osteomalacia bone alkaline phosphatase may be markedly increased without an

increase in bone mineralization because of a mineralizing defect. Measurement of bone alkaline

phosphatase has several advantages over osteocalcin measurement. Because of its relatively half

life in vivo (1 to 3 days), it is relatively unaffected by diurnal variation. Bone alkaline

phosphatase is more useful in individuals with impaired renal function because it is not screened

by glomerular filtration. (Jones G 2010 (4) ).




MATERIALS & METHODOLOGIES

Materials Required

Preparation of Reagent Integrals

The preparation of reagent integral includes two steps:

Re Suspension of Magnetic Particles

Magnetic particles must be completely re-suspended before the integral is placed on the

instrument. The following steps are followed to ensure complete suspension:

Before the seal is removed, rotate the small wheel at the magnetic particle compartment

until the color of the suspension has changed to brown. Gentle and careful side to side

mixing may assist in the suspension of the magnetic particles (avoid foam

formation).Visually check the bottom of the magnetic particles vial to confirm that all

settled magnetic particles have re-suspended.

Repeat as necessary until the magnetic particles are completely re-suspended.

Magnetic Particles (24 ml)

Magnetic particles coated with antibody against

25 OH Vitamin D, protein, phosphate buffer,

< 0.1% Sodium Azide.

Assay Buffer (28.0 ml)

Buffer with 10% ethanol, surfactant and 0.1%

ProCin 300.

Conjugate (4.5 ml)

25 OH Vitamin D conjugated to an isoluminol

derivative, in phosphate buffer with 10% ethanol,

EDTA, Surfactant and Preservatives.

Calibrator 1 (1.0 ml)

Human Serum, Tris buffer,<0.1% Sodium azide and 25

OH Vitamin D. The calibrator concentrations (ng/ml) are

referenced to Standard preparations containing highly

purified 25 OH Vitamin D.

Calibrator 2 (1.0 ml)

Human Serum, Tris buffer,<0.1% Sodium Azide

and 25 OH Vitamin D. The calibrator concentrations

(ng/ml) are referenced to standard preparations

containing highly purified 25 OH Vitamin D.




Foaming Of Reagent

In order to ensure optimal performance of the integral, foaming of reagent should be

avoided.Adhere to the recommendation below to prevent this occurrence:

Visually inspect the reagent, calibrators in particular (position two and three following the

magnetic particles vial) to ensure there is no foaming present before using the integral. If the

foam is present after re-suspension of the magnetic particles, place the integral on the instrument

and allow the foam to dissipate. The integral is ready to use once the foam has dissipated and the

integral has remained onboard and mixing for a minimum of 30 minutes.

Estimation of Bone Specific Alkaline Phosphatase Level in Serum Sample

Bone Specific Alkaline Phosphatase is estimated by enzyme immunoassay for determination of

mass of the bone specific isoenzyme. The enzyme assay is done by the immunometric method

(or immunocapture method) in a Beckman Coulter immunochemistry analyzer.Magnetic beads

are coated with anti bone specific alkaline phosphatase. The beads are incubated with serum

sample to capture the bone specific alkaline phosphatase or parathyroid hormone. The captured

ostase bind the second antibody tagged with alkaline phosphatase. The bound second antibody

was assayed using chemiluminescent substrate, ProClin 300.

Estimation of Total Serum Alkaline Phosphatase Level in Serum Sample

Total serum alkaline phosphatase is estimating using the dry chemistry method. A drop of

patients sample is deposited on the slide and is evenly distributed by the spreading layer to the

underlying layers. The spreading layer contains the p-nitro phenyl phosphate substrate and other

components needed for the reaction. The ALKP in the sample catalyzes the hydrolysis of the p-

nitro phenyl phosphate to p-nitro phenol at alkaline pHs. The p-nitro phenol diffuses in to the

underlying layer, and it is monitored by reflectance spectrophotometery. The rate of change in

reflection density is converted to enzyme activity.

Reaction Sequence

Alkaline Phosphatase

p-nitro phenyl phosphate p-nitro phenol + H₃PO₄

Mg², AMP




The method is performed using the VITROS ALKP Slides, using the VITROS Chemistry

Calibrator Kit on VITROS Chemistry autoanalyser 5.1. The VITROS ALKP Slide is a multi

layered, analytical element coated on a polyester support.

Vitamin D

25 OH Vitamin D TOTAL Assay uses chemiluminescent immunoassay (CLIA) technology for

the quantitative determination of 25-hydroxyvitamin D and other hydroxylated vitamin D

metabolites in human serum. EDTA (Ethylene Diamine Tetra Acetic Acid)-plasma or lithium

heparin plasma to be used in the assessment of Vitamin D sufficiency.Assay results should be

used in the conjunction with other clinical or laboratorial data to assist the clinician in making

individual patient management decisions in an adult population.

