Laboratory Investigation of the Porphyrias
By: Brian M. Gilfix MDCM, PhD, FRCPC, DABCC, FACB
MUHC-Royal Victoria Hospital
Montreal, Quebec
BACKGROUND
In the hospital laboratory, we often receive small urine samples in clear containers and nothing more written on the accompanying requisition than “porphyrins” or “porphyria”. When later questioned, these physicians are often investigating patients with recurrent abdominal pain, whether seen in the Emergency Room or after being admitted to hospital, for porphyria. But the diagnosis of porphyria is more complex than this requiring the correct sample type but also the correct porphyrin or porphyrin precursor being measured in the sample.
The term porphyria refers to a group of conditions resulting from defects in heme biosynthesis. Heme is the iron-containing ring structure in hemoglobin to which oxygen binds. Hemoglobin is found in the red blood cells and so converts them into oxygen carrying packets, thus providing a means to transport oxygen to various organs and cells of the body.
One useful classification of the porphyrias is acute or non-acute. The acute porphyrias are associated with acute attacks of abdominal and neurological symptoms (e.g. nausea, vomiting, abdominal pain). Some types of acute porphyrias are also associated with photosensitivity. In contrast, patients with non-acute porphyria suffer photosensitivity but not acute attacks.
The hallmark of the acute porphyrias is the accumulation of porphobilinogen (PBG) and, more rarely, 5-aminolaevulinic acid (ALA). PBG and ALA are termed porphyrin precursors. This accumulation reflects a block in the pathway of heme biosynthesis and increased activity of the steps in the pathway prior to the block. The result is an increase in the PBG and/or ALA found in the urine. Depending on where the block is in heme biosynthesis, there can also be excess porphyrin production leading to photosensitivity.
In the non-acute porphyria, there is excessive production and accumulation of porphyrins (uro- and coproporphyrins), but not porphyrin precursors. Accumulation of porphyrins causes photosensitivity.
The different types of porphyrias and the porphyrins and porphyrin precursors that accumulate in each are shown in the Table. In making the diagnosis of porphyria, one has to pay attention as to where each type of porphyrin accumulates: red blood cells, urine, or stool. The more water-soluble porphyrins are excreted in the urine and the less in the stools.
I would briefly like to review the sample types we require in the laboratory and how we analyze them for the porphyrin precursors and porphyrins present. I shall not discuss enzyme assays (measuring the actual synthesis of porphyrins) or testing of DNA for mutations causing porphyria, which will be the subject of other articles.
SAMPLE COLLECTION
The single most important step in the diagnosis of the porphyrias is the obtaining of the correct sample type. Without it diagnosis can be both confused and delayed.
The acute porphyrias are characterized by the accumulation of the porphyrin precursors, PBG and ALA that are almost exclusively measured in the urine. (See Table.) Urine collected over a 24-h period is the best indication of overall excretion and is considered the “gold standard”. Single urine specimens taken at a random time can be used under certain circumstances. The latter values obtained are adjusted using urine creatinine to take into account daily fluctuations in excretion of porphyrin precursors. An abnormal value is an indication for a 24-h collection not for a final diagnosis. In both instances, a specimen associated with a symptomatic episode offers the best possibility of a diagnosis.
The most important aspect of specimen preservation is refrigeration (4°C). Urine specimens to measure PBG and ALA are stable at 4°C in the dark for several days. For this reason the patient is given an opaque container; if not, the container should be covered with aluminum foil. To further enhance PBG recovery (as PBG is produced in all the acute porphyrias except the very rare ALAD deficiency porphyria), sodium carbonate or sodium bicarbonate (baking soda) is sometimes added to the container given to the patient, and the patient told to keep the specimen refrigerated during and after collection to insure proper sample preservation.
Refrigeration is again the key to preserving specimens for porphyrin analysis. To enhance porphyrin recovery, sodium carbonate or sodium bicarbonate (baking soda) is sometimes added to the container given to the patient. Urine collected over a 24-h period is the best indication of overall excretion.
Porphyrins are not routinely measured in blood although there are some laboratory physicians who advocate its use as an aid to diagnosis. The exception is the measurement of protoporphyrin for the diagnosis of erythropoietic protoporphyria. You may hear the physician refer to the FEP content of the blood – they are referring to the protoporphyrin content of the blood. To measure the protoporphyrin content, blood is drawn from the patient into a special tube (it will have a lavender top); it is stable for several weeks if refrigerated. A drop of this blood (after suitable preparation) is placed in a special laboratory device called a “fluorometer” which produces a light signal proportional to the protoporphyrin content.
