| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
1
Department of Laboratory Medicine, Box 359743, University of Washington, Seattle, WA 98104.
2
Department of Pediatrics, Room S-214, Stanford
University, Stanford, CA 94305-5119.
a Author for correspondence. Fax 206-731-3930; e-mail boblabbe{at}ix netcom.com.
 |
Abstract |
|---|
 Top Abstract IntroductionHistorical BackgroundFormationMetabolismClinical ApplicationsChemical and Physical PropertiesAnalytical ProceduresSummary and Future DirectionsReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionHistorical BackgroundFormationMetabolismClinical ApplicationsChemical and Physical PropertiesAnalytical ProceduresSummary and Future DirectionsReferences |
|---|
). Earlier brief reviews of both protoporphyrin (1)
and ZnPP (2) have appeared; this more comprehensive and
updated review is intended to highlight important advances and their
clinical relevance, primarily during the past decade. In view of this
latest research, additional diagnostic and new therapeutic applications
of ZnPP can be anticipated concomitant with a need for its
determination in the clinical laboratory.
|
the ZnPP/FREE ERYTHROCYTE PORPHYRIN ENIGMA
Much confusion in terminology between ZnPP and erythrocyte
protoporphyrin (EP) arose during early studies of blood porphyrins and
was a result of analytical procedures whereby ZnPP was for many years
unknowingly converted to EP during analysis. Unfortunately, this
history of inconsistent and/or inaccurate terminology relating to ZnPP
persists and has impaired its clinical utilization. ZnPP and EP, also
referred to as free EP, are terms often used interchangeably. ZnPP and
EP are not equivalent; they are different metabolites that arise under
different clinical conditions. The practice of considering ZnPP and EP
as equivalent has obscured the biochemical and analytical differences
between these two metabolites and detracted from the clinical utility
of both porphyrins, each of which has its own diagnostic application
when used appropriately. Although analytical results may sometimes be
similar, attempts to equate ZnPP and EP should be avoided to prevent
confusion, either in the selection of a test or in the interpretation
of a result.
Clinical applications of blood porphyrin analysis have been further clouded by the different reporting units that have been used in the medical literature by investigators and incorporated by manufacturers. A trend can now be seen for the increasing use of SI units and for use of a metabolic ratio in lieu of concentration, i.e., micromoles of porphyrin per mole of heme (3).
|
Historical Background |
|---|
|
TopAbstractIntroductionHistorical BackgroundFormationMetabolismClinical ApplicationsChemical and Physical PropertiesAnalytical ProceduresSummary and Future DirectionsReferences |
|---|
Metal-free protoporphyrin in erythrocytes has been associated with lead poisoning and iron deficiency since the early clinical studies of porphyrins. We now know that many of these reports were, in fact, describing the metabolically formed zinc chelate that was being converted to the metal-free protoporphyrin during sample processing (see Analytical Procedures). ZnPP per se received virtually no attention until 1974, when its presence in circulating erythrocytes was identified as a toxic response to lead (6), a biochemical change that found widespread use in screening young children for chronic lead exposure. Within a decade, during which investigators discovered that ZnPP quantification was not sufficiently sensitive to identify all at-risk cases of lead exposure, interest quickly waned in the clinical applications of ZnPP. In this era, many investigators presumed that ZnPP was formed nonenzymatically from Zn2+ and protoporphyrin, thereby giving it no particular metabolic purpose.
After an hiatus of several years, new discoveries began drawing attention again to ZnPP. One proposed physiological function considered that ZnPP enters the free heme pool in neonates to control the catabolism of heme until bilirubin (BR) conjugation becomes activated (7). Another hypothesis suggested that ZnPP may alter brain metabolism through modulation of CO production (8). The value of ZnPP in assessing iron status and diagnosing iron disorders continues to be elucidated, whereas discoveries of its unique role in heme metabolism, its association with CO and NO metabolism, and its therapeutic potential in hyperbilirubinemia all serve to support a broad clinical potential that has contributed to the sharp increase in research interest. This latest biochemical and clinical research now permits several new topics to be considered in some detail, including an evaluation of the overall clinical potential for ZnPP and its laboratory determination.
