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We report an 11-y-old boy (J) with severe growth failure, hepatomegaly, vasculitis, osteoporosis, and immune deficiency. He has been treated symptomatically, but with no definitive diagnosis. Adventitious investigation showed gross hyperzincemia. On further investigation a previously undescribed plasma zinc-binding protein with mass 140-300 000 and abnormalities of zinc metabolism were found.

METHODS

Patient. The patient (J) was born in 1984 at 36-wk gestation by cesarean section because of fetal distress. Neonatal problems included early tachypnea and jitteryness, conjugated hyperbilirubinemia, hepatosplenomegaly, and hypoglycemia. Tests for inborn errors of metabolism and congenital infection were negative. The parents are unrelated, and there is no family history of growth defects, liver disease, or any other symptoms reported here. He has an elder sister who is of normal stature and is in good health.

In early childhood J was small and had marked head lag. An intermittent vasculitic type of rash was noted. There were associated low Hb, neutrophils, and platelets, raised IgA with high normal IgG and IgM, low T cells, and reduced neutrophil mobility but normal phytohemagglutinin antigen stimulation test. At age 7 T cells were normal, but monocytes and B cells were low. Treatment was with multiple blood and platelet transfusions with prednisolone to control symptoms of vasculitis and asthma. Any attempts to withdraw or reduce this treatment since have not been successful, as he develops wheezing, worsening vasculitis, and swollen hands. The current steroid dose is 10 mg and 5 mg on alternate days. Other current medication includes azothioprine (25 mg daily), Septrin (Burroughs Wellcome) (5 mL daily), salbutamol, and budesonide.

His anemia has persisted, with a predominantly microcytic picture and low serum iron concentrations; bone marrow aspiration shows a reactive picture and secondary dyserythropoiesis. White cell and platelet counts are consistently low and have left him subject to recurrent infections. Immunoglobulin infusions (Immuno Ltd., Maidenhead, Berks., UK, current dose 12 g every 3-4 wk) were started as a prophylactic measure at age 3, and have been continued to the present. Height and weight have remained below the 3rd centile (Fig. 1). Bone age is minimally delayed only. A skeletal survey showed osteoporosis and collapse of L3 vertebra developed by 7 y. The bone condition is worsening, and there is a danger of further vertebral collapse or even vertebral transection.

Figure 1
figure 1

Height and weight growth curves for J shown with median and lower 3% centile.

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The hepatosplenomegaly has persisted, with increased γ-glutamyl transpeptidase, but with normal or only slightly raised transaminases and total ALP (Table 1). ALP isoenzyme studies show predominantly liver isoform, with the biliary isoform at 16% of total ALP activity, so the bone isoform activity is reduced. Abdominal ultrasound shows an enlarged liver and spleen but no ascites. There is a small lesion in the liver, suggestive of a hemangioma, and a cystic lesion in the right kidney. Low gonadotrophin and testosterone concentrations suggest delayed puberty. All other endocrine investigations are normal. A glucagon stimulation test showed a normal rise in growth hormone and cortisol.

Table 1 Results of biochemical and hematologic investigations
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Zinc deficiency was suspected during an admission in September 1993 when J had worsening rash and diarrhea. Plasma Zn concentrations greater than 40μmol/L (reference range 11-18 μmol/L) were initially dismissed as probably due to sample contamination. A plasma Zn concentration of 205μmol/L (Table 2) was eventually accepted as correct. Plasma Cu concentration is consistently raised at 25-30 μmol/L (reference range 12-20 μmol/L), with an appropriately raised ceruloplasmin concentration of 0.4-0.45 g/L (reference range 0.2-0.4 g/L). Urine Zn excretion is less than 2 μmol/L (reference range 5-10 μmol/L), but erythrocyte Zn concentration is 230 μmol/L (reference range 200-250μmol/L). The serum α2-macroglobulin concentration of 1.9 g/L is below the reference range (2.8-6.7 g/L). Plasma Zn and Cu concentrations in the patient's mother and healthy elder sister were within the reference range.

