Specialist Biochemistry

Porphyrin Section
Dr Joanne Marsden
 Who we do it for – our users – internal and
external
 Why we do it – clinical reasons for requesting –
conditions etc
 What we do – range of assays – sample types -
conditions
 What we do – analytical methodology – principles –
instrumentation - limitations
 (short example if time)
 Users
2004 2005 2006*
Number of
External 776 826 447
referrals
Internal 255 246 116

Total 1031 1072 563

Type of
porphyria 2004 2005 2006*
AIP 8 10 4
VP 4 4
HCP 1
PCT 15 16 8
EPP 10 5 5
TOTAL 32 36 21
* April - Oct 06
 Internal users

April 2005 - March 2006

 Porphyria Clinic – 23%

 Monitoring haem arginate therapy – 27%
 Neurosciences, Maudsley & general Medicine – 46%
 Dermatology – 2%
 Haematology – 2%
 Internal users
(3 month snapshot)
August 2006 - October 2006 (57 referrals)

 Porphyria Clinic – 28%

 Monitoring haem arginate therapy – 0%
 *Neurosciences, Maudsley & General Medicine – 23%
 Dermatology – 2%
 Haematology – 42%
 Trial (EPO-GCSF) – 5%

* Neuropathy (x2), epilepsy, metabolic, muscle weakness,

unexplained abdo pain (x4), GBS, GI symptoms, schizophrenia
(x2)
 External users

Eastern
South East
5.9%
13.6%

Scotland
1.7%

Northern&Yorks
8.2%

North West
0.2%

London
70.3%

Regional distribution of porphyrin requests 2005

 External users
(3 months snapshot)
August 2006 - October 2006 (208 referrals)

 Acute symptoms – 32%

 Skin symptoms – 32%
 No clinical details – 36%
Clinical presentation of porphyria
 Acute symptoms
Cardiovascular
Neurological Tachycardia
Hypertension
Pain in extremities - 4
Muscle weakness -6
Paralysis - 4
Mental changes - 4
Convulsions - 2

Gastrointestinal
Abdominal pain - 36
Vomiting - 1
Constipation
Diarrhoea
 Clinical presentation of porphyria
 Skin lesions

Blisters - 10
Fragility
Scarring - 1
Erosions
Pigmentation - 3
Milia
Hirsuitism - 1
Clinical presentation of porphyria
Acute photosensitivity

Erythema - 2
Urticaria - 21
Itching
Burning - 1
Oedema
 Glycine + Succinyl CoA
ALA synthase
Aminolevulinic acid (ALA)
ALA dehydratase Plumboporphyria
Porphobilinogen (PBG)
PBG deaminase AIP (Acute)
Uroporphyrinogen I Uro 1
Uroporphyrinogen III
CEP
synthase Copro 1
Uroporphyrinogen III
Uroporphyrinogen PCT
decarboxylase Isocopro
Coproporphyrinogen
Coproporphyrinogen HC (Acute)
oxidase 7 COOH
6 COOH Protoporphyrinogen
5 COOH
Protoporphyrinogen VP (Acute)
oxidase Protoporphyrin
EPP
Ferrochelatase
 Porphyria Symptoms

AIP (Acute) Acute neurovisceral attacks

CEP Skin lesions

PCT Skin lesions

HC (Acute) Acute neurovisceral attacks

skin lesions

VP (Acute) Acute neurovisceral attacks

skin lesions
EPP Acute photosensitivity
 Initial investigations

CLINICAL PRESENTATION SPECIMEN TEST

ACUTE NEUROVISCERAL ATTACKS URINE PBG

Abdominal pain, vomiting, neuropathy (during attack)
Tachycardia, psychiatric

SKIN LESIONS URINE PORPHYRINS

Fragility, bullae, hypertrichiasis FAECES
Pigmentation, scarring, milia BLOOD
(serum/plasma)
ACUTE PHOTOSENSITIVITY BLOOD PROTO-
PORPHYRIN

Tetrapyrrole
 Glycine + Succinyl CoA
ALA synthase
Aminolevulinic acid (ALA)
ALA dehydratase Plumboporphyria
Porphobilinogen (PBG)
PBG
ALAdeaminase
& PBG (urine) AIP (Acute)
Uroporphyrinogen I Uro 1
Uroporphyrinogen III
Uro I & Copro I (urine) CEP
synthase Copro 1
Uroporphyrinogen III
Uro I, Hepta (urine)
Uroporphyrinogen PCT
Isocopro (faeces)
decarboxylase Isocopro
Coproporphyrinogen
Coproporphyrinogen HC (Acute)
Copro, ALA & PGB7(urine)
COOH
oxidase
6 COOH Protoporphyrinogen
ALA & PBG (urine)
5 COOH
Protoporphyrinogen VP (Acute)
Proto oxidase
& Copro (faeces) Protoporphyrin
EPP
Proto (faeces)
Ferrochelatase
 Methodology

