Alkaline phosphatase

Laboratory Perspective

Professor William D Fraser

Professor of Medicine

University of East Anglia,

Norwich, UK

W.Fraser@uea.ac.uk

Disclosures – Prof. Fraser

Unrestricted funding for assay development from Roche,

Siemens, and IDS

Holds patents with IDS regarding assays in development

Consultant fees from Alexion Pharmaceuticals

Alkaline phosphatase (ALP)

A hydrolase enzyme (EC 3.1.3.1)

Removes phosphate groups from several

molecules:

Proteins, nucleotides and alkaloids

Optimal activity in vitro in an alkaline environment1

1. Vroon DH and Israili Z. Alkaline phosphatase and gamma glutamyltransferase.

In: Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition. Boston 1990.

Alkaline phosphatase (ALP)

In humans, the following isoenzymes1 are commonly detected:

Tissue non-specific ALP (liver, bone, kidney)

Intestinal ALP

Placental ALP

Germ cell ALP

Highest concentrations are found in liver, bone, placenta, intestine

and kidney

Several techniques exist for the quantification of ALP and the

isoenzymes

1. Whyte MP. Hypophosphatasia. In: Thakker RV, Whyte MP, Eisman J, Igarashi T, ed.

Genetics of Bone Biology and Skeletal Disease. Amsterdam: Elsevier/Academic Press; 2013.

Assessing total ALP concentration by

enzymatic colorimetric methods

Relatively cheap and fully automated

Following the cleavage of PO4 groups from the substrates,

molecules with specific quantifiable absorbance characteristics

are generated, e.g.

Disodium p-nitrophenyl phosphate to p-nitrophenol

4-nitrophenyl phosphate to 4-nitrophenoxide

Zn and Mg are important co-factors required for the action of ALP

in these reactions

Rearranges at

alkaline pH

O

P O- O

O-

N

O

O- O N

O

P O- O

O-

O O

HOH

N

O

P O- O

O-

O O

+ ALP

Zn Mg2+

pH 10.3

+

4-Nitrophenyl phosphate

(colourless)

4-Nitrophenoxide

(colourless

benzenoid form)

4-Nitrophenoxide

(yellow, quinonoid form)

Rate of formation at 37C

Buffers (amino alcohols):

Diethanolamine (DEA) Germany/Scandinavia

2-amino-2-methyl-1-proponol (AMP) IFCC/USA/France

HEDTA

Method N Mean CV, % Um

Roche, AMP buffer IFCC 499 16.259 6.8 0.06

AMP, optimised to IFCC 379 19.707 12.8 0.16

Diethanolamine buffer, DEA 111 39.079 17.8 0.82

AMP, non-optimised 92 18.612 7.7 0.19

Ortho Viros MicroSlide Systems 89 26.132 8.5 0.29

Dade Dimension, AMP buffer 80 27.277 22.4 0.86

Other AMP kits 65 18.063 7.8 0.22

Tris/carbonate buffer, KA units 44 31.591 31.8 1.89

AMP, optimised to NVKC/SFBC 6 21.167 8.5 0.92

Agappe – DGKC-SCE 4 45.000 28.7 8.08

Colorimetric 4 18.050 14.9 1.68

AMP, reduced interference 4 16.500 6.1 0.63

- Select - 2 20.500 3.4 0.62

ALP (WEQAS Results)

WEQAS: Wales External Quality Assessment Scheme

provider in the UK

N Mean CV, % Um SDP

A

Exc.

All methods 1372 19.705 25.1 0.17 1.80 164

AMP, optimised to IFCC 379 19.707 12.8 0.16 1.80 41

Siemens ADVIA

1200/1650/1800/2400

42 19.049 8.0 0.3 1.74 3

ALP, 37°C

U/L

Num

ber

of la

bora

tories

The most common approach for isolating isoenzymes

Electrophoresis followed by specific substrate-staining methods

Detection of bone-specific ALP

Specific antibody-employing immunoassays for bone ALP1

Measure activity of ALP

Measure the mass of the molecule

Disadvantages

Cross reactivity with liver ALP in ~3–16% of these assays2,3,4

1. Manufacturers Pack inserts (IDS, Oxford Biosystems).

2. Fraser WD. Data on file.

3. Gomez, et al. Clin Chem. 1995;41(11):1560-1566.

4. Broyles, et al. Clin Chem. 1998;44(10):2139-2147.

Detection of ALP

Measurement of ALP

Historically, the vast majority of ALP assays have been

performed to investigate diseases in which ALP is

increased

10–12% of the test results were due to abnormal liver function,

vitamin D deficiency, metabolic bone disease (e.g. Paget’s

disease)