Table 2 Plasma trace element concentrations
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As a result of these findings a detailed investigation of Zn metabolism was undertaken. Blood samples for speciation studies and the stable isotope experiments were taken with the informed consent of the patient's mother and with approval of the medical ethics committee at the Chelsea and Westminster Hospital. Stable isotope studies were also performed on the patient's mother. A liver and muscle biopsy was performed under general anesthesia, and samples were taken for general histology, metal analysis, and MT staining.

Procedures. Plasma Zn and Cu and erythrocyte Zn concentrations were measured by flame AAS (Perkin-Elmer 3030, Perkin-Elmer, Beaconsfield, Bucks, UK). Serum ceruloplasmin and α2-macroglobulin were measured by immunonephelometry (Beckman Array, Beckman, High Wycombe, Bucks, UK).

Zinc speciation studies. Plasma zinc-binding proteins were separated by gel filtration using Sepharose 6B (Pharmacia Biotech, St. Albans, Herts., UK). Proteins were separated on a 1.5 × 100-cm column packed with Sepharose 6B and eluted with 30 mmol/L Tris-HCl buffer, pH 8.6 and by ion exchange with DEAE-Sepharose CL6B. The molecular weight of the eluted proteins was estimated by comparison with a calibration graph obtained from proteins of known molecular weight (data not shown). The absorbance of the eluate at 280 nm was monitored with a Pharmacia UV-1 monitor. Zn and Cu concentrations in the fractions were measured by electrothermal atomization AAS (Perkin-Elmer 3030 with HGA 600 furnace, Perkin-Elmer, Beaconsfield, Bucks, UK). Fractions with a high Zn content were pooled, concentrated by ultrafiltration(Centriplus-30, Amicon, Stonehouse, Glos, UK), and further fractionated by ion-exchange chromatography. Ion exchange separations were performed with DEAE-Sepharose CL6B in a 1.5 × 30-cm column. Elution was with a gradient of 0-250 mmol/L NaCl in 30 mmol/L Tris-HCl, pH 8.6. Samples of the immunoglobulin infusate were fractionated by gel filtration.

Proteins in the fractions from gel filtration and ion exchange columns were further characterized by agarose and SDS-PAGE. Agarose electrophoresis was performed with the Beckman Paragon electrophoresis system (Beckman, High Wycombe, UK) using Beckman SPE gels, stained with Titan Blue. SDS-PAGE was with Excel-gel SDS 8-18 (Pharmacia) using the Pharmacia Multiphor electrophoresis system.

Antisera to the isolated protein were raised in New Zealand White rabbits. Antisera were tested by the double diffusion technique using micro-Ouchterlony films (Calbiochem, San Diego, CA). Antisera to common serum proteins were tested at the same time. Gels were kept at 4 °C for 48 h for diffusion before washing and staining with Titan Blue. Immunoprecipitation studies were performed on Li-heparin plasma samples from J and a normal subject using antisera to HRG and α2-macroglobulin (Sigma Chemical Co., Poole, Dorset, UK). Protein-antibody complexes were precipitated after incubation for 24 h at 4 °C overnight in 50 mM Tris-HCl buffer, pH 7.5, by addition of PEG-6000 to a final concentration of 5.5%. Zinc concentration in the precipitate and supernatant were measured as above.

CZE of the purified protein was performed using a P/ACE 5000 system(Beckman Instruments, High Wycombe, UK). Samples were separated at 10 kV, and components were detected at 200 nm using a diode array detector and an untreated polyimide-coated silica capillary (57 cm × 75 μm inside diameter, Composite Metal Services, Hallow, UK). For CZE, the electrolyte was 100 mmol/L sodium borate, pH 8.4, and for MECC the electrolyte additionally contained 10 mmol/L SDS. Sudan III was injected into the sample as a migration marker to facilitate comparison of electropherograms. The migration of purified human serum albumin was studied independently or as a spiked addition to the Zn-binding protein or plasma samples.

Stable isotope studies. Stable isotope absorption tests were performed on J and his mother (M). After an overnight fast 3 mg of 67Zn was given in orange juice with breakfast containing 1.1 mg of endogenous Zn. One hour later, 1 mg of 70Zn was given by i.v. injection. Blood samples were taken at frequent intervals over 3 h. A further sample was obtained from J at 5 d. Urine and fecal collections were requested over the following 4 d, but there was some doubt as to whether all collections were complete, and results could not be used in the kinetic analysis.