Vitamin D assay is direct competitive Chemiluminescence Immunoassay (CLIA) for

quantitative determination of total 25 OH Vitamin D in serum or plasma. During the first

incubation,25 OH Vitamin D is dissociated from its binding proteins and binds to the specific

antibody on the solid phase. After 10 minutes the tracer (Vitamin D linked to an isoluminol

derivative) is added .After a second 10 minutes incubation the unbound material is removed

with a wash cycle. Subsequently the starter reagents are added to initiate a flash

chemiluminescent reaction. The light signal is measured by a photomultiplier as relative light

units (RLU) and is inversely proportional to the concentration of 25 OH Vitamin D present in

calibrators, controls or samples.

Student t-test Method

The t distribution is mainly based on the degree of freedom of a distribution i.e. n-1.The Degree

Of Freedom (DF) of a distribution is defined as the number of variants that can be entered in that

distribution before the values of the reminder of the variants are fixed by the necessity to produce

a certain total. The t distribution takes into account the increased space under the sampling

distribution curve with the decrease in the DF. The t-distribution can be used for the same

purpose for which it is applied for the Z values, such as the estimation of population mean in

terms of confidence interval, and hypothesis testing comparison of sample statistic with

population parameter and of two samples means. In addition-testing of matched pair data

analysis is also a useful application in experimental biology (D.G.Altman,Practical Statistics For

Medical Research.1991)




The Equation for t-testing method of data:

Where,

Pooled Variance

n = No Of Observations

= Standard Deviation of Variable 1 & 2

SE of difference between means is calculated as:

Calculation of t-value as:

Table t at specified LS and degrees of freedom (one or two tailed test)

Correlation Estimation Methods

A. Karl Pearson’s Correlation Coefficient Method

Coefficient of Correlation (r) is quantitative measure of the correlation between two variables.

The correlation coefficient is calculated on the basis of the following assumptions about the two

variables,(D.G.Altman. Practical Statistics For Medical Research.1991 & N.Gurumani.

Introduction To Biostatistics,2005)

1. The correlation between the two variables is linear.

2. The two variables are related to each other in a cause and effect relationship.

3. The values of the two variables are affected by factors that are common to both the variables.

The equation for getting Karl Pearson’s coefficient (r) is:

Coefficient of Correlation, X = Variable X, Y = Variable Y n = Number of

Pairs of Variables

Interpretation of Karl Pearson’s Coefficient Of Correlation

The value of r always lies between +1 and -1 when,

1. r = +1,the correlation between the two variables is perfect positive.

2. r = -1,the correlation is perfect negative.

3. r = a positive value lying between 0 and +1,the correlation is positive.

4. r = a negative value lying between -1 and 0,the correlation is negative.

5. r = 0,there is no linear correlation between the two given variables.




B. Testing of Significance of Correlation

Whether the observed coefficient of correlation (r) is significant or not can be tested using

a sampling distribution of sample r s, if it is a large sample (n>30) and t-distribution, if it is a

small sample (n<30).If it is a large sample, the SE of r is computed as:

, n=number of pairs of values.

(For smaller samples)

ANOVA (Analysis Of Variance)

Suppose one should compare more than two groups (k groups) with n1, n2…nk samples and x1,

x2… xk means (1, 2). These k groups might be experimental groups with different “treatments”

or might be samples from populations which are different in some specific feature. Whatever be

their nature, they are always assumed to have come from different populations about which

inference are to be drawn. The observed differences between these groups will consist of two

components. (N.Gurumani. Introduction To Biostatistics,2005)

1. A natural variation (error)

2. Variation due to treatment or any other factor.

The procedures of separation of the total variance into within variance and between variance

involve computation with which we are very familiar, such as the calculations of arithmetic

mean and variance. The calculations involves Sum of Sqs (SS) values viz., SS total, SS between,

SS within : Generally Sum of Squares (SS) =

Let a,b.c,…….,k be the k groups to be compared among themselves n1,n2,n3,…………..,nk

samples in each groups and N, the grand total of all the samples. Let the means of the k groups

be X1, X2, X3… Xk and X be the grand mean, i.e. combined mean of all the groups. Now if X is

the variants in all the groups.

SS total =

SS between =

=

SS within =

= , where i is the variate of one of the k groups.




The above computations can be simplified as follows

SS total =

SS between =

SS within = SS total-SS between

Source Of

Variation

Degree

Of Freedom (DF)

Sum Of Sq

(SS)

Mean Of

Squares (MS) F Ratio

Between k-1 SS between SS between/(k-1)=

MS between

F=

MS between/MS

within

Within

(error) N-k SS within

SS within/(N-K)=

MS within --

Total N-1 SS total -- --

RESULTS

Two sets of data may be compared by student t-testing & correlation method. Total Serum

Alkaline Phosphatase & Bone Specific Alkaline Phosphatase are grouped into high and low

groups and the level of other parameters was compared. There was a significant relation between

high and low Total Serum Alkaline Phosphatase with high & low Bone Specific Alkaline

Phosphatase.Karl Pearson’s Correlation Coefficient had, r=0.5 and the two values were found to

be significantly correlating. X-Y Scatter Diagram also demonstrated a positive correlation.