As a result, less attention has been given to low ALP

concentrations (0.4% of results)

ALP reference ranges may also contribute to the

underestimation of the prevalence of low ALP

Representative reference ranges for ALP

(Europe and the USA)

Specimen Age group/age Concentration or enzyme

activity Concentration (SI

units) Source

Adult 36–92 U/L 0.5–1.5 µkat/L Merck [1] Serum Adult 30–120 U/L 0.5–2.0 nkat/L USA [2] Plasma

1–11 m

1–3 y

10–11 y

≥20 y

Male

70–350 U/L

125–320 U/L

150–470 U/L

40–120 U/L

Female

70–350 U/L

125–320 U/L

150–530 U/L

40–120 U/L

ARUP [3]

Plasma Neonate

Infant

1–14 y

14–16 y

Adult

73–391 U/L

59–425 U/L

76–308 U/L

49–242 U/L

30–130 U/L

Europe [4]

1. Wians FH. Merck manuals. Available at http://www.merckmanuals.com/professional/appendixes/normal-laboratory-values/blood-

tests-normal-values.

2. Kratz A, et al. N Engl J Med 2004;351:1548–63.

3. ARUP National Laboratory. Alkaline Phosphatase Isoenzymes, Serum or Plasma. Available at http://ltd.aruplab.com.

4. Sheffield children’s NHS foundation trust. Laboratory handbook. April 2014.

Reference ranges

A recent survey of 26 laboratories in the UK revealed:

The lower limit was stated as being zero (0) U/L by

two laboratories

No gender-specific reference range by eight laboratories1

Often a lack of recognition that paediatric reference

ranges are significantly higher than adult ranges

1. Fraser WD. Data on file held at the University of East Anglia.

Canada:

CALIPER

CALIPER: Canadian Laboratory Initiative on Paediatric Reference Intervals

(http://www.sickkids.ca/caliperproject/index.html)

Age (years)

CALIPER

CALIPER Cohort samples have been used to produce

paediatric reference ranges for most of the major

manufacturers machines/methods. (Adeli K)

Survey of child and adolescent health:

KiGGS ALP levels, boys

• IFCC standard

method (Hitachi 917)

KiGGS:a long-term study conducted by the Robert Koch Institute

http://www.kiggs-studie.de/english/home.html

Survey of child and adolescent health:

KiGGS ALP levels, girls

• IFCC standard

method (Hitachi 917)

KiGGS:a long-term study conducted by the Robert Koch Institute

http://www.kiggs-studie.de/english/home.html

Representative reference ranges for

bone ALP (Europe and the USA)

1. ARUP National Laboratory. Alkaline Phosphatase Isoenzymes, Serum or Plasma. Available at ttp://ltd.aruplab.com/Tests/Pub/0021020.

2. ARUP National Laboratory. Bone Specific Alkaline Phosphatase. Available at http://ltd.aruplab.com/Tests/Pub/0070053.

Specimen Age group/age Concentration or

enzyme activity

(conventional units)

Concentration (SI units) Source

Serum

6 m–2 y

3–6 y

7–9 y

10–12 y

≥25 y

Premenopausal female

Male

31.6–122.6 µg/L

31.3–103.4 µg/L

48.6–140.4 µg/L

48.8–155.5 µg/L

6.5–20.1 µg/L

Female

33.4–145.3 µg/L

32.9–108.6 µg/L

36.3–159.4 µg/L

44.2–163.3 µg/L

4.5–16.9 µg/L/7.0–22.4 µg/L

ARUP [1]

1–6 y

7–9 y

10–15 y

16–19 y

Females ≥16 y and males ≥20 y

Male

0–208 U/L

0–264 U/L

0–340 U/L

0–165 U/L

0–55 U/L

Female

0–189 U/L

0–246 U/L

0–340 U/L

0–91 U/L

0–55 U/L

ARUP [2]