|
Formation |
|---|
|
TopAbstractIntroductionHistorical BackgroundFormationMetabolismClinical ApplicationsChemical and Physical PropertiesAnalytical ProceduresSummary and Future DirectionsReferences |
|---|
-aminolevulinic acid (ALA) condense to form the
monopyrrole porphobilinogen. Four molecules of this porphyrin precursor
cyclize and undergo several side-chain modifications to yield the
tetrapyrrole protoporphyrin. Ferrochelatase then catalyzes the
chelation of a ferrous ion by protoporphyrin as the terminal reaction
in heme formation (Fig. 2
).
|
It was a long-held view that an excess of metal-free protoporphyrin, reported to accumulate in iron deficiency, nonenzymatically chelated zinc ions to form ZnPP. However, subsequent research on ferrochelatase revealed that this enzyme catalyzes zinc as well as iron chelation by protoporphyrin (12)(13). The reaction with zinc is linked to and occurs as a byproduct of heme biosynthesis during states of suboptimum iron availability (2)(14). This secondary reaction occurs to a trace extent in the bone marrow during normal heme biosynthesis and cell maturation, whereas enhanced ZnPP accumulation appears in circulating erythrocytes during states of iron deficiency in the marrow (15)(16). ZnPP remains bound within circulating erythrocytes during their life span, unlike metal-free protoporphyrin, which can leak from the cells (17).
As a metabolic byproduct that forms during hemoglobin synthesis in the
developing erythrocyte, ZnPP is found in blood in healthy individuals
at a ratio of ~50 ZnPP molecules per 1 x
106 heme molecules (Fig. 2
). The minor non-heme
porphyrins in healthy erythrocytes consist of ~95% ZnPP and 5% EP.
An important exception to these ratios occurs with protoporphyria, in
which an inherited ferrochelatase deficiency leads to a massive
overproduction and accumulation of EP (11).
|
Metabolism |
|---|
|
TopAbstractIntroductionHistorical BackgroundFormationMetabolismClinical ApplicationsChemical and Physical PropertiesAnalytical ProceduresSummary and Future DirectionsReferences |
|---|
Administration of ZnPP to rats leads to its deposition in a variety of organs (plasma, liver, spleen, kidney, lung, and brain), causing decreased HO activity and CO formation (21). Hepatic HO activity in control rat neonates peaked between 1 and 4 days (22). ZnPP administration (40 nmol/g intraperitoneal) led to tissue ZnPP concentrations of 27–38 nmol/g, whereas HO activity in the liver was inhibited 27–51% between 1 and 4 days after dosing. This inhibition produced a 23–28% reduction in serum BR concentration. Furthermore, hepatic HO-1 protein concentrations were only transiently (24 h) and slightly increased. HO inhibition in the brain may interfere with the hypothesized role of CO as a neuronal messenger (23)(24)(25). However, ZnPP does not appear to cross the blood brain barrier in most studies (20)(26)(27); but one study using a sensitive fluorometric method reported finding barely detectable concentrations of ZnPP (22). These results may lead us to conclude that ZnPP could be an effective and safe compound for the treatment of severe neonatal jaundice. However, this conclusion may be questioned until a recent report citing potent inhibition of hematopoiesis in animal and human bone marrow cell cultures (28) can be clarified.
As first described by Maines (18) in 1981, when administered subcutaneously twice daily for 2 consecutive days, 40 nmol/g ZnPP was shown to inhibit by 40–60% hepatic, splenic, and renal HO activity in 5-day-old neonatal rats. Later studies with newborn rhesus monkeys showed that little ZnPP was excreted in the urine and bile; however, erythrocyte ZnPP increased dramatically 4 days after administration so that by day 11, ~46% of the administered dose had accumulated in the erythrocytes (20).
Through measurements of serum BR and hepatic and splenic HO activity, the duration of action of intraperitoneally administered ZnPP (40 nmol/g) to neonatal rats was found to be less than 1 week. Most of the ZnPP was scavenged and sequestered by the liver with minor amounts in spleen and kidney (22). Essentially, administered ZnPP is relocated from the initial tissue-binding sites to circulating reticulocytes where the porphyrin does not seem to affect further maturation of the erythrocytes. These findings suggest that the presence of the ZnPP and the consequent inhibition of HO do not lower serum BR concentrations by altering the turnover of fetal erythrocytes.