Stool specimens are of use less in the diagnosis of porphyria than in distinguishing among the types of porphyrias particularly the coproporphyrias. Only a small random sample of stool (1 g) is needed, but refrigeration or preferably freezing is required. The porphyrins are extracted and analyzed by high performance liquid chromatography. (See below.)
IN THE LABORATORY
Assuming the correct sample type has arrived in the laboratory, what happens next? Screening tests formerly used are now considered obsolete as they can produce both over- and under diagnoses of the porphyrias as many substances found in urine can interfere with the test (e.g. Watson-Schwartz and Hoesch tests, Wood’s light). (Screening tests are laboratory tests that tell us if excess porphyrins are present or absent, but not how much.)
Now, if an acute porphyria is suspected the first step is a quantitation of the amount of PBG present in a urine sample (i.e. measuring how much is present). The urine sample is made alkaline and applied to an ion-exchange column. An ion-exchange column is a small tube only a few centimeters long filled with a fine powder that has charged groups on its surface. ALA and PBG will stick to the column while impurities will travel through the column and be washed out. Adding acid to the column will wash out the PBG. A special reagent added to this “acid wash” will react with PBG forming a coloured product. The amount of colour formed is proportional to the amount of PBG present and can be used to calculate the actual amount of PBG present. ALA is measured in an analogous manner. The quantitation of PBG and ALA has been simplified by the availability of kits containing all the required materials (Bio-Rad Laboratories, Montreal, QC). Elevation of urinary PBG by two or three times the upper reference limit is diagnostic of an acute porphyria.
If a non-acute porphyria is suspected, the first step is a non-specific measurement of the total uro- and coproporphyrins present in urine. Acid is added to the urine and the amount of light absorbed by the sample as measured in a laboratory instrument called a spectrophotometer. The amount of light absorbed is proportional to the amount of total uro- and coproporphyrins present and can be used to calculate the actual amount present. If the total amount is elevated than a sample of the urine must be evaluated by high performance liquid chromatography (HPLC) in order to make the diagnosis of porphyria and more importantly to identify the type of porphyria.
In HPLC, a urine sample suitably prepared is placed on a column. The column is a narrow stainless steel tube (25 x 0.5 cm) containing very fine specially treated particles of silica. As the urine sample is pumped through the column at very high pressure (4000 psi), the various types of uro- and coproporphyrins literally stick to the column each in a characteristic way. The result is the various uro- and coproporphyrins leave the column at different times. The quantity of each can be measured in a device called a spectrofluorimeter. The HPLC apparatus consisting of special high-pressure pumps, automatic sampling device, and spectrofluorimeter costs on the over of $50,000.
The porphyrin thus identified in the urine indicates the type of porphyria. For example, if both excess uro- and coproporphyrins were present, the one would diagnose erythropoietic porphyria. If only excess uroporphyrins are present, the one would diagnose porphyria cutanea tarda.
Version:1.0 StartHTML:0000000155 EndHTML:0000658471 StartFragment:0000000476 EndFragment:0000658454 body, div, table, thead, tbody, tfoot, tr, th, td, p { font-family: "Geneva"; font-size: x-small; } Porphyrias Symptoms Red Blood Cell Urinary Stool
Porphyrins Porphyrins Porphyrins
& Precursors
Acute
acute intermittent porphyria N none ALA, PBG none
variegate porphyria N, P none ALA, PBG coproporphyrin
coproporphyrin protoporphyrin
herediatry coproporphyria N, P none ALA, PBG coproporphyrin
coproporphyrin
ALAD deficiency porphyria N protoporphyrin ALA none
Non-Acute
erythropoietic porphyria P uroporphyrin uroporphyrin coproporphyrin
coproporphyrin coproporphyrin
erythropoietic protoporphyria P protoporphyrin none protoporphyrin
porphyria cutanea tarda P none uroporphyrin isocoproporphyrin
hepatoerythropoietic porphyria P protoporphyrin uroporphyrin isocoproporphyrin
N = neurological
P = photosensitizing
The table is a modification of Adriaans, 1995.
