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Original Communication
Association of alkaline phosphatase phenotypes with
arthritides
Padmini. A*, Ushasree. B*, Ravi Babu**, Pratibha. N*
*Department of Genetics, Osmania University, Hyderabad. **AVVP Hospital, King Kothi, Hyderabad. India.

Arthritides, a symmetrical polyarticular disease of the bone

are a heterogenous group of disorders in which hereditary
and environmental factors in combination with an altered
immune response appear to play a causative and
pathogenic role in its occurrence. Alkaline phosphatase
(ALP) is an enzyme found in all tissues, with particularly
high concentrations of ALP observed in the liver, bile ducts,
placenta, and bone.Alkaline phosphatase is an
orthophosphoric monoester phosphohydrolase catalyzing
the hydrolysis of organic esters at alkaline pH, indicating
that alkaline phosphatase is involved in fundamental
biological processes.1 The present study envisages on
identifying the specific electromorphic association of
alkaline phosphatase with arthritides. Phenotyping of serum
samples was carried out by PAGE (Polyacrylamide gel
electrophoresis) following Davies (1964)2 protocol on 41
juvenile arthritis, 150 rheumatoid arthritis and 100 osteo
arthritis apart from, 25 normal children and 100 adult
healthy subjects. Phenotyping of alkaline phosphatase
revealed an increase in preponderance of p+ and p++
phenotypes in juvenile, rheumatoid and osteo arthritic
patients. However a significant association of these
phenotypes was observed only with rheumatoid arthritis
condition (c2:17.46). Similarly, a significant increase of p+
phenotypes in female rheumatoid arthritis patients was
observed (c2:14.973), suggesting that the decrease in p
(tissue non specific) synthesis/secretion of alkaline
phosphatase could be associated with decreased
mineralization and ossification process in arthritis condition.
Key Words: Alkaline phosphatase, Juvenile arthritis,
Rheumatoid arthritis, Osteo arthritis, phenotypes

Introduction

Arthritides categorised as juvenile, rheumatoid

arthritis and osteo arthritis, are a heterogeneous group

of disorders, predominantly affecting the joints and

causes inflammation of the synovial membrane leading
to erosion & destruction of the joint cartilage. Heredity
as well as environmental factors, in combination with
humoral response may play a causative and pathogenic
role in the aetiology of arthritides, with the common mode
of inheritance reported to be multifactorial in nature.
Juvenile ar thritis is a condition that causes
inflammation in one or more joints and begins before
the age of 16. Rheumatoid arthritis, is an inflammatory
disease in which the immune system attacks the tissues,
particularly the joints & synovium, resulting in swelling,
stiffness & loss of function in the joints and the age at
onset was observed to be usually between 20-40 years.3
Osteo arthritis is the most common form of arthritis in
which a tilt in normal balance between cartilage
synthesis and degradation is observed 4 with the
incidence found to be increased between 40-70 years
of age.
Although alkaline phosphatase is found in many
tissues, the highest concentrations are found in the liver,
biliary tract epithelium, and bone. Alkaline phosphatase
is a nonspecific phosphomonoesterase that hydrolyze
phosphate monoesters and is found attached to the
plasma membranes where extensive transport takes
place, indicating that alkaline phosphatase is involved
in fundamental biological processes.1 Since it is found
localized in the plasma membrane of the osteoblastic
cells, its role in bone mineralization is justified.5 The
tissue specific alkaline phosphatase gene is found to

Address for correspondence: Dr. Pratibha Nallari, Associate Professor, Department of Genetics, College of Science, Osmania University,
Jamai Osmania Post office, Hyderabad - 500 007, India. E-mail: prathinallari@yahoo.com
Indian Journal of Human Genetics January-June 2004 Volume 10 Issue 1

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Association of alkaline phosphatase phenotypes with Arthritides

be located on chromosome 2q34-37 while the tissue

non-specific gene is assigned to 1p34-36.1
chromosomal region.6
Three distinct isozymes p, p+ and p++ of alkaline
phosphatase encoded by three structural genes is
reported with characteristic type of several tissues
involved in its secretion like the bone/kidney, intestinal
and placental types respectively.
Since no information with regards to alkaline
phosphatase and its electromorph association with
arthritis is available, such a study is imperative which
may help in risk prediction and in delineation of genetic
heterogeneity of the condition.
Therefore, the present study aims at identifying
specific electromor ph association of alkaline
phosphatase in the etiology of Arthritides.