Isotope enrichment in the samples was determined by thermal ionization quadrupole mass spectrometry. Sample preparation and analytical conditions used have been described elsewhere(1). Zinc kinetics were determined using a two-compartment model(2)(Fig. 2). Plasma volume was estimated from a nomogram of age, sex, and weight(3). Speciation of the plasma Zn after stable isotope administration was performed using fast protein liquid chromatography with inductively coupled plasma mass spectrometry as described by Owen et al.(4).

Figure 2
figure 2

Two-compartment model used for analysis of zinc kinetics after administration of stable isotopes. Compartment P is the plasma pool and compartment E is the labile pool identified primarily with liver. Q is excretion or loss from the system. Rate constants for movement between the pools are shown.

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Tissue analysis. Zinc and Cu concentrations in the biopsy sample were determined by AAS after digestion with nitric acid. Results were recorded as μg/g wet weight. Certified reference materials were analyzed at the same time (BCR 185, bovine liver and BCR 186, pig kidney, IRMM, Geel, Belgium). Liver and muscle sections were examined using routine histologic methods. Formalin fixed sections of liver were stained for MT using techniques described elsewhere(5).

RESULTS

Plasma protein studies. The results of gel filtration of J's plasma are shown in Figure 3. A major Zn-containing fraction eluted at a relatively high molecular weight (peak A). This fraction was not present in normal plasma or in a sample of the immunoglobulin infusate. Normal amounts of Cu eluted as expected with the major ceruloplasmin and albumin containing fractions (peak B). Unusually, there was no zinc associated with the albumin containing fraction. The molecular weight range of the zinc containing fraction was estimated to be 150-300 000. Separation of normal plasma loaded with 200 μmol/L Zn showed that all of the exogenous Zn was recovered with the albumin or in a low molecular weight fraction. Ion exchange separation of peak A gave a major Zn containing fraction which bound to the column and was well resolved from the albumin containing fraction.

Figure 3
figure 3

Gel filtration of 1) J's plasma and 2) normal plasma on Sepharose 6B eluted with 30 mmol/L Tris-HCl pH 8.6 as described in the methods section. ―, Zn, J's plasma; ._._., Zn, normal plasma (scale expanded times 10); ·····, protein absorbance at 280 nm;peak A, high molecular weight zinc binding protein; peak B, albumin.

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The immunoglobulin infusate had a Zn concentration of 10 μmol/L. Gel filtration of the infusate gave a single protein band which did not contain Zn, which was associated with a lower molecular weight fraction-presumably citrate and other low molecular weight chelators.

CZE of the purified protein, with or without mercaptoethanol in the electrolyte, yielded two major components, one migrating at an earlier time than purified human serum albumin and the second migrating after albumin. Spiking the patient's plasma with the purified Zn-binding protein confirmed that the two components did not co-migrate with serum albumin. MECC separations revealed three major and several minor components (Fig. 4), and from similar separations of protein sample spiked with albumin or the patient's plasma, it was quite clear that albumin had much longer migration times than any of the major components in the Zn-binding protein.

Figure 4
figure 4

MECC capillary electrophoresis of the purified protein. The electrolyte was 100 mmol/L sodium borate, pH 8.4, with 10 mmol/L SDS using a polyimide-coated silica capillary. ―, Purified protein (left-hand scale);- - - - -, J's whole plasma (right-hand scale).

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Antibodies raised in rabbits to the partially purified protein were tested by double diffusion in agarose gels (Fig. 5). The antibodies showed reactivity to one component in the protein preparation, and gave a line of identity with the α2-macroglobulin antibody. The protein also showed reactivity to antibodies to α1-acid glycoprotein. The immunoprecipitation studies show that no Zn was precipitated from the serum of the patient with antibodies to α2-macroglobulin or HRG, but Zn was precipitated from the normal serum (Fig. 6). Other antibodies were tested, including that raised to the purified protein, but results were inconclusive due to high background Zn concentrations in the antisera.