Low ALP

Several clinical conditions may result in a low ALP

An algorithm has been developed that should help to

guide the investigation of a low ALP

ALP decision

algorithm

ECG: electrocardiogram; ESRF: end stage renal failure; FBC: full blood

count; PTH: parathyroid hormone; TSH: thyroid stimulating hormone

Metal

analysis

low Zn, Mg,

high Cu

Disease-associated

hypothyroidism,

hypoparathyroidism,

severe anaemia

Vitamin abnormality

low vitamin C, B12,

B6, folate,

excessive vitamin D

Post-cardiac surgery

post liver transplant

ESRF

osteodystrophy

achondroplasia

cretinism

Drug effect

estrogen,

bisphosphonate,

clofibrate,

omeprazole,

lansoprazole

Abnormal

investigate

and correct

Measure TSH,

PTH, FBC

Correct

abnormality

Measure and

correct

abnormality

Possible genetic cause for HPP

PEA and PLP measurement

Genetic testing of ALPL

Review medical

history for low ALP

Protein

calorie

malnutrition

Drug therapy

may unmask

genetic cause

for low ALP

Total protein

albumin

Non-biochemical tests

radiology, ECG muscle studies

ALP remains below reference range after

correction of abnormality

Artefact

EDTA, oxalate (blood transfusion)

Confirm low on serum sample

ALP lower than appropriate

reference range

Pathophysiology of HPP

PPi

Ca

Pi

ALP

Pi

HPP is characterised by accumulation of PPi,

which suppresses hydroxyapatite crystal formation

NPP1

(ENPP1)

ATP

Inhibition of

hydroxyapatite crystal

Formation

Promotion of

hydroxyapatite crystal

formation

Bone mineralisation

Alkaline phosphatase gene (ALPL)

Over 280 known loss-of-function mutations in the alkaline

phosphatase gene (ALPL)

Autosomal recessive versus autosomal dominant

transmission determines the clinical severity

http://www.sesep.uvsq.fr/03_hypo_mutations.php

Serum ALP activity in HPP

Normal mean and range (± 2 SD mean)

Children 166 (80–342); Adults 51 (28–91)

O = odontohypophosphatasia

Whyte MP. In: Bilezikian JP, Raisz LG, Martin TJ, eds. Principles of Bone Biology. Vol 1. 3rd ed. San Diego, CA, USA, Academic Press. 2008;1573–98.

Clinical Features of Hypophosphatasia

Perinatal/Infantile Severe Hypomineralisation/ Skeletal Deformities

Ricketic Type Lesions

Fractures

Respiratory Failure

Poor Feeding/Weight Gain/ Failure to Thrive

Hypotonia

Vitamin B6 Responsive Seizures

Hypercalcaemia/Hypercalciuria/Nephrocalcinosis

Craniosynostosis

Clinical Features of Hypophosphatasia

Juvenile (6 months to 18y)

Skeletal Deformities

Ricketic Type Lesions

Recurrent Fractures/Poor Healing Fractures

Low BMD

Short Stature

Muscle Weakness

Waddling gait

Premature Tooth Loss

Adult Hypophosphatasia (≥18y)

Liverpool 12 patients

Norwich 26 patients – Age 17-86

– 26 detected by biochemical testing after presenting with bone aches (16) joint pains (14) or recurrent fractures (10)

– Low Total ALP in all cases (many (10) had abnormal ALP in childhood missed –used wrong reference ranges)

– Family connections in 8 (cousins, uncle and niece, brothers)

– Misdiagnosis: Multiple Sclerosis (2)

Symptoms

Bone aches(18)

Fracture pain (6)

Joint aches (16), Osteoarthritis (14), Articular Chondrocalcinosis (3)

Abdominal Pain (9)

Joint Laxity, Recurrent Dislocation - especially younger female patients 17-34 y (5)

• Difficulty with household tasks, concentrating, working (hairdressing x3)

An Adult Case – 86 y Old Female

PMH

Asthma

Hypertension

Cholecystectomy

2 humeral # as child

Also # x3 wrist, x2 ankle, left femoral

DHx

• Felodipine

• Montekukast

• Steroid inhaler

• Omeprazole

• Mirtazapine

• Bendroflumethiazide

• Domperidone + erythromycin

• Alendronic acid ( 8 months )

SH

• Never smoked

• Infrequent alcohol

Fracture History

2000 – FOOSH – smith’s right wrist

2006 – leg gave way - midshaft femur

2007 – Fall – 2nd metatarsal

2011 – Fall – left distal radius

5/2012 – Fall – right distal radius

Further Investigations

FBC U+E LFT LFT Bone Other

Hb 114 Cr 87 Bil 7 aCa2+ 2.44 TSH 1.59

MCV 95 eGFR 54 Tot Prot 62 PO4- 1.24 FSH 36.3

Wbc 5.5 Na 136 Alb 35 ALP 22 LH 15.9

Plt 273 K 3.5 ALT 14 Vit D - 42 Prolac

1397

Dexa Scan

T score – 1.7 & Z score of 1.2 lumbar spine

21% increase since last scan in 2000

What can we learn from this case?

