Robert F. Labb#{233},1Clement A. Finch,2 Nathan J. Smith,3 Roscius N. Doan,3 S. K. Sood,4 and ... supply; or (c) a decrease in ferrochelatase ..... moot point.
CLIN. CHEM.
25/1.
87-92
(1979)
ErythrocyteProtoporphyrin/Heme Ratio in the Assessment of Iron Status Robert F. Labb#{233},1 Clement A. Finch,2 Nathan J. Smith,3 Roscius N. Doan,3 S. K. Sood,4 and Nishi Madan4
The protoporphyrinemia of iron deficiency is well recognized. Clinically, information on the protoporphyrin/heme molar ratio in whole blood offers certain advantages over protoporphyrin measurement alone. A procedure for determining this ratio is reported. Protoporphyrin is extracted, solubilized,
and
measured
hemin chloride) is precipitated precipitate
is dissolved
fluorometrically.
Heme
(as
with the blood proteins, the
in an alkaline/pyridine
solvent,
and
the resulting bispyridine ferriprotoporphyrin is measured spectrophotometrically. The molar ratio of these two metabolites correlates well with values for plasma ferritin, plasma iron, trarisferrin saturation, hemoglobin, and hematocrit. In some cases the ratio increases detectably while the other variables, especially hematocrit and hemoglobin, remain normal. Evidently it is a more sensitive index to iron status. For healthy men and women, the mean ratio is 16.0 (SD, 5.3). The mean + 3 SD, or a ratio of 32, is distinctly abnormal, as shown by a confirmatory test. We validated the test by surveying routine blood specimens obtained from several population groups.
Quantitatively, the most striking example of protoporphyrinemia can be found in erythropoietic protoporphyria, a rare inborn error of metabolism with decreased ferrochelatase activity (1, 2). So-called free erythrocyte protoporphyrin concentration (FEP) has been the customary measure of protoporphyrinemia. We considered the possibility that a more accurate idea of iron status could be gained from knowledge of the molar ratio of protoporphyrin to heme, that is, the ratio of reactant to product in the ferrochelatase-catalyzed reaction (Figure 1). Accordingly, to facilitate the determination of this ratio, we developed the procedure reported here, along with our clinical evaluation of it.
Materials and Methods Reagents All chemicals are reagent grade. De-ionized water is used. Hydrochloric acid. Prepare a 1.5 mol/L solution by diluting 128 mL of concentrated hydrochloric acid to 1 L with water.
AdditionalKeyphrases: normal values body
anemia
.
iron status of the
protoporphyrinemia
Protoporphyrin is converted to heme by the chelation of a ferrous ion with the concomitant loss of two protons from the porphyrin base (Figure 1). During normal erythropoiesis, the rate of protoporphyrin formation and the transfer of iron to its site of utilization are so well-balanced that mature circulating erythrocytes contain only about one molecule of excess protoporphyrin for each 30 000 molecules of heme formed. Hypothetically, three biochemical factors could alter this equilibriumto cause protoporphyrinemia:(a)an insufficiency of substrateiron,with normal protoporphyrin formation;(b) an increased production of protoporphyrin, with a normal iron supply; or (c) a decrease in ferrochelatase (EC 4.99.1.1) activity. An insufficient supply of iron available to the normoblast during hemoglobin synthesis is the most frequent cause of protoporphyrinemia, the principal example being nutritional iron deficiency. Protoporphyrinemia may also develop during marrow hyperplasia, an otherwise adequate iron supply producing a condition of relative iron-deficient erythropoiesis.
Departments of Laboratory Medicine,’ Medicine,2 and Pediatrics,3 University of Washington, Seattle, WA 98195. ‘ Department of Pathology, All-India Institute of Medical Sciences, Ansari Nagar, New Delhi 16, India 110016. Received July 6, 1978; accepted Oct. 24, 1978.
Hydrochloric acid/n-butanol. Dissolve one volume of nbutanol in 11 volumes of 1.5 mol/L hydrochloric acid, mixing adequately to clear. The butanol must be completely dissolved; otherwise, a separate layer of this component will concentrate the protoporphyrin and give erroneously low results. The solvent appears to be stable for several weeks, but does develop a new odor, tentatively identified as that of butyl chloride. Sodium tungst ate. Prepare a 0.33 mol/L solution by dissolving 10 g of the salt in 100 mL of water. Potassium hydroxide. Prepare a 0.5 mol/L solution by dissolving 28 g of potassium hydroxide in water and diluting to 1 L. Potassium hvdroxide/pvridine. Mix these solvents in a ratio of 25:1 by adding 20 mL of pyridine to 500 mL of 0.5 mol/L potassium hydroxide, giving a pyridine concentration of 0.5 mol/L. This solution appears to he stable indefinitelv. Coproporphyrin standard. Coproporphyrin suitable for preparing a fluorescence standard is obtained from either Sigma Chemical Co., St. Louis, MO 63178, or Porphyrin Products, Logan, UT 84321. The porphyrin as supplied is dissolved by adding dilute (1.5 mol/L) HCI to the container and either allowing it to stand overnight or heating it in boiling water for 5 mm. Determine the concentration spectrophotometrically (molar absorptivity = 4.88 X i0 at 399 nm) and then dilute the porphyrin to give a 0.010 nmol/mL solution. This standard is stable for many months, even at room temperature. CLINICAL CHEMISTRY, Vol. 25, No. 1, 1979
87
Ferroche
+
CH2
CH2COOH
CH2 COOH
Proto
CHZCH2
;.V-