By: Brian M. Gilfix MDCM, PhD, FRCPC, DABCC, FACB
MUHC-Royal Victoria Hospital
Montreal, Quebec
BACKGROUND
In the hospital laboratory, we often receive small urine samples in clear containers and nothing more written on the accompanying requisition than “porphyrins” or “porphyria”. When later questioned, these physicians are often investigating patients with recurrent abdominal pain, whether seen in the Emergency Room or after being admitted to hospital, for porphyria. But the diagnosis of porphyria is more complex than this requiring the correct sample type but also the correct porphyrin or porphyrin precursor being measured in the sample.
The term porphyria refers to a group of conditions resulting from defects in heme biosynthesis. Heme is the iron-containing ring structure in hemoglobin to which oxygen binds. Hemoglobin is found in the red blood cells and so converts them into oxygen carrying packets, thus providing a means to transport oxygen to various organs and cells of the body.
One useful classification of the porphyrias is acute or non-acute. The acute porphyrias are associated with acute attacks of abdominal and neurological symptoms (e.g. nausea, vomiting, abdominal pain). Some types of acute porphyrias are also associated with photosensitivity. In contrast, patients with non-acute porphyria suffer photosensitivity but not acute attacks.
The hallmark of the acute porphyrias is the accumulation of porphobilinogen (PBG) and, more rarely, 5-aminolaevulinic acid (ALA). PBG and ALA are termed porphyrin precursors. This accumulation reflects a block in the pathway of heme biosynthesis and increased activity of the steps in the pathway prior to the block. The result is an increase in the PBG and/or ALA found in the urine. Depending on where the block is in heme biosynthesis, there can also be excess porphyrin production leading to photosensitivity.
In the non-acute porphyria, there is excessive production and accumulation of porphyrins (uro- and coproporphyrins), but not porphyrin precursors. Accumulation of porphyrins causes photosensitivity.
The different types of porphyrias and the porphyrins and porphyrin precursors that accumulate in each are shown in the Table. In making the diagnosis of porphyria, one has to pay attention as to where each type of porphyrin accumulates: red blood cells, urine, or stool. The more water-soluble porphyrins are excreted in the urine and the less in the stools.
I would briefly like to review the sample types we require in the laboratory and how we analyze them for the porphyrin precursors and porphyrins present. I shall not discuss enzyme assays (measuring the actual synthesis of porphyrins) or testing of DNA for mutations causing porphyria, which will be the subject of other articles.
SAMPLE COLLECTION
The single most important step in the diagnosis of the porphyrias is the obtaining of the correct sample type. Without it diagnosis can be both confused and delayed.
The acute porphyrias are characterized by the accumulation of the porphyrin precursors, PBG and ALA that are almost exclusively measured in the urine. (See Table.) Urine collected over a 24-h period is the best indication of overall excretion and is considered the “gold standard”. Single urine specimens taken at a random time can be used under certain circumstances. The latter values obtained are adjusted using urine creatinine to take into account daily fluctuations in excretion of porphyrin precursors. An abnormal value is an indication for a 24-h collection not for a final diagnosis. In both instances, a specimen associated with a symptomatic episode offers the best possibility of a diagnosis.
The most important aspect of specimen preservation is refrigeration (4°C). Urine specimens to measure PBG and ALA are stable at 4°C in the dark for several days. For this reason the patient is given an opaque container; if not, the container should be covered with aluminum foil. To further enhance PBG recovery (as PBG is produced in all the acute porphyrias except the very rare ALAD deficiency porphyria), sodium carbonate or sodium bicarbonate (baking soda) is sometimes added to the container given to the patient, and the patient told to keep the specimen refrigerated during and after collection to insure proper sample preservation.
Refrigeration is again the key to preserving specimens for porphyrin analysis. To enhance porphyrin recovery, sodium carbonate or sodium bicarbonate (baking soda) is sometimes added to the container given to the patient. Urine collected over a 24-h period is the best indication of overall excretion.
Porphyrins are not routinely measured in blood although there are some laboratory physicians who advocate its use as an aid to diagnosis. The exception is the measurement of protoporphyrin for the diagnosis of erythropoietic protoporphyria. You may hear the physician refer to the FEP content of the blood – they are referring to the protoporphyrin content of the blood. To measure the protoporphyrin content, blood is drawn from the patient into a special tube (it will have a lavender top); it is stable for several weeks if refrigerated. A drop of this blood (after suitable preparation) is placed in a special laboratory device called a “fluorometer” which produces a light signal proportional to the protoporphyrin content.