Not all atypical femoral fractures are BP induced

Always consider hypophosphatasia in these patients

Do BPs bring out phenotype in previously subclinical

hypophosphatasia?

Treatment

Pathophysiology of HPP

PPi

Ca

Pi

ALP

Pi

HPP is characterised by accumulation of PPi,

which suppresses hydroxyapatite crystal formation

NPP1

(ENPP1)

ATP

Inhibition of

hydroxyapatite crystal

Formation

Promotion of

hydroxyapatite crystal

formation

Bone mineralisation

PEA

ALP substrates

A lack or low activity of tissue non-specific ALP (TNSALP)

results in accumulation of ALP substrates:

Pyridoxal 5’-phosphate (PLP)

Pyrophosphate (PPi)

Phosphoethanolamine (PEA)

Vitamin B6-responsive

seizures

Inhibits bone mineralisation,

causing rickets or osteomalacia

PLP, PPi and PEA in HPP

Measured by HPLC or tandem mass spectrometry or

spectrophotometric methods

Poor sensitivity and specificity as a diagnostic tool

May help in a small number of cases

May be of value in following enzyme therapy, monitoring

changes with treatment

HPLC: high-pressure liquid chromatography;

PEA: phosphoethanolamine;

PLP: pyridoxal 5´-phosphate

Representative reference ranges for PLP, PO4

and 25-hydroxy vitamin D (Europe and the USA) Specimen Age

group/age Concentration or enzyme activity (conventional units)

Concentration (SI units) Source

Pyridoxal–5'–phosphate (PLP)

Plasma 5–50 µg/L* USA [1] 5–30 ng/mL 20–121 nmol/L USA [2]

Phosphorus, inorganic Serum 3.0–4.5 mg/dL 0.97–1.45 mmol/L Merck [3] 3–4.5 mg/dL 1.0–1.4 mmol/L USA [2]

0-11 months

1-4 y

8-13 y

16-17 y

≥18 y

Males

NA**

4.3-5.4 mg/dL

3.1-4.7 mg/dL

2.5-4.5 mg/dL

Females

NA**

4.0-5.2 mg/dL

3.1-4.7 mg/dL

2.5-4.5 mg/dL

[4]

Phosphate, fasting Plasma Neonate

Infant

Child

Adult

1.0–2.7 mmol/L

1.1–2.4 mmol/L

0.8–1.9 mmol/L

0.8–1.5 mmol/L

Europe [5]

25-hydroxy vitamin D

(vitamin D3; 25-

hydroxycholecalciferol)

Serum 15–80 ng/mL

>20 ng/mL

37–200 nmol/L

>50 nmol/L

Merck [3]

UK (NOS, USA IOM)

Plasma 10–68 ng/mL 24.9–169.5 nmol/L USA [2]

1. Mayo Clinic, Mayo Medical Laboratories (http://www.mayomedicallaboratories.com/test-catalog/Clinical+and+Interpretive/60295.

2. Kratz A, et al. N Engl J Med 2004;351:1548–63.

3. Wians FH. Merck manuals. Available at http://www.merckmanuals.com/professional/appendixes/normal-laboratory-values/blood-tests-normal-value.

4. Mayo Clinic. http://www.mayomedicallaboratories.com/test-info/pediatric/refvalues/reference.php. 5. Sheffield children’s NHS foundation trust. Laboratory

handbook. April 2014..

Conclusions

Biochemical measurement of ALP is relatively cheap and

readily available

Reference ranges are extremely variable with age and

gender

The lower limit of normal range is often poorly defined

Paediatric reference ranges are higher than adult

An algorithm for investigation of low ALP is valuable to

ensure the correct diagnosis is made in all cases and

appropriate therapy can be commenced