 UV-Vis spectrophotometry
Total urine and faecal porphyrins

 Ion-exchange chromatography
Urinary ALA and PBG

 Fluorimetry
Plasma scans, red cell protoporphyrins and red cell HMBS
activity

 HPLC with fluorimetric detection

Urine and faecal porphyrin isomers
 UV-vis spectrophotometry
 A spectrophotometer measures the amount of light that a
sample absorbs
 It operates by passing a beam of light through the sample
and measuring the intensity of light reaching the detector
Perkin Elmer Lambda 20 UV-vis spectrophotometer

Printer
 UV-vis spectrophotometry
Halogen lamp
Optical path for Lambda 20

M2

M1

Deuterium lamp Optical filter wheel

Reference Lens
M5
Photodiode
Slit 1 Detector

M3
Slit 2
Beam splitter

Sample Lens

Photodiode
Grating
Detector
M4
(monochromator)
 UV-vis spectrophotometry
 In the UV-visible absorption spectrum the highly conjugated
porphyrin shows intense absorption at around 400 nm (the
“soret” band) followed by several weaker absorptions (Q
bands) at higher wavelengths (450-700 nm)
 Total urine porphyrins

2 ml aliquot 1 -2ml aliquot for urine

for urine creatinine BSL
10-15 ml random urine or 24hr porphyrins
aliquot - covered (store at 4 C)

1ml conc HCL,

vortex

Scan in UV-vis spectrophometer

350 to 450 nm
 Total faecal porphyrins

+ 1ml +3ml +3ml

conc HCL diethyl ether water

Vortex, centrifuge
Vortex vortex 3000rpm for 10 mins

~5g random specimen 0.025 -0.1g

- covered faeces in
weighed glass
10ml tube

Scan in UV-vis
spectrophometer
350-450 nm
 Calculation of results

Draw a baseline by
constructing a tangent corrected peak
between ~390 nm and height (mm)
425 nm. Measure in mm (h)

the vertical height of

the peak in the 400nm Absorbance
region

390 425
Wavelength nm

URINE: FAECES:
Total porphyrin (nmol/L) = h x 50 Porphyrin excretion nmol/g = h x 0.269
Porphyrin excretion nmol/24hr = Total porphyrin Wt faeces(g)
x vol (L)
Porphyrin excretion nmol/mmol creatinine = total
porphyrin/urine creatinine
 APPENDIX 1 – URINE SCAN SHEET APPENDIX 2 – FAECAL PORPHYRIN SCAN SHEET

450 450
440 440
430 430
PORPHYRIA
NORMAL 420
CUTANEA
420

TARDA
410 410
400  400 
390 390
380 380
370 370
360 360
350 350
3.0 2.0 A 1.0 0.0
3.0 2.0 A 1.0 0.0
Name: _____________ Lab No: _______________ Name: ______________ Lab No: ___________

Urine Vol: ______Urine Creatinine : _______mmol/L

Wt of tube: ___Wt of tube+faeces: ___Wt of faeces___
Date of Sample_________Date Run: ________

ALA: ___μmol/ mmol creat PBG: ____µmol/mmol creat Porphyrin content = Peak H (mm) x 0.269 = ----x 0.269
Wt of faeces
ALA:_____µmol/24hr PBG:____ µmol/24 hr = _________

Urine Total Porphyrins:________nmol/mmol creat

Faecal Total Porphyrins:____________ nmol/g stool
Urine Total Porphyrins: _______nmol/24hrs
 Calculation of results

Draw a baseline by
constructing a tangent corrected peak
between ~390 nm and height (mm)
425 nm. Measure in mm (h)

the vertical height of

the peak in the 400nm Absorbance
region

390 425
Wavelength nm

URINE: FAECES:
Total porphyrin (nmol/L) = h x 50 Porphyrin excretion nmol/g = h x 0.269
Porphyrin excretion nmol/24hr = Total porphyrin Wt faeces(g)
x vol (L)
Porphyrin excretion nmol/mmol creatinine = total
porphyrin/urine creatinine
 Principle of calculation
Urine

• Where does the 50 come from?