ZnPP AND HEME CATABOLISM
ZnPP and other metalloporphyrin (MP) analogs of heme can play a
prominent role in the catabolism of heme by membrane-bound HO in
conjunction with cytochrome P450 reductase, NADPH, and
O2 (29). This reaction (Fig. 2
) leads
to the formation of one molecule each of biliverdin, CO, and
Fe2+ (29). The biliverdin is
subsequently reduced by biliverdin reductase to form the linear
tetrapyrrole BR (30)(31).
Although BR has been identified as a potent antioxidant that may serve as an adjunct to bolster the immature antioxidant status of a neonate during the transitional phase from 5% (in utero) to the 21% (ex utero) O2 environment (32)(33), hyperbilirubinemia [>428 µmol/L (25 mg/dL)] is an important risk factor during the neonatal period (34). Excessive BR concentrations can lead to neurodevelopmental deficits and even death (35)(36)(37).
Current therapies for hyperbilirubinemia, such as phototherapy and exchange transfusion, are being applied after diagnosis of the problem (38)(39). A more desirable strategy, however, would be to prevent hyperbilirubinemia. Because HO is the rate-limiting enzyme in BR formation, its inhibition may afford important advantages for the control of BR formation (18)(40)(41). MP analogs of heme, such as the endogenous ZnPP and synthetic derivatives, whose central metal ion and/or 2- and 4-ring substituents have been replaced, are potent competitive HO inhibitors in vitro as well as in vivo (18)(19)(42). Maines (18) first described this property for ZnPP in studies using isolated neonatal rat tissue supernatants. In contrast to cobalt and iron protoporphyrin, ZnPP was found to affect neither heme biosynthetic enzymes nor cellular actions that depend on hemoproteins. These findings led to the conclusion that ZnPP may be useful as an experimental tool for the selective suppression of heme degradation (41)(43). Many subsequent in vitro and in vivo animal studies have further described the characteristics of ZnPP and other MP inhibitors of HO (26)(44)(45). Despite its therapeutic promise (46)(47)(48)(49), ZnPP has yet to be administered to humans.
techniques for in vivo studies of ZnPP
Although measurements of BR in plasma and tissue preparations have
been used as the principle indicator of ZnPP function
(26)(50), these measurements in whole organisms
are considerably more difficult to interpret because BR is distributed
into body compartments that are difficult to access
(51)(52). BR is lipophilic and accumulates
preferentially in lipid-containing tissues. BR quantification, except
for transcutaneous spectrophotometric measurements
(53)(54), is invasive. Finally, the
determination of free and conjugated BR in neonatal blood samples is
tedious and subject to errors (55).
The equimolar generation of the unique and volatile product, CO, from
heme catabolism (Fig. 2
), has led to the development of nontraditional
analytical and clinical methods and devices to assess and predict the
outcome of heme degradation
(50)(56)(57). CO production in
mammals is at least 85% attributable to the HO-mediated degradation of
hemoprotein heme (58)(59). The remainder may
derive from processes such as lipid peroxidation and/or bacterial
metabolism (60).
Thus, measurements of CO under controlled and steady-state conditions can be used for the in vitro (61) and ex vivo (62)(63) determination of cellular and tissue HO activity and for the assessment of HO inhibitors such as ZnPP (21). Furthermore, CO measurements permit the noninvasive in vivo estimation of the rate of heme degradation and BR formation as well as monitoring the efficacy of ZnPP as an HO inhibitor (22)(64)(65)(66)(67).
Under steady-state physiological conditions, most of the total body CO is produced in the spleen, which sequesters and breaks down senescent erythrocytes. The released heme is then catabolized locally, or presumably, when the spleen capacity is exceeded, is transported to the liver. The generated CO binds to hemoglobin to form carboxyhemoglobin (COHb), which is transported to the lungs, where CO is exchanged for O2 and expired with the breath (68)(69). Thus, total body heme degradation and BR production can be estimated not only through COHb measurements in blood by CO-oximetry (70), or more accurately by gas chromatography (71)(72)(73), but also noninvasively through CO measurements in expired air (60).
The latter is performed most accurately by measuring the CO in the outlet air of a chamber that contains the subject (64)(68). Alternatively, and more conveniently, CO can be measured in end-tidal breath for an estimation of the rate of heme degradation and BR formation (74)(75)(76). COHb and breath CO measurements must be corrected for inhaled CO in ambient air (69).