Figure 5
figure 5

Double diffusion of purified protein and antibodies in mico-Ouchterlony gels. Center wells a and b, purified protein; c, normal plasma. Outer wells, 1 and 4, rabbit antibody to purified protein, 2, anti-α2-macroglobulin; 3, anti-ceruloplasmin;5, anti-transferrin; 6, anti-α1-acid glycoprotein; 7, anti-α1-macroglobulin; 8, anti-albumin; 9, anti-prealbumin; 10, anti-α1-antitrypsin.

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Figure 6
figure 6

Immunoprecipiation of Zn-binding proteins from patient's plasma and control plasma. The results are shown as percentage of the total plasma Zn in solution. ⋄, J, anti-α2-macroglobulin;▪, J, anti-HRP; □, control, anti-α2-macroglobulin; ♦, control, anti-HRP.

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Agarose and PAGE of the fractions from ion-exchange consistently show the presence of several components. These are all present in the same relative amounts in each fraction and are so well separated that they should be readily resolvable by ion-exchange and are not consistent with the high molecular mass from the gel filtration. The major component shows reactivity toα2-macroglobulin antibody.

Stable isotope studies. Results of the kinetic modeling using the two-compartment model are shown in Table 3. The data from M are comparable with data from normal subjects (S. Fairweather-Tait, unpublished observations). The results from J are clearly abnormal, with increased plasma (10.2 mg) and exchangeable (230 mg) pools, rapid movement from the plasma to the exchangeable pool (Kq 0.339 min-1) and a relatively slow movement out of the system(Kqp 0.022 min-1). Turnover of the plasma(P) pool was rapid (t½(P) 1.92 min), but turnover of the exchangeable (E) pool (t½(E) 46.5 min) was normal or slightly decreased.

Table 3 Kinetic parameters derived from the two compartment model of zinc metabolism (Fig. 2)
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The interpretation of results from the fecal and urine collections is difficult. In the two fecal samples provided by the patient, 45% of the oral dose was recovered. Without knowing the sample times it is difficult to ascribe the high recovery to poor absorption of Zn or efficient enterohepatic excretion. The appearance of 70Zn in feces supports the latter view. Urinary Zn excretion by J was less than 1 μmol/d, and by M was 2-5μmol/d. Excretion of the administered isotopes by M was within the expected range.

Fast protein liquid chromatography-inductively coupled plasma mass spectrometry studies of serum after stable isotope administration show that, in M, 5 min after administration of Zn all of the endogenous 66Zn was bound to a protein of approximate mass 38 kD, and not to albumin. The70 Zn i.v. dose is associated with a low molecular weight fraction, possibly citrate. Because the i.v. dose of 70Zn also contains 3-8% of66 Zn and 67Zn peaks of both isotopes are found at the same elution volume as the main 70Zn peak.

In J after 90 min the i.v. administered 70Zn was found in a fraction of mass 180 kD, and a negligible amount in a low molecular weight fraction. After 5 d the distribution pattern was changed, with the Zn in a peak of mass 110 kD. The orally administered 67Zn showed the same distribution. No Zn was found with the 67 000 molecular weight peak (albumin). The isotope ratios in the Zn peak compared with total plasma and natural abundances(Table 4) showed that the exogenous Zn may be capable of displacing bound endogenous Zn.

Table 4 Zn isotope ratios in protein fractions-comparison of results from thermal ionization quadrupole mass spectrometry (TIQMS) and inducively coupled plasma mass spectrometry (ICPMS) determinations of total plasma Zn and Zn in the main peak from fact protein liquid chromatography protein separations
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Biopsy analysis. The results of trace element analysis of the biopsy samples are given in Table 5. Zinc and Cu concentrations are increased in both tissues, but not enough to account for the exchangeable pool identified in the stable isotope studies. Routine histologic examination shows no significant abnormalities in the liver, but the muscle shows mildly myopathic changes. Metallothionein staining showed a generalized increase in basal hepatocyte MT expression, with accentuation in the hepatocytes in the immediate periportal region.