Beer-Lambert Law (Beer’s law) is the linear relationship between
absorbance and concentration usually expressed as:

A = measured absorbance
A = bc
b = path length
C = analyte concentration
 = molar absorptivity coefficient

• Milli-molar absorptivites of the porphyrins derived by Rimington:

Uroporphyrin = 541, Coproporphyrin = 489, Protoporphyrin = 275
• Assume urine specimen contains normal proportion of porphyrin 7:1

mM = (7x489) + 541 = 495.5 mmol/L

8
 Principle of calculation
Conc = A = A mmol/L
(mM) 495.5

Adjusting for dilution by HCL (2ml urine + 0.5 ml HCL)

Conc = A x 2.5 mmol/L

495.5 x 2

= A x 2.5 x 1,000,000 nmol/L

495.4 x 2

= A x 2523 nmol/L

Finally:
Porphyrin Conc = h x 2523 nmol/L
50.5
(h = peak height measured in mm, assuming 0 – 3 absorbance scale and
50.5 nm = 1A)
= h x 50 nmol/L
 Principle of calculation
Faeces

• Where does the 0.269 come from?

Beer-Lambert Law (Beer’s law) is the linear relationship between
absorbance and concentration usually expressed as:

A = measured absorbance
A = bc
b = path length
C = analyte concentration
 = molar absorptivity coefficient

• Milli-molar absorptivites of the porphyrins derived by Rimington:

Uroporphyrin = 541, Coproporphyrin = 489, Protoporphyrin = 275
• Assume faecal specimen contains normal proportion of porphyrin 3:1

mM = 489 + (3 x 275) = 328.5 mmol/L

4
 Principle of calculation
Conc = A = A mmol/L
(mM) 328.5

Assuming that the volume of extract is 4.5 ml then the amount of porphyrin in
the extract is:
A x 4.5 mmol/L
328.5 x 1000

This amount of porphyrin was extracted from the weight of faeces, so that the
porphyrin content per g of faeces is given by:

A x 0.0000136
W
To convert to nmol/g multiply by 1,000,000:

Porphyrin Conc = A x 0.0000136 x 1,000,000

W

= A x 13.6 nmol/g faeces

W
= h x 13.6 = h x 0.269 mol/g faeces
W X 50.5 W
Normal ranges and limitations of assay
Normal ranges
 Total urine porphyrin < 35 nmol/mmol creatinine
 Total faecal porphyrin < 50 nmol/g faeces

Limitations of the assay

 Semi-quantitative
 Dilute urines (<2.5 mmol/L) can produce misleading
results.
 Further analysis needed for excess porphyrins i.e HPLC
fractionation for diagnosis of porphyria
 Ion-exchange Chromatography
 An ion exchanger consists of an insoluble matrix to which
charged groups have been covalently bound
 The separation is obtained by reversible adsorption
 Affinity can be controlled by varying conditions such as ionic
strength and pH

- +
-
- - + +
- + + - +
- - -
++ + -
- + +
+ + - -

Anion exchanger Cation exchanger

 Urinary ALA and PBG
Bio-Rad quantitative method

Dowex-2
anion PBG
exchanger

7 ml sodium
acetate pH 6.7
4 ml acetic
acid
Dowex-50
cation
ALA exchanger

ALA

PBG

4 ml 4 ml
Ehrlich’s Ehrlich’s
reagent reagent Read at
ALA Read at
553 nm 553 nm
PBG
 Calculation of results
 The blank value (eluting solution + Ehrlich’s reagent) for ALA
or PBG is subtracted from the absorbance readings for each
urine.
 The corrected absorbance values (Test absorbance – blank)
are multiplied by the appropriate factor to give the ALA or
PBG concentration in mol/L.

ALA mol/L = corrected absorbance x 458

PBG mol/L = corrected absorbance x 389
(The calculation factors are quoted by Bio-Rad)

 For random urines divide the ALA & PBG by mmol/L of

creatinine
 For 24 hr urines, multiply the result of the ALA & PBG by
the volume of the 24hr urine in litres.
Normal ranges and limitations of assay
Normal ranges
 ALA < 3.8 mol/mmol creatinine or < 46 mol/24hrs
 PBG < 1.5 mol/mmol creatinine or <9 mol/24hrs

Limitations of the assay

 The method is linear up to 500 mol/L.
 For results > 500 mol/L the urine must be diluted 1/5
with distilled water and repeated.
 Dilute urines (<2.5 mmol/L) can produce misleading
results.
 Urinary PBG
Alpha-labs qualitative method

PBG PBG PBG PBG

Expel urine and Expel water and add
elution reagent Expel into tube
wash with water containing
Ehrlich’s reagent

Compare
intensity of
1ml fresh urine 1ml water 1ml elution reagent colour to the
colour chart

PBG Test kit Standard Chart

+ PBG approx 25-50 mol/L
++ PBG approx 50-100 mol/L
+++ PBG > 100mol/L
 Fluorimetry
 Fluorescence is based on the ability of a compound to be
subjected to a specific wavelength of light (excitation) and to
re-emit this light energy at one or more higher wavelengths
(emission).