CO-measuring methodology is being applied in animals and humans with
considerable success, not only for the diagnosis of hemolytic disease
(57) but also increasingly for monitoring the efficacy of HO
inhibitor administration for the control of BR formation (Table 1
) (65). These measurements reflect total body HO
activity as one or both isoenzymes catalyze heme turnover in many body
compartments (77).
|
techniques for in vitro studies of ZnPP
Many in vitro studies have been performed to elucidate the role of
ZnPP in HO-mediated heme degradation
(18)(26)(40). Similarly, for in vivo
studies, BR and CO are the products most frequently quantified. In
general, measurements of BR are most frequently used because they can
be made with the more readily available spectrophotometric
(44)(78) and HPLC instrumentation
(31). However, these methods require highly purified
membrane preparations (microsomes) free of interfering substances, such
as light-dispersing particles and hemoglobin (44). Thus,
sample preparation tends to be tedious and time-consuming. Measurements
of CO, on the other hand, require special gas handling techniques and
gas chromatographic instrumentation (61). However, once this
technique has been mastered, the method lends itself to more specific,
rapid assays for which the enzyme matrix, and thus the tissue type or
the degree of purification, is not a factor (79).
Furthermore, the addition of colored porphyrin compounds, such as ZnPP,
does not interfere with this measurement technique (21)
(Table 2
).
|
|
Clinical Applications |
|---|
|
TopAbstractIntroductionHistorical BackgroundFormationMetabolismClinical ApplicationsChemical and Physical PropertiesAnalytical ProceduresSummary and Future DirectionsReferences |
|---|
assessment of nutritional iron status
Numerous tests that measure different iron indices (storage,
transport, end product, and receptor) are available for the assessment
of nutritional iron status. It is noteworthy that each of these tests
measures a different facet of iron metabolism, and therefore, they
should not be expected to correlate with one another. These tests range
widely in specificity and sensitivity, and no one test adequately
diagnoses iron deficiency (80). The generally accepted
"gold standard" test for iron deficiency is the determination of
bone marrow iron stores, but the test is too costly and invasive for
routine screening or monitoring. This limitation underscores a key
benefit of ZnPP, which in fact has been shown to reflect the iron
status in the bone marrow (16). Storage iron as reflected in
serum ferritin concentration is often considered the most suitable
index for iron status. But, because ferritin is an acute phase protein,
its concentration is used to best advantage in combination with the
ZnPP/heme ratio (ZnPP/H) for the evaluation of many iron disorders
(Table 3
).
|
A cost-effective approach for the assessment of iron status is to first
determine ZnPP/H; if the result is within the reference range, then the
marrow and peripheral tissues are assumed adequately supplied with
iron, regardless of the ferritin concentration, which may show low iron
stores in the presence of adequate tissue supplies. On the other hand,
if ZnPP/H is increased, some follow-up is always indicated because of
the many factors that can influence iron utilization in the marrow.
Table 3
summarizes the interpretation of ZnPP/H, especially when used
in combination with serum ferritin concentration. Hastka et al.
(81) suggested combining the ZnPP data with ferritin and
hemoglobin in assessing iron deficiency.
iron status in pediatrics
Hematocrit and hemoglobin can, by definition, diagnose iron
deficiency only at the stage of anemia. The practice of using these
tests to screen children for iron status misses many who are preanemic
but iron depleted and probably should be receiving iron supplementation
(82)(83). Although hemoglobin and hematocrit are
simple tests to perform, they are neither as sensitive nor as specific
as ZnPP/H, which can be used effectively in routine pediatric practice
(84)(85). Avoiding iron deficiency anemia is
especially important in young children during stages of rapid growth
and development because low iron may lead to impaired motor and
cognitive development (86) as well as exacerbate any
exposure to lead (87).
