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Zinc protoporphyrin: a metabolite with a mission.

The future importance of zinc protoporphyrin (ZnPP)[3] as an essential metabolite promises to far exceed historical experience. Although ZnPP determination currently is used in some clinical applications to assess iron status and diagnose iron disorders, this focus may be but the tip of the iceberg. Recent discoveries concerning ZnPP metabolism reach beyond iron status, extending to roles in physiological chemistry, metabolic regulation, and therapeutic application, all of which have been responsible for the enhanced interest in ZnPP (Fig. 1). 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.

[FIGURE 1 OMITTED]

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

Fischer and his colleagues succeeded in the complete syntheses of protoporphyrin and heme in 1929, for which he subsequently received the Nobel Prize. In 1932, van den Bergh and co-workers first identified metal-free protoporphyrin as a constituent of blood, an observation soon reported by others as being responsible for the "fluorocytes" found among circulating erythrocytes. Darrien and Cristol published a 1930 report entitled Zincoporphyrinurie, which became the first identification of a naturally occurring zinc porphyrin. However, clinical studies of blood porphyrins were really promoted by the development of analytical methods, beginning with that of Grinstein and Watson (4) in 1943. The use of EP for the detection of preanemic iron deficiency was described as early as 1966 by Dagg et al. (5).

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 [Zn.sup.2+] 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

During the two decades of the 1950s and 1960s, the porphyrin and heme biosynthetic pathway was developed in detail, and this aspect of biochemistry has been well summarized, including porphyrin metabolism disorders (9-11). Through a series of reactions, two molecules of [delta]-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 [10.sup.6] 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).

[FIGURE 2 OMITTED]

Metabolism

FATE of ZnPP

The presence of ZnPP in erythrocytes as the result of errant heme synthesis leads to its deposition in the spleen, and possibly the liver, when the aged erythrocytes are sequestered and the heme and ZnPP are released and bound to heme oxygenase (HO) in these organs, leading to the subsequent slower degradation of heme. Because ZnPP does not bind [O.sub.2], it does not undergo the HO-mediated oxidative degradations characteristic of heme (18). In vitro assays with rat liver and spleen homogenates revealed no evidence of CO production with 50 [micro]mol/L ZnPP as substrate (19). Minor excretion of ZnPP in bile, but not in urine, has been reported for neonatal rhesus monkeys. However, only 0.12% of administered ZnPP was excreted via bile during the 28-h study period (20). Thus, the metabolic fate of neither the HO-ZnPP complex nor ZnPP is known.

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 (23-25). 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 [O.sub.2] (29). This reaction (Fig. 2) leads to the formation of one molecule each of biliverdin, CO, and [Fe.sup.2+] (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) [O.sub.2] environment (32, 33), hyperbilirubinemia [>428 [micro]mol/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 (35-37).

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 (46-49), 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, 64-67).

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 [O.sub.2] 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 (71-73), 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 (74-76). 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

In the discussion that follows, the published work cited frequently refers to the porphyrin under investigation as EP or free EP and reports results in concentration units. Because these substances are most often formed as an artifact of porphyrin analysis and reflect instead the presence of ZnPP, the conclusions cited are not necessarily invalidated. Furthermore, although concentration units are not invalid, the porphyrin/heme ratio is now preferred (3).

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 (91-93). 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 (99-101).

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 [micro]mol/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 [micro]mol 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

A comprehensive resource is available for readers wishing detailed information about the chemical and physical properties of porphyrins and MPs (117). Perhaps the one preferred resource for ZnPP in the clinical laboratory is the NCCLS Approved Guideline (3). Below are described primarily those properties of ZnPP that may be of special interest for clinical applications.

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 [B.sub.12], 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.sup.-1] * [cm.sup.-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

METHODOLOGY

Three different types of analytical procedures are in common use for ZnPP determination in biological materials, which is typically blood for clinical purposes: (a) extraction of both ZnPP and EP with an ethyl acetate/ acetic acid solvent, back-extraction into dilute HCl, and then measurement of the total metal-free protoporphyrin by either fluorometry or absorptivity (3, 4); (b) extraction of ZnPP with a neutral or weakly acidified organic solvent, separation using HPLC, and then measurement of ZnPP by fluorometry (22,105,118,124); or (c) direct measurement of ZnPP in whole blood or washed erythrocytes by hematofluorometry (3). Many variations of all of these procedures have been published and are in use. Two particular resources are especially recommended for the clinical laboratory, namely, the 1989 report by Gunter et al. (121) and the 1996 NCCLS report on erythrocyte protoporphyrin testing (3).