Table 5 Zinc and copper concentrations in biopsy tissue samples from J and reference materials
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DISCUSSION

The symptoms of J are similar to those of the patients Prasad et al.(6) described in 1961 in Iran with dwarfism, severe anemia, hypogonadism, hepatosplenomegaly, and dry, scaly skin. Although the anemia in these cases was corrected by Fe supplementation, this did not explain the other symptoms. Similar patients in Egypt were found to be Zn-deficient(7). Further studies with other subjects in the Middle East showed that a daily supplement of 18-40 mg of Zn was sufficient to restore normal growth and gonadal function in these patients(8–10). The Zn and Fe deficiency were attributed to metal binding by dietary phytic acid, which is abundant in cereals and many other vegetables, and effectively diminishes gastrointestinal uptake of many metal ions(11). The presentation in this case, however, suggests that J has a hitherto unknown defect in Zn metabolism. The symptoms described are distinct from those of chronic or acute Zn toxicity: nausea or vomiting, abdominal pain, lethargy and loss of muscle co-ordination. J suffers from a chronic inflammatory response, with a raised plasma C-reactive protein concentration, and the low plasma Fe and raised plasma Cu concentrations may be in part due to the acute phase response, which can result in very low plasma Fe concentrations(12). This may be compounded by Fe deficiency anemia.

We cannot at this time be certain that we are dealing with a new inborn error of Zn metabolism, but there is no other immediate explanation for the findings in this case. The plasma Zn in the patient is bound to a high molecular weight protein(s) whose identity remains unknown. The stable isotope speciation studies suggest that either there is more than one protein involved in Zn metabolism, or that there may be a redistribution of the exogenous Zn after oral or i.v. administration. The finding of Zn bound to a low molecular weight protein in the patient's mother was unexpected, given the finding of a normal plasma Zn concentration, and has not yet been confirmed with further studies. It is possible to speculate that the 110- and 180-kD proteins found in J may be trimers and pentamers of the 38-kD protein observed in the mother, but there is no evidence for this.

The only recognized inborn error of Zn metabolism is acrodermatitis enteropathica(13). Patients have very low plasma Zn concentrations. This disease develops in babies early in life, manifesting as a skin rash with diarrhea, and may be lethal if untreated. The defect is in intestinal Zn transport, involving the intestinal mucosa. Malabsorption leads to weight loss, but the disorder is readily treated with Zn supplements.

The finding of raised plasma Zn concentration to the levels noted in this case has not previously been described. In most cases in which a moderately increased Zn concentration is found, this can usually be attributed to sample contamination from the collection tubes(14), hemolysis, or prolonged storage before separation of plasma(15). Hyperzincemia can also be caused by excessive Zn content of parenteral solutions and, rarely, by in vivo hemolysis and tissue catabolism. In all such cases there is a marked hyperzincuria.

There has only been one previously reported case of familial hyperzincemia(16). There were no associated symptoms, with plasma Zn concentrations in the range of 30-50 μmol/L. The abnormality was ascribed to high affinity Zn binding by albumin(17). Hyperzincemia was reported associated with a case of pyoderma gangrenosum(18). Zn was in a plasma protein fraction associated with α2-macroglobulin.

About 70-85% of plasma Zn is normally bound to albumin, with 10-30% bound to α2-macroglobulin and 5-10% as low molecular weight complexes(19–23). The zinc bound toα2-macroglobulin is not available for exchange as it is an integral part of the protein structure. Albumin is the normal transport protein for Zn. The molar ratio of Zn to albumin in normal plasma is 1:30, demonstrating the functioning of a strict homeostatic mechanism. Zinc will also bind to transferrin with a lower affinity. Another plasma protein with a high affinity for Zn is HRG(24). This protein, which contains a histidine/proline-rich metal and ligand binding domain, is capable of binding 10-15 metal atoms. The normal in vivo composition of HRG is not known, but its role may be more closely linked to its plasminogen-binding capacity(25). Although the antibody raised to the purified protein detected α2-macroglobulin, the immunoprecipitation experiment suggests that the protein is notα2-macroglobulin or HRG.

The high molecular weight Zn-binding protein found in this patient remains unidentified. The Zn bound to this protein is apparently unavailable metabolically, as the urinary Zn excretion is very low. It cannot be certain whether the zinc is accumulating as a result of long-term immunoglobulin influsions, although studies on samples of the infusate suggest that this is unlikely.