Materials and Methods

The present study includes arthritides patients

referred to out patient unit of orthopedic department at
Andhra Vaidya Vidhan Parishad, King Koti, Hyderabad.
Cases of juvenile arthritis (JA), rheumatoid arthritis (RA)
and osteo arthritis (OA) referred for the first time and
confirmed radiologically based on small and large joint
involvement were considered. For comparative
purposes, juvenile and adult healthy individuals matched
for age and sex were considered from voluntary blood
donors of various blood camps organized by Lions Club
and dental checkup camps by schools in and around
Hyderabad city.
Blood samples from 41 juvenile, 150 rheumatoid, 100
osteo arthritic patients, 25 juvenile children and 125
healthy individuals matched for age and sex were
collected for the serum analysis of alkaline phosphatase.
5ml of venous blood was collected from each patient/
individual in sterilized vials, for obtaining serum samples.
The samples were centrifuged at 2000rpm for about
10minutes and the clear supernatant serum from clotted
blood was collected in eppendorff tubes and were
analyzed for alkaline phosphatase phenotypes.
A 7%, vertical slab PAGE gel was prepared in the
ratio of 1:1:2 wherein 1 volume of each contained
solutions of Tris-borate (hydroxy methyl) methylamine

and Acryl amide/ bis acryl amide solution, and two

volumes contained Ammonium per sulfate following
Smithies et al7 protocol. Later the unit was connected to
the anodic and cathodic ends of the DC power supply,
with the tank buffer being Tris borate (pH 9.5) diluted 1:
4 folds
30ml of the serum sample was mixed with a drop
of bromophenol blue indicator and layered in each
sample slot/well. The electrophoresis was carried at
a constant current of 20mA for 1hour at 4oC until the
indicator reached the other end of the gel. Later the
gel was stained with a staining solution containing 2mg
b naphtyl disodium phosphate and 1mg fast blue (BB)
salt dissolved in 1ml location buffer of boric acid and
magnesium chloride (pH 9.7). The gel was kept in the
incubator maintained at 37 o C until pink bands
appeared and based on their eletrophoretic mobility
and intensity of staining the phenotypes of alkaline
phosphatase were identified as p, p+ and p++
respectively.
The phenotypic frequencies were computed and 2
test of analysis (2x2 contingency), Woolfs test of relative
association was carried out to interpret the results
statistically.8
Results

Table 1 presents the frequency distribution of alkaline

phosphatase phenotypes in the control and disease
groups. The difference of these phenotypes between
juvenile arthritis and juvenile controls was statistically
insignificant (2 -3.509). A significant difference was noted
in the of alkaline phosphatase phenotypes in rheumatoid
arthritis condition (2 _20.650; p<0.001) as compared to
controls. Whereas no significant variation was observed
Table 1: Frequency distribution of alkaline phosphatase
phenotypes in control and disease groups
Condition

pO

p+

p++

Total

14

56.0

24.0

20.0

25

Juvenile Arthritis 20

48.8 17

41.5

9.8

41

Control

89

71.2 30

24.0

4.8 125

Rheumatoid

69

46.0 54

36.0 27

18.0 150

65

65.0 27

27.0

Juvenile Control

2
value
3.509
20.650**

Arthritis
Osteo Arthritis
**p<0.01

8.0 100

1.424

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N. Pratibha, et al

Discussion

between osteoarthritis and controls (2 _1.424).

To test for an association, relative risk estimates of
alkaline phosphatase in control and juvenile arthritis
group is computed and presented in table 2. The relative
risk estimates were not found to vary with juvenile
arthritis condition.
The relative risk estimate of alkaline phosphatase
phenotypes in rheumatoid arthritis in comparison to
control group is presented in table 3. A significant
association of alkaline phosphatase phenotypes was
observed only with rheumatoid arthritis condition. The
relative risk estimate of alkaline phosphatase
phenotypes in osteo arthritis in comparison to the control
group is presented in table 4. No significant variation in
the phenotypic distribution of alkaline phosphatase was
observed in osteo arthritic condition.
Table 5 gives the frequency distribution of alkaline
phosphatase phenotype with respect to sex in disease
and control groups. Phenotypes of alkaline phosphatase
did not seem to vary in juvenile arthritis though
predominance of p+ and p++ phenotypes in female
patiens (35.7% and 14.3% respectively) were observed
when compared to the control group (22.2% and 11.1%
respectively). Similarly p+ phenotypes were found to be
predominant in male patients (53.0%) compared to male
control subjects (25.0%). Predominance of p+ phenotype
and decreased predisposition of p0 phenotypes in female
patients of rheumatoid arthritis were found to be
significantly associated (2 : 14.973), while in male
rheumatoid arthritis and osteo arthritis patients, an
increased predisposition of p+ and p++ phenotypes was
observed with the association being insignificant.