iron status in pregnancy
Because iron-deficiency anemia can develop during pregnancy, the
iron status of these patients commonly is monitored by determinations
of hemoglobin, hematocrit, and/or ferritin (88). During the
course of pregnancy, iron status monitored by these methods is based on
concentration and, accordingly, may often be inaccurate because of
dilution by plasma volume expansion. Schifman et al. (89)
concluded that ZnPP concentration measurements had sufficient
diagnostic sensitivity and predictive value for iron status to be used
effectively in pregnant patients. The value of the test is further
enhanced by use of ZnPP/H, which obviates the dilution problem, because
both ZnPP and hemoglobin (or heme) are diluted equally, thereby
avoiding the misinterpretation of laboratory results that may occur
with plasma volume change (90).
iron status of blood donors
Potential blood donors are screened routinely for iron deficiency,
primarily by a very simple standardized copper sulfate/hemoglobin
precipitation test. Although this test detects iron-deficiency anemia,
it does not accurately reflect iron status because some frequent donors
will have low iron stores (ferritin) and yet peripheral tissues can be
receiving adequate iron as shown by a normal ZnPP/H. This application
of the test has been evaluated and described repeatedly
(91)(92)(93). In our experience (results not published), the use
of ZnPP/H as a screen would cause fewer potential donors overall to be
deferred because of poor iron status. Not only would more volunteers
become eligible for donation, but they may be different donors because
ZnPP/H is equivalent to bone marrow iron available (16)
rather than the principal end-product of iron utilization (hemoglobin).
Thus, ZnPP/H can provide an improvement over current procedures to
screen blood donors for iron status. As Finch (94) has
discussed regarding iron regulators, blood donation creates an
imbalance between the iron needs of the marrow and the iron supplied to
the marrow, leading to increased ZnPP. In polycythemia patients treated
with phlebotomy, a situation somewhat similar to blood donation, EP (or
ZnPP) was found to increase in correlation with a decrease in ferritin
(95), again demonstrating the linkage of iron status with
ZnPP.
diagnosing other disorders in iron metabolism
Relative iron deficiency is a condition in which iron is being
delivered to the marrow at a rate insufficient to meet the demands of
accelerated erythropoiesis (94). Examples where this may
occur include ineffective erythropoiesis or hemolytic anemia, cases in
which iron requirements for erythrocyte production become exaggerated.
Sideroblastic anemia is a metabolic defect in iron utilization that
produces a deficiency-like response with increased ZnPP/H
(11). Impaired iron utilization is commonly found in anemia
of chronic disease and leads to increased ZnPP/H, which can be used to
identify such anemias (96). As a rule, a greater proportion
of hospitalized patients can be expected to have increased ZnPP/H
because of the numerous etiologies that impair iron utilization in the
bone marrow. Despite this apparent lack of specificity, ZnPP is very
specific when defined in terms of marrow iron requirements rather than
in terms of iron stores (serum ferritin) or the products of iron
utilization (hemoglobin and hematocrit). Given a clear understanding
and accurate interpretation of results, evidence shows that ZnPP/H is a
good screening tool for iron deficiency even in hospitalized patients
(97).
Thalassemia is characterized by a disordered globin chain formation. However, thalassemia also suggests iron deficiency by virtue of its characteristic low mean corpuscular volume. The latter is explained by an extreme erythroid hyperplasia in thalassemia that creates a state of relative iron deficiency (98). ZnPP can be used to differentiate this apparent iron deficiency based on low mean corpuscular volume from that attributable to impaired hemoglobin (or globin) synthesis in thalassemia (99)(100)(101).
Renal disease is a case in which erythropoietin may be administered to stimulate hematopoiesis; this stimulation of erythrocyte production can then lead to a state of relative iron deficiency. In comparing indicators of iron status in hemodialysis patients, Fishbane and Lynn (102) concluded that ZnPP offered the greatest utility for predicting the need for iron therapy. Braun et al. (103) did not find a ZnPP response to iron therapy in hemodialysis patients, although they did note a ZnPP increase that correlated with blood lead concentrations. Similarly, Baldus et al. (104) could find no correlation of ZnPP with other indicators of iron status. Hematopoiesis in renal patients may be a special situation in which iron status indicators are not always interpretable as for other conditions.