Stool specimens are of use less in the diagnosis of porphyria than in distinguishing among the types of porphyrias particularly the coproporphyrias. Only a small random sample of stool (1 g) is needed, but refrigeration or preferably freezing is required. The porphyrins are extracted and analyzed by high performance liquid chromatography. (See below.)
IN THE LABORATORY
Assuming the correct sample type has arrived in the laboratory, what happens next? Screening tests formerly used are now considered obsolete as they can produce both over- and under diagnoses of the porphyrias as many substances found in urine can interfere with the test (e.g. Watson-Schwartz and Hoesch tests, Wood’s light). (Screening tests are laboratory tests that tell us if excess porphyrins are present or absent, but not how much.)
Now, if an acute porphyria is suspected the first step is a quantitation of the amount of PBG present in a urine sample (i.e. measuring how much is present). The urine sample is made alkaline and applied to an ion-exchange column. An ion-exchange column is a small tube only a few centimeters long filled with a fine powder that has charged groups on its surface. ALA and PBG will stick to the column while impurities will travel through the column and be washed out. Adding acid to the column will wash out the PBG. A special reagent added to this “acid wash” will react with PBG forming a coloured product. The amount of colour formed is proportional to the amount of PBG present and can be used to calculate the actual amount of PBG present. ALA is measured in an analogous manner. The quantitation of PBG and ALA has been simplified by the availability of kits containing all the required materials (Bio-Rad Laboratories, Montreal, QC). Elevation of urinary PBG by two or three times the upper reference limit is diagnostic of an acute porphyria.
If a non-acute porphyria is suspected, the first step is a non-specific measurement of the total uro- and coproporphyrins present in urine. Acid is added to the urine and the amount of light absorbed by the sample as measured in a laboratory instrument called a spectrophotometer. The amount of light absorbed is proportional to the amount of total uro- and coproporphyrins present and can be used to calculate the actual amount present. If the total amount is elevated than a sample of the urine must be evaluated by high performance liquid chromatography (HPLC) in order to make the diagnosis of porphyria and more importantly to identify the type of porphyria.
In HPLC, a urine sample suitably prepared is placed on a column. The column is a narrow stainless steel tube (25 x 0.5 cm) containing very fine specially treated particles of silica. As the urine sample is pumped through the column at very high pressure (4000 psi), the various types of uro- and coproporphyrins literally stick to the column each in a characteristic way. The result is the various uro- and coproporphyrins leave the column at different times. The quantity of each can be measured in a device called a spectrofluorimeter. The HPLC apparatus consisting of special high-pressure pumps, automatic sampling device, and spectrofluorimeter costs on the over of $50,000.
The porphyrin thus identified in the urine indicates the type of porphyria. For example, if both excess uro- and coproporphyrins were present, the one would diagnose erythropoietic porphyria. If only excess uroporphyrins are present, the one would diagnose porphyria cutanea tarda.
Version:1.0 StartHTML:0000000155 EndHTML:0000658471 StartFragment:0000000476 EndFragment:0000658454 body, div, table, thead, tbody, tfoot, tr, th, td, p { font-family: "Geneva"; font-size: x-small; } Porphyrias Symptoms Red Blood Cell Urinary Stool
Porphyrins Porphyrins Porphyrins
& Precursors
Acute
acute intermittent porphyria N none ALA, PBG none
variegate porphyria N, P none ALA, PBG coproporphyrin
coproporphyrin protoporphyrin
herediatry coproporphyria N, P none ALA, PBG coproporphyrin
coproporphyrin
ALAD deficiency porphyria N protoporphyrin ALA none
Non-Acute
erythropoietic porphyria P uroporphyrin uroporphyrin coproporphyrin
coproporphyrin coproporphyrin
erythropoietic protoporphyria P protoporphyrin none protoporphyrin
porphyria cutanea tarda P none uroporphyrin isocoproporphyrin
hepatoerythropoietic porphyria P protoporphyrin uroporphyrin isocoproporphyrin
N = neurological
P = photosensitizing
The table is a modification of Adriaans, 1995.