Alkaline phosphate (ALP), a phosphomonoester was

examined for its isoenzyme variation in arthritides in
the present study. An increased prevalence of p +
Table 2: Relative Risk (RR) estimates of alkaline
phosphatase phenotypes in juvenile control and juvenile
arthritis
ALP Phenotype

Juvenile Arthritis
2
RR

Control

pO

14

20

pO vs others

11

21

0.748

0.324

pO vs p+

17

0.504

2.075

pO vs p++

1.786

0.588

p+ vs p++

3.54

2.37

Table 3 : Relative Risk (RR) estimates of alkaline

phosphatase phenotypes in rheumatoid arthritis in
comparison to control group.
ALP Phenotype
p

Control

89

Rheumatoid Arthritis
n

RR

2
17.46**

69

pO vs others

36

81

0.345

pO vs p+

30

54

0.431

9.21*

pO vs p++

27

0.172

13.51**

p+ vs p++

27

0.4

3.28

**p<0.01, *p< 0.05.

Table 4: Relative Risk (RR) estimates of alkaline

phosphatase phenotypes in control and osteo arthritis
ALP Phenotype
p

Control

89

Osteo Arthritis
n

RR

65

pO vs others

36

35

0.751

0.988

pO vs p+

30

27

0.811

0.458

pO vs p++

0.548

1.14

0.675

0.425

p vs p

++

Table 5: Frequency distribution of alkaline phosphatase phenotypes with respective to sex in control and disease
groups
pO

p+

MALE
p++

Total

pO

p+

FEMALE
p++

Total
Total

2
Male

Female

4.85

0.78

Sex
Juvenile Control

16

25

Juvenile Arthritis

13

14

10

28

41

Control

13

19

76

25

106

125

Rheumatoid arthritis

19

15

10

44

50

39

17

106

150

4.2

14.9**

Osteo Artritis

19

29

46

20

71

100

0.4

1.1

**p<0.01

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Association of alkaline phosphatase phenotypes with Arthritides

phenotypic individuals in juvenile arthritis (41.5%)

compared to juvenile control groups (24.0%) was
observed. Similarly, p+ and p++ phenotypes were found
to be significantly higher in individuals of rheumatoid
arthritis (2 : 20.65) only indicating preponderance of
intestinal component may predispose an individual to
the disease condition and/or deficiency of p phenotype
of alkaline phophatase may contribute to a decrease
in bone mineralization and bone formation processes.9
Alternatively, individuals of p+ and p++ phenotypic
individuals could be predisposed to increased risk to
develop arthritides, indicating that the intestinal
secretion/synthesis of alkaline phosphatase could be

Implication for future research

enhanced, as alkaline phosphatase is needed for the

References

The study highlights the tissue specific role of ALP.

However, the role of tissue specific component of ALP,
in relation to calcium is to be evaluated in the ossification
and resorption of bone processes especially with
reference to arthritides.
Estimation of ALP levels and its individual components
may be of relevance for drug discovery and clinical trials.
Acknowledgement
We acknowledge the financial support from UGC- New Delhi.

mineralization and ossification processes of the bone,

which could be mediated by calcium. Further, the
reduced alkaline phosphatase p phenotypic frequency
in the diseased condition may attribute to decreased
series of tissue non specific (bone/kidney/liver)
component confirming the decreased ossification
process. Studies indicate that during adulthood, activity
of alkaline phosphatase bone fraction is constant and
no significant differences were observed between
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phosphatase activity.10 However, alkaline phosphatase
phenotypic distribution was found to be associated with
predominance of intestinal component in our study,
correlating the obser vation to decreased bone
mineralization and ossification and an increased bone
resorption.
Hence, to maintain serum homeostasis of alkaline
phosphatase, a simultaneous increase of intestinal
component secretion of alkaline phosphatase can be
observed, which is also found to correlate with the increase
in p+/p++ phenotypes in rheumatoid arthritis patients.
In conclusion, deficiency of alkaline phosphatase p
isoenzymes observed in arthritides confirms decreased
bone mineralization and low bone formation in the
disease process.

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