Excitation
Monochromator

Light source
Emission
Sample Photomultiplier
monochromator

Fluorescent Recorder
signal

LIGHT RED FLUORESCENCE

( Max ~400nm) (max ~620 nm)
 Fluorimetry

Perkin Elmer LS30 Luminescence Spectrometer fitted with a red filter wheel

Printer paper

Set Excitation and

emission wavelengths

Aspirate sample
 Plasma scans

 The emission maximum helps to differentiate between the

types of porphyria:
AIP, CEP, PCT, HC = 615 – 620 nm
VP = 624 – 627 nm
EPP = 626 – 634 nm

Methodology

Dilute 0.5ml serum with 4.5 ml phosphate buffered saline

a) Determine emission maximum
 Excitation maximum 405 nm
 Scan emission spectrum 550–700nm
a) Determine excitation maximum
 Scan excitation spectrum 350-450nm
 Plasma scans
Typical scans
100
VARIEGATE
NORMAL
PORPHYRIA
50

630 700 nm 630 700 nm

Plasma scans
 Plasma scans

Limitations of the assay

 In general, moderate lipaemia, haemolysis or icterus, do not
cause a problem as the plasma/serum samples are diluted
1:10 prior to scanning, however gross levels of lipaemia,
haemolysis or icterus may interfere with this test

 Other substances present in the sample that absorb at

400nm can potentially be identified as porphyrins; the
selection of a specific excitation wavelength (405nm) reduces
that possibility
 Red cell protoporphyrin

 Free protoporphyrin accumulates in the red cells in patients

with EPP
 Zinc protoporphyrin accumulates in red cells in patients with
iron deficiency anaemia and lead toxicity

Methodology

Scan the extract in the LS30

fluorimeter
Vortex while Centrifuge
adding 5ml 3000rpm 10 mins Excitation wavelength 405 nm
ethanol
Emission range 550 – 700 nm

50l packed cells

+ 200l water
 Red cell protoporphyrin
100

50

0
550 590 630 670 710

Peak at 586 nm = zinc protoporphyrin

Peak at 630 nm = free protoporphyrin and a contribution of any zinc
protporphyrin present such that:
Corrected peak height (PH)630nm = PH630nm – (0.34 x PH586nm)
 Calculation of results
Manual calculation of red cell protoporphyrin

 Free protoporphyrin (nmol/L cells)

= Corrected PH630nm extract x std conc(nmol/L) x scale factor(std) x 105
Std PH630nm x Scale factor (extract)

Where 105 allows for the dilution of 0.05ml cells to 5.25ml

 Zinc protoporphyrin (nmol/L cells)

= Extract PH586nm x std conc(nmol/L) x scale factor(std) x 150
Std PH 630nm x Scale factor (extract)

Where 150 allows for the dilution of 0.05ml cells to 5.25ml and the
fluorescence of zinc protoporphyrin is 0.7 that of free protoporphyrin at
630 nm
 Normal range and limitations of assay
Normal ranges
 Free protoporphyrin = 0 – 200 nmol/L cells
 Zinc protoporphyrin = 0 – 800 nmol/L cells

Limitation of the assay

 The assay must be completed promptly after the ethanol has
been added to the red cells due to degradation of
protoporphyrin
 Red cell hydroxmethlybilane
synthase (HMBS) activity
 AIP is caused by partial deficiency of HMBS
COOH A = acetate

CH2
COOH P = propionate

P= CH2 CH2 =A A P A P A P A P
HMBS

-4NH3
CH2- NH2
OH CH2
NH
NH NH NH NH

Porphobilinogen Hydroxmethylbilane
(PBG)
UNSTABLE

Uroporphyrinogen III spontaneous

synthase

Uroporphyrinogen III Uroporphyrinogen I

 Hydroxmethylbilane

Uroporphyringen
III synthase spontaneous

Uroporphyrinogen III Uroporphyrinogen I

oxidation

Uroporphyrin I Uroporphyrin I
 Red cell hydroxmethlybilane
synthase (HMBS) activity
• Methodology