diagnosing lead toxicity
The effects of lead on the porphyrin/heme biosynthetic pathway
have been studied extensively. These findings have demonstrated that
lead toxicity and iron utilization are inextricably linked
(87), at least in bone marrow. As a toxic substance, lead
profoundly impairs heme biosynthesis although ZnPP increases. Before
porphyrin formation, lead inhibits porphobilinogen synthase (Fig. 2
),
which leads to excessive urinary excretion of ALA. The common
perception that lead also inhibits ferrochelatase is not entirely
accurate; in vitro lead binds to the sulfhydryl groups of
ferrochelatase, but in vivo the inhibition evidently occurs only during
extreme, acute toxicity (105). This phenomenon may explain
the lead inhibition of ferrochelatase observed in cultured hepatocytes
(106). If the effect of lead in marrow during the state of
chronic lead toxicity were through inhibition of ferrochelatase, this
enzyme inhibition would be expected to increase metal-free
protoporphyrin as found in protoporphyria, an inherited deficiency of
ferrochelatase (11). Moreover, the chelation of iron and
zinc appears to be catalyzed by the same ferrochelatase
(107), which would be unlikely to cause increased ZnPP
production by lead. Thus, lead evidently impairs iron delivery to or
utilization in immature erythrocytes, thereby inducing the iron
deficiency-like response with increased ZnPP appearing in mature
erythrocytes.
In 1991, the CDC pronounced that neither metal-free protoporphyrin nor ZnPP/H was suitable as a screening test for chronic lead exposure in infants and young children (108), a conclusion that the medical community has subsequently extrapolated to all ages. Nevertheless, ZnPP/H can provide valuable information when used in combination with blood lead concentration as a monitor of tissue toxicity in individuals of all ages with a large body burden of lead (109). ZnPP/H has the added benefit of detecting iron deficiency, a concomitant of lead poisoning in young children (87)(109). ZnPP/H can also be a convenient and cost-effective screening test for lead exposure in adults, especially in the workplace (110) because adults do not suffer the infant-type permanent neurologic damage. The effects of chronic exposure in adults are generally reversible with treatment.
ZnPP AS A THERAPEUTIC AGENT
Although ZnPP has diagnostic applications, it has also therapeutic
potential. Because neonatal jaundice is a transitional and temporary
syndrome, an effective chemopreventive agent with a relatively short
duration of action would be desirable (22)(52).
Clinical efficacy studies in humans have been performed only with tin
proto- and mesoporphyrin because their potencies relative to ZnPP were
greater (78). Because ZnPP weakly up-regulates HO-1 activity
no more than twofold (111) and because it lacks
photosensitizing properties, this compound may be suitable for the
management of hyperbilirubinemia. In addition, ZnPP is naturally
occurring, and thus the body is believed to have the mechanisms to
dispose of the injected drug. Of possible benefit, ZnPP does not cross
the neonatal blood brain barrier
(20)(26)(27).
dosage and routes of administration
ZnPP administration can be achieved via several routes, each of
which has advantages and disadvantages (112). Enteral
administration would be clinically advantageous; however, the
physicochemical characteristics of ZnPP preclude this route of
administration. ZnPP is precipitated and probably stripped of zinc in
the highly acidic environment of the stomach. Even if it survives this
barrier intact, ZnPP could not become functional because it is not
resolubilized in the dilute bicarbonate milieu of the upper intestine.
Thus, enteral administration of 40 µmol/L ZnPP/kg to neonatal and
adult rats did not affect local (enteric) or systemic (hepatic and
splenic) HO activity (113). For animal studies
intraperitoneal administration of ZnPP has been used frequently because
this route appears to ensure relatively rapid systemic absorption and
distribution (18)(22)(114).
Subcutaneous and intramuscular ZnPP administration at 40 µmol ZnPP/kg reduced BR production rates by ~20% during hours 2–12 in adult rats as indexed by postpeak BR concentrations (20). Intramuscular administration was used in the majority of MP studies in neonates (48)(49). However, this method has the limitation that only relatively small volumes can be administered to neonates because their skin is thin and fragile.