Analysis of the purified protein by CZE and MECC as well as agarose and PAGE showed that it was composed of several components. This result was surprising and indicated that one or more large proteins may have dissociated into several constituent components during electrophoresis, suggesting that the protein may be composed of subunits.

The effects of zinc deficiency on immune function are well known. Nutritional Zn deficiency has several effects on the immune system, including reduced T cell numbers, reduced response of T lymphocytes to mitogens, decreased natural killer cell activity, and decreased functional activity(26). The patient shows some, but not all, of these indicators, with current evaluation showing normal T cell subsets, CD25 expression on CD4 high, but possibly low CD45RACD4 and DR expression low. Monocytes and B cells are low, and IgA is raised. Natural killer cells were previously normal, but are now low. Phytohemagglutinin antigen stimulation is normal.

The liver biochemistry suggests a hepatocellular disorder. We have found raised γ-glutamyl transaminase and raised liver ALP isoenzyme activity. The raised liver ALP isoenzyme in the presence of normal for age total ALP activity suggests that bone ALP may be reduced, possibly a reflection of the low bone age and lack of growth shown by the patient.

The stable isotope experiments show that there is a very large exchangeable Zn pool. It was originally thought that this would be identified as liver, but the measured liver Zn content does not support this. The site of Zn storage remains unknown. There is a very slow return from this pool to the plasma, and a negligible urinary excretion of Zn. It was not possible to quantify fecal Zn, so we cannot comment on the gastrointestinal absorption or biliary excretion of Zn. The results quoted here for Zn metabolism in normal subjects are in broad agreement with those reported in other studies(2, 27, 28).

There are parallels with other disorders of trace element metabolism: hemochromatosis, Wilson's disease, and Indian childhood cirrhosis. Hemochromatosis is characterized by hepatic accumulation of Fe with a high plasma Fe concentration and transferrin saturation and increased urinary Fe excretion. In Wilson's disease hepatic Cu accumulation is accompanied by hypocupremia and increased urinary Cu excretion(14). Symptoms include hepatomegaly and liver necrosis leading to fulminant liver failure and also neurologic symptoms. Indian childhood cirrhosis is of unknown etiology, but excessive Cu ingestion has been implicated. It is characterized by hepatic Cu accumulation with increased plasma concentrations and urinary Cu excretion. This case may not be an exact parallel as there is no storage of Zn in the liver and no demonstrable liver pathology, despite the abnormal liver function tests. The demonstration of increased concentrations of MT in the liver and the pattern of staining is reminiscent of early Wilson's disease, although there are many other stimuli to MT synthesis(29). However, J suffers from a chronic inflammatory condition, with a consistently raised plasma C-reactive protein concentration. The induction of liver MT and ceruloplasmin are to be expected in this circumstance, and may not be directly related to the hyperzincemia.

We have considered several therapeutic options. Any possible therapy must take into account the full range of symptoms, especially his deteriorating lung function and progressing osteoporosis, which may lead to further spinal collapse. Chelation therapy, to reduce the plasma Zn concentration and remove any stored Zn, may be difficult to justify in the absence of any positive evidence for systemic Zn accumulation. Administration of chelated Zn to correct systemic Zn deficiency may be a means of overcoming a possible transport defect, but as no such defect has been demonstrated there can be no basis for this treatment. Plasmapheresis could reduce the plasma concentration of the Zn-binding protein, but may only be an effective course of action if there were a supposition that the presence of this protein in the plasma was itself causing symptoms. In this current condition it is felt that this may be too hazardous a procedure to undertake. A more drastic option to consider is a liver transplant, as this may allow normal Zn metabolism and permit a“cure” of the condition. However, the patient's other problems dictate that if this were to be contemplated a lung transplant would also be required, and this is unlikely to occur. The results of the liver biopsy suggest that the liver may not be the primary site of Zn deposition, and this suggestion has been abandoned.

We are currently undertaking a more detailed characterization of the protein and studies of Zn transport in cultured fibroblasts. It is hoped that these will give us more information on the biochemical defect in the patient, and possibly give a more definitive pointer to possible therapeutic strategies. The investigation of this defect may lead to a greater insight into the biochemistry and physiology of zinc transport and metabolism.