In the water bath:

Incubate at 37C
for 10 mins
1.4ml assay buffer +
50l packed cells (a
blank is included with
assay buffer alone).
Add 50l PBG substrate to each
tube at 30 sec timed intervals
Add 1.5ml trichloroacetic acid
solution to each tube after 30 min
incubation
Centrifuge the tubes for 10mins at
3000 rpm
Measure luminescence at:
Excitation wavelength 404 nm
Emission wavelength 599 nm
 Calculation of results

Manual calculation of HMBS activity

HMBS activity = sample fluorescence x 301 x 0.12

(nmols/Uro/ml standard fluorescence (factor)
rbc/hr at 37C

where 301 is the standard concentration in nmol/L

 Calculation of results
• Where does the 0.12 come from?

• Amount of Uro/ml = Conc Uro(nmol/L) x 3.0

1000
Where 3.0 is the total volume per tube=(1.4 + 0.05 + 0.05 + 1.5)

• Amount of Uro/ml rbc = Conc Uro(nmol/L) x 3.0

1000 x 0.050

• HMBS activity = Conc Uro (nmol/L) x 3.0 x 2

1000 x 0.050

= conc Uro (nmol/L) x 0.12

in nmol Uro/ml rbc/hr at 37C
 Normal range and limitations of assay
Normal range
 HMBS activity = 20 – 42 nmol uroporphyrin/ml red cells/h at
37C

Limitations of the assay

 Diluted red cells and clots can result in a lower result
 Interpretation of the result:
 The test is invalid if the patient has recently received blood
transfusions
 HMBS activity falls as red cells age
 Increased erythropoiesis e.g anaemia can cause an increase in
activity
 Chronic alcohol abuse can cause a similar effect
 It is important to consider haematological factors when
interpreting HMBS activity
 There is a rare variant of AIP in which the defect is not
expressed in red cells
 There is a small overlap between normals and individuals with AIP
 HPLC with fluorimetric detection
 Total urine porphyrin > 35 nmol/mmol creatinine
 Total faecal porphyrin > 50 nmol/g faeces

• Standard mixture of porphyrins containing known amounts of

uroporphyrin I & III, hepta-, hexa- and pentacarboxylate
porphyrin, coproporphyrin I & III and dicarboxylate
porphyrin.

• Reverse phase C1-Hypersil column with gradient elution and

flow rate of 1 ml/min, 100 l injection volume, fluorimetric
detector.

• Mobile Phase A - 10% acetonitrile in ammonium acetate

pH 5.16
• Mobile Phase B - 10% acetonitrile in methanol
HPLC fractionation of a porphyrin mixture
 Small case
 Urine, blood and faeces received from external customer on 54 year
old female with no clinical details on the form
Random urine (24.10.06)
5-aminolevulinic acid (ALA) = 1.6 mol/mmol creat (Normal: <3.8)
Porphobilinogen (PBG) = 0.9 mol/mmol creat (Normal: <1.5)
Total porphyrin = 25 nmol/mmol creat (Normal: <35)
Creatinine = 8.1 mmol/L

Blood (24.10.06)
Erythrocyte hydroxymethylbilane synthase (HMBS; formerly called PBG-deaminase or uroporphryin-I
synthase) = 35 nmol uroporphyrin/ml red cells/h @37C (Reference Range: 20 – 42)

Erythrocyte free protoporphyrin = 83 nmol/L cells (Normal: <200)

Erythrocyte zinc protoporphyrin = 119 nmol/L cells (Normal <800)

Flourescence emission spectrophotometry of plasma shows a peak characteristic of porphyrin:

Excitation maximum = 411 nm
Emission maximum = 631 nm

Faeces (24.10.06)
Total porphyrin = 22 nmol/g faeces (Normal: <50)
HPLC fractionation of faeces
Coproporphyrin-I = 11 nmol/g dry wt
Cproporphyrin-II = 21 nmol/g dry wt
Total coproporphyrin (I+III) = 32 nmol/g dry wt (Normal: <46)
Dicarboxylate porphyrin (“protoporphyrin”) =151 nmol/g dry wt

These data are consistent with a diagnosis of VARIEGATE

PORPHYRIA (VP).

Follow up
The GP was contacted by phone and a conversation confirmed that the
patient had a family history of porphyria and was asymptomatic
The end