Intravenous administration for other MPs to human neonates (47)(115) produces rapid systemic distribution and inhibition of HO in liver, kidney, and spleen within a few hours of administration (27)(28). This advantage may need to be offset by the relatively rapid disappearance of the compound from the circulation. A slower, more gradual release via intraperitoneal, subcutaneous, or intramuscular administration may be more effective clinically. The route of administration may contribute to the clinical efficacy or exacerbation of potential side effects (28). Thus, when ZnPP is to be used as a drug or chemical agent, consideration must be given to the target tissue or organ, so that ZnPP packaging (mode of delivery, dose, and lifetime) and desired effect will be optimized (63)(116).
|
Chemical and Physical Properties |
|---|
|
TopAbstractIntroductionHistorical BackgroundFormationMetabolismClinical ApplicationsChemical and Physical PropertiesAnalytical ProceduresSummary and Future DirectionsReferences |
|---|
stability and solubility
Porphyrin solubility is related to the number of free carboxyl
groups. Protoporphyrin, bearing only two such groups is lipophilic, as
is ZnPP, which shares numerous chemical properties with other MPs. For
example, it is soluble and stable in strongly alkaline aqueous
solution. MPs are also very soluble in basic organic solvents such as
pyridine and ethanolamine as well as in some surfactants. In contrast,
a particularly important property of ZnPP is its rapid loss of zinc
upon exposure to strong acid. Preserving ZnPP during extraction from
biological materials typically depends on use of a neutral or weakly
acidic organic medium (105)(118).
In blood, ZnPP evidently is bound to a heme site on globin (119). Hirsch et al. (120) further studied the interaction of ZnPP with oxyhemoglobin using microcalorimetry, front-face fluorometry, absorption spectroscopy, oxygen equilibration, and isoelectric focusing. Based on such detailed observations, they concluded, "ZPP binds to intact, tetrameric hemoglobin at non-heme pocket sites in a nonspecific, weak, noncovalent interaction". Thus, ZnPP binding details in erythrocytes may not yet be fully elucidated.
spectrum and fluorescence
Among the physical properties that distinguish ZnPP from most
other MPs of natural origin is its fluorescence. One exception is
chlorophyll, which is a magnesium-containing porphyrin-like structure.
Ferrous protoporphyrin (heme) and vitamin B12, a
cobalt-containing pyrrole structure or corrin, are nonfluorescent.
The absorptivity of ZnPP usually has not been determined directly but
rather indirectly after its conversion to metal-free protoporphyrin,
which is accomplished by dissolving ZnPP in HCl. This approach is used
because the free-base protoporphyrin or its methyl ester can be more
easily prepared in highly purified form. A longstanding controversial
issue had been the correct molar absorptivity of protoporphyrin, often
used as a reference material. The definitive study to resolve this
issue was reported by Gunter et al. (121) in 1989. Using
highly purified material, they determined that the absorptivity for
free-base protoporphyrin at the Soret maximum is 297
L · mmol-1 · cm-1.
Spectral characteristics of both compounds are, however, known
(122). The fluorescence characteristics, including
excitation and emission peaks, of protoporphyrin in various forms
relevant to ZnPP determination are summarized in Table 4
. These spectral properties have been used in the diagnosis of
porphyrin metabolism disorders (123). Although both
ZnPP and protoporphyrin fluoresce, albeit at slightly different
wavelengths, only protoporphyrin in the free-base form is known to be
phototoxic, causing cellular and tissue damage on exposure to porphyrin
excitation wavelengths (17).
|
|
Analytical Procedures |
|---|
|
TopAbstractIntroductionHistorical BackgroundFormationMetabolismClinical ApplicationsChemical and Physical PropertiesAnalytical ProceduresSummary and Future DirectionsReferences |
|---|
Hematofluorometry is the fastest and easiest means of determining ZnPP in blood specimens. A hematofluorometer is a dedicated instrument that measures directly in whole blood or in washed erythrocytes the ratio of ZnPP fluorescence to heme (hemoglobin) absorption and presents the result as a ratio of these two factors (3)(125) Although the determination is simple and rapid, it is not without pitfalls. The plasma interference, most of which is attributable to BR, gives falsely increased values (126). Other potentially interfering substances include some drugs (127) as well as high plasma riboflavin concentrations. A common solution to eliminate interference requires washing the erythrocytes free of plasma (127)(128), although alternatives to washing have been offered (129). A second potential problem is the need for complete oxygenation of hemoglobin, lack of which gives falsely low values because of a shift in hemoglobin absorption. One solution to this problem is to use a reagent that converts the hemoglobin to cyanmethemoglobin (128)(130). Hemolysis has also been considered to give erroneous values, but this is now questioned and means of resolving most potential problems have been described (129). Although many other reporting units have been in use, the currently recommended unit is micromoles of ZnPP per mole of heme (3). Molar concentrations are recommended as SI units, and the ratio of metabolites is recommended in part to eliminate the effects of dilution by changes in plasma volume. Some hematofluorometers, especially older units, may not report results in SI units or as a ratio.