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

ZnPP should be distinguished from EP or free EP. ZnPP is a product of disordered heme biosynthesis, and the concentration appearing in circulating erythrocytes is closely linked to iron status in the bone marrow. ZnPP measured as a molar ratio to heme (ZnPP/H) has found its greatest value in assessing iron nutrition status and in diagnosing iron metabolism disorders. The clinical importance of ZnPP is not limited to iron, however, because its accumulation can influence heme catabolism, leading to modulation of BR and CO production. The therapeutic potential of ZnPP as a preventive measure for hyperbilirubinemia has yet to be tapped with clinical studies founded on established biochemical knowledge. Of possible physiological importance, CO in particular may influence nerve cell function.

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.

This investigation was supported in part by National Institutes of Health Grants DK35816 (R.F.L.), HD14426 (H.J.V. and D.K.S.), and HL58016 (H.J.V. and D.K.S.) and the Mary L. Johnson Research Fund (H.J.V. and D.K.S.). We acknowledge the invaluable assistance of Ronald J. Wong during the preparation of this manuscript.

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ROBERT F. LABBE, [1] HENDRIK J. VREMAN, [2] and DAVID K. STEVENSON [2]

[1] Department of Laboratory Medicine, Box 359743, University of Washington, Seattle, WA 98104. [2] Department of Pediatrics, Room 5-214, Stanford University, Stanford, CA 94305-5119.

[3] Nonstandard abbreviations: ZnPP, zinc protoporphyrin; EP, erythrocyte protoporphyrin; BR, bilirubin; ALA, [delta]-aminolevulinic acid; HO, heme oxygenase; MP, metalloporphyrin; COHb, carboxyhemoglobin; and ZnPP/H, zinc protoporphyrin/heme ratio.

* Author for correspondence. Fax 206-731-3930; e-mail boblabbe@ix.netcom.com.

Received July 1, 1999; accepted August 23, 1999.
Table 1. Effect of heme loading (30 nmol/g) and ZnPP (40 nmol/g)
treatment at age 12 h on indices of heme degradation in 24-h-old
rhesus neonates (n = 4). (a)

Treatment group VeCO, (b) [micro] L COHb, %-tHb
 x [h.sup.-1] x
 [kg.sup.-1]

Baseline 20.9 [+ or -] 0.9 0.52 [+ or -] 0.07
Heme 44.2 [+ or -] 7.1 1.00 [+ or -] 0.23
Heme+ ZnPP 19.0 [+ or -] 3.8 0.50 [+ or -] 0.14

 Plasma BR

Treatment group mg/dL [micro] mol/L

Baseline 5.4 [+ or -] 1.0 92 [+ or -] 17
Heme 10.4 [+ or -] 1.9 178 [+ or -] 32
Heme+ ZnPP 6.9 [+ or -] 1.6 118 [+ or -] 27

(a) Adapted from Vreman et al. (21).

(b) VeCO, rate of total body CO excretion.

Table 2. HO activity at t = 24 h in 13 000g tissue homogenate
fractions from rhesus neonates (n = 4) treated at t = 12 h with
heme loading (30 nmol/g) and ZnPP (40 nmol/g). (a)

 HO activity, nmol CO x [h.sup.-1] x mg [protein.sup.-1]

Group Liver Spleen

Control 0.19 [+ or -] 0.10 0.62 [+ or -] 0.20
Heme 0.33 [+ or -] 0.23 0.78 [+ or -] 0.14
Heme+ ZnPP 0.08 [+ or -] 0.06 0.28 [+ or -] 0.03

 HO activity, nmol CO x [h.sup.-1] x mg [protein.sup.-1]

Group Kidney Brain

Control 0.12 [+ or -] 0.07 0.46 [+ or -] 0.14
Heme 0.32 [+ or -] 0.15 0.68 [+ or -] 0.28
Heme+ ZnPP 0.30 [+ or -] 0.13 0.60 [+ or -] 0.26

 HO activity, nmol CO x [h.sup.-1] x mg [protein.sup.-1]

Group Skin Intestine

Control 0.04 0.30 [+ or -] 0.12
Heme 0.12 [+ or -] 0.02 0.41 [+ or -] 0.42
Heme+ ZnPP 0.10 [+ or -] 0.12 0.23 [+ or -] 0.12

(a) Adapted from Vreman et al. (21).