ANOVA (Analysis Of Variance) showed that groups owned by high & low Vitamin D and high

& low Total Serum Alkaline Phosphatase were found to be forming Bone Sp. Alkaline

Phosphatase groups which were significantly different as the f –ratio of MS between was 4.4

time than MS within. Various statistical methods were used to analyze serum parameters such as

Total Serum Alkaline Phosphatase, Bone Specific Alkaline Phosphatase & Vitamin D. The

distribution of Total Serum Alkaline Phosphatase & Bone Specific Alkaline Phosphatase showed

a positive skewness. Various methods of transformation were incorporated into the distribution

of these parameters. The major transformations used were logarithmic transformation,

reciprocative transformation, square rooting transformation & natural logarithmic

transformation.




Post t-Test Graph

Post Correlation & ANOVA Graph

Tabulated ANOVA Results

Source Of

Variation

Degree Of

Freedom

(D.F)

Sum of Squares

(SS)

Mean of

Squares (M.S) F Ratio

Between 3 860.7308 286.9103 4.393397

Within

(error) 75 4897.866 65.30489

--

Total 78 5758.597 --

--




Normal Distribution (N.D) Graphs

DISCUSSION

Two sets of related data may be compared by student t-test and correlation. The data was

grouped into two groups of low and high Total Serum Alkaline Phosphatase. In these two groups

mean & standard deviation of Bone Specific Alkaline Phosphatase was compared (Stumpf ,1981

(5) ). .It was found that the level of Bone Specific Alkaline Phosphatase was significantly

different in these two groups. At 0.05 Level Of Significance (L.O.S), the calculated t value was

3.064 and the critical value from table is 2.0. Similarly Bone Specific Alkaline Phosphatase was

grouped into two groups of high and low levels. The mean & standard deviation was calculated

(Gomez B Jr, Ardakani,1995 (6) ). The calculated t value at 0.05 Level Of Significance (L.O.S)

was 4.25 which was higher than the critical value from table. The relationship between two sets

of values is compared by correlation method. The relation is expressed as Karl Pearson’s

Correlation Coefficient, r = 0.58. The maximum possible value of r is 1.0.The calculated Z value

(Z=6.3) was very high & therefore relationship between 2 groups was significant. X-Y Scatter




Diagram demonstrated the positive correlation. Analysis Of Variance (ANOVA) is done to

compare more than two groups. Four groups with high & low Vitamin-D and Total Serum

Alkaline Phosphatase were constructed and Bone Specific Alkaline Phosphatase values were

estimated in these groups. The MS between was found to be more than 4.4 times the MS within

of 6.33.The F ratio being higher than 1, four groups of Total Serum Alkaline Phosphatase was

significantly different. Total Serum Alkaline Phosphatase estimated values (n=154) where

distributed in Class Interval of 20 Units. The distribution was found to be positively skewed.

Similarly, the distribution of Bone Specific Alkaline Phosphatase was studied and was also

found to be positively skewed. Transformation of data was performed to bring the distribution to

normal. A positively skewed sample usually becomes normal after logarithmic transformation.

But here log transformation converted the positively skewed distribution of Total Serum

Alkaline Phosphatase to negatively skewed one. Another method of transformation and

distribution to normal is by reciprocating data. When this is done for Total Serum Alkaline

Phosphatase, the sample was found to be distributed less positively skewed. The third method of

transformation was with natural logarithmic transformation .This method transformed the data to

normal distribution (D.G.Altman,1991 (7) & N.Gurumani,2005 (8) ).

CONCLUSION

A highly skewed data became less positively skewed by reciprocated transformation, normal by

natural log transformation & negatively skewed by log transformation. These method shows the

extend of transformation that can be done. The studies suggest that the level of normal serum

alkaline phosphatase and vitamin D are significantly related to bone specific alkaline

phosphatase in normal human being.

ACKNOWLEDGEMENT

It’s my privilege to thank the Management & Principal, Dr K. Sundararaman of Sri Krishna

Arts & Science College, Coimbatore for the inspiration provided to undergo this research

project to get exposed into a practical world.

I thank Dr S.F. Maleeka Begum, Head of Department, Department Of Biotechnology, Sri

Krishna Arts & Science College, Coimbatore for her valuable suggestion in scrutinization of

report to make necessary rectifications.




Special thanks to Dr. Jose Jacob MD, Head of Department, Department Of Biochemistry,

Amala Institute of Medical Sciences, Thrissur for his indespensible contribution in

computation & detailed analysis of serum variables which forms the basis of this project.

REFFERENCES

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basic and clinicalaspects, Cohn DV ed, Elsevier Science, Amsterdam;1987, p 419-25.

3. Robins SP. Collagen crosslinks in metabolic bone disease. Acta Orthop Scand Suppl.

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Documents

International Journal of Innovative Pharmaceutical ... · PDF filereferenced to Standard preparations containing highly ... foam is present after re-suspension of the magnetic particles,