proficiency testing
For laboratories planning to set up a ZnPP test, a federally
sponsored national program is available for participation. This is the
Erythrocyte Protoporphyrin Proficiency Testing Program, which can be
contacted at Toxicology Section, Wisconsin State Laboratory of Hygiene,
2601 Agriculture Drive, P.O. Box 7996, Madison, WI 53707-7996 (phone
608-224-6252; e-mail toxpt@slh.wisc.edu).
cost considerations
Whether for screening, diagnosis, or therapeutic monitoring, ZnPP
determination should become a routine laboratory test. The
determination of ZnPP/H by hematofluorometry is the easiest available
method. Purchase of an hematofluorometer for measuring ZnPP/H directly
in whole blood or erythrocytes costs less than $5000. Currently, two
different hematofluorometers are on the market. One is manufactured by
Aviv Associates, Inc. (Lakewood, NJ) and the other is the ProtoFluor Z
manufactured by Helena Laboratories (Beaumont, TX). Only manual test
equipment is currently being manufactured.
The actual cost of a ZnPP/H determination can be influenced by many factors, but typically ~$3.00 per test is realistic with use of the ProtoFluor-Z reagent (130) and without washing the cells. A common practice of not using the reagent but washing the cells to remove interfering substances and cause oxygenation of hemoglobin (127) eliminates one cost factor while adding another. Thus, the cost can be relatively low, but it is affected by the method chosen as well as test volume. A series of tests can be run at the rate of approximately one per minute with current hematofluorometers.
|
Summary and Future Directions |
|---|
|
TopAbstractIntroductionHistorical BackgroundFormationMetabolismClinical ApplicationsChemical and Physical PropertiesAnalytical ProceduresSummary and Future DirectionsReferences |
|---|
Opportunities abound for further investigations and developments relating to ZnPP. Beyond basic biochemistry, these include diagnostics and therapeutics. A few examples can illustrate the potential.
Given the tight coupling of ZnPP with heme formation, does ZnPP play a role in heme metabolism and, if so, what role? Providing a sparing action to limit heme catabolism is supported by experimental results, but this does not adequately explain the close linkage of ZnPP to iron status. Possibly associated with iron utilization is the mechanism, still unknown, by which lead causes ZnPP to accumulate in erythrocytes.
If CO produced via HO activity has neuronal tissue effects, as has been suggested, does ZnPP then affect nerve function in a secondary manner by modulating CO production? Alternatively or additionally, ZnPP may affect nerve cell metabolism directly and without CO intervention through its inhibition of nitric oxide synthase and soluble guanylyl cyclase enzyme activity (116).
The metabolic fate of either endogenous or exogenous ZnPP is unknown. Although animal studies have shed some light on ZnPP disposition, human studies remain to be performed. Of course, such knowledge underlies any therapeutic uses of ZnPP.
Development of an automated method for ZnPP/H determination would contribute toward broader acceptance of this diagnostic test. A ZnPP/H result provided as a component of the hemogram panel would be ideal. As a complement to these other hemogram tests, ZnPP/H would add a cost-effective, less invasive indication of marrow iron status in addition to end-product (hemoglobin or hematocrit) status.
A test worthy of evaluation for monitoring ZnPP therapy in neonates is measurement of the ratio of CO (from COHb) to ZnPP/H. The result could provide a measure of ZnPP inhibition of HO activity and, in turn, diminished BR formation. A modification of this concept might be measurement of the ratio of free BR to ZnPP/H in which a decrease may reflect inhibited HO activity.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
-aminolevulinic acid; HO, heme oxygenase; MP, metalloporphyrin; COHb, carboxyhemoglobin; and ZnPP/H, zinc protoporphyrin/heme ratio. 
|
References |
|---|
|
TopAbstractIntroductionHistorical BackgroundFormationMetabolismClinical ApplicationsChemical and Physical PropertiesAnalytical ProceduresSummary and Future DirectionsReferences |
|---|