Table 3. Interpretation of ZnPP/H (a) in the diagnosis of iron
disorders.

Lab results Interpretation

Low ZnPP/H Adequate systemic iron supply.
 (<60 [micro] mol/mol) No further deficiency
 work-up needed.

Midrange ZnPP/H Possible nonreplete iron status.
 (60-80 [micro] mol/mol) Consider inadequate diet, blood
 loss, anemia of chronic disease,
 and other causes.

High ZnPP/H-low ferritin An indication of iron-deficient
 [>80 mmol/mol ZnPP/H plus erythropoiesis attributable to
 low to normal ferritin low marrow iron supply,
 (<20 [micro] mol/mol)] possibly related to depleted
 iron stores.

High ZnPP/H-high ferritin Severe inflammatory block, anemia
 [>80 [micro] mol/mol ZnPP/H of chronic disease, other
 plus normal to high ferritin etiologies of impaired iron
 (>200 [micro] mol/mol)] utilization.

Lab results Recommendations

Low ZnPP/H Iron stores can be determined
 (<60 [micro] mol/mol) using serum ferritin
 concentration.
 If ZnPP/H is <40 [micro] mol/mol,
 consider test for iron
 overload and/or
 hemochromatosis using serum
 ferritin
 concentration and/or
 percentage of transferrin
 saturation.

Midrange ZnPP/H Hgbbor Hct from CBC may
 (60-80 [micro] mol/mol) support a state of iron
 depletion.
 If clinically indicated,
 obtain serum ferritin
 concentration
 to differentiate between low
 iron stores and
 inflammatory block of iron
 release from stores.
 Latter may be identified using
 C-reactive protein.

High ZnPP/H-low ferritin Iron supplementation indicated.
 [>80 mmol/mol ZnPP/H plus Monitor therapy with decrease
 low to normal ferritin in ZnPP/H and/or
 (<20 [micro] mol/mol)] increase in reticulocyte count.
 Consider etiologies to
 include: poor diet, abnormal
 blood loss, anemia of chronic
 disease, chronic lead exposure.

High ZnPP/H-high ferritin Correct cause of impaired iron
 [>80 [micro] mol/mol ZnPP/H utilization.
 plus normal to high ferritin Effectiveness of iron
 (>200 [micro] mol/mol)] supplementation limited.
 Consider severe chronic lead
 poisoning.

(a) ZnPP/H is an indicator of iron supplied to developing erythrocytes
and available for heme formation. A ratio >80 [micro] mol/mol is
biochemical evidence of relative iron-deficient erythropoiesis
attributable to any etiology.

(b) Hgb, hemoglobin; Hct, hematocrit; CBC, complete blood count.

Table 4. Fluorescence excitation and emission maxima for different
states and forms of protoporphyrin. (a)

Protoporphyrin Excitation, nm Emission, nm

Erythrocytes in iron deficiency 424 594
Erythrocytes in chronic lead 424 594
 toxicity
Zinc protoporphyrin-globin 423 594
Magnesium protoporphyrin-globin 420 594
Protoporphyrin-globin 403 625
Erythrocytes in erythropoietic 397 625
 protoporphyria
Acid-extracted protoporphyrin from 405 606
 any of the above
Ferrous protoporphyrin (heme) No fluorescence No fluorescence

(a) Adapted from Gambino R. Protoporphyrins. Lab Rep Physicians
1986;8:9-13.
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Author:Labbe, Robert F.; Vreman, Hendrik J.; Stevenson, David K.
Publication:Clinical Chemistry
Article Type:Clinical report
Date:Dec 1, 1999
Words:10389
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