Transferrin Loss into the Urine with Hypochromic, Microcytic Anemia

Transcription

1 Transferrin Loss into the Urine with Hypochromic, Microcytic Anemia DANIEL E. HANCOCK, M.D., JOHN W. ONSTAD, M.D., AND PAUL L. WOLF, M.D. Department of Pathology, Division of Clinical Pathology, University of California, San Diego, School of Medicine, Lajolla, California ABSTRACT Hancock, Daniel E., Onstad, John W., and Wolf, Paul L.: Transferrin loss into the urine with hypochromic, microcytic anemia. Am J Clin Pathol 65: 73-78, Anemia developing during the course of chronic renal disease is a frequent complication often necessitating periodic transfusion therapy. A number of etiologic factors have been implicated, including decreased production of erythropoietin; decreased erythrocyte life span secondary to uremia and splenomegaly; increased bleeding tendency due to platelet dysfunction; and acquired lack of folic acid and iron. This paper concerns the problem of acquired hypochromic, microcytic anemia secondary to heavy urinary loss of iron and transferrin in a child with the nephrotic syndrome. The patient had microcytic, hypochromic anemia with serum iron, 12 /xg. per dl. and a serum iron-binding capacity of 12 fig. per dl. There was no evidence of major bleeding resulting in a chronic hemorrhagic anemia. Urinary iron was 64 fig. per dl., with a urinary iron-binding capacity of 366 fig. per dl. Renal biopsy showed mesangio-proliferative glomerulonephritis. Evaluation of any patient with the nephrotic syndrome should include careful analysis of the various serum and urinary proteins and determination of serum and urinary iron and iron-binding capacity. This information would offer a more precise evaluation of the underlying cause of anemia in the nephrotic patient who may develop urinary loss of iron and transferrin and subsequent hypochromic, microcytic anemia. (Key words: Nephrotic syndrome; Iron loss in urine; Renal anemia; Transferrin; Urinary iron excretion.) HYPOCHROMIC, MICROCYTIC ANEMIA serum iron and decreased serum ironusually is related to iron deficiency due binding capacity. to excessive loss from chronic hemorrhage, This paper concerns a child who develdecreased nutritional intake, or decreased oped hypochromic, microcytic anemia absorption as a result of gastrointestinal during the course of the nephrotic syndisease. The serum iron is decreased while drome. The anemia was secondary to acthe serum iron-binding capacity is elevated, quired transferrin deficiency from urinary Hypochromic, microcytic anemia may also loss of the iron-binding protein, be seen infrequently in chronic disease states that are associated with decreased Report of a Case Received April 2, 1975; accepted for publication A 7-year-old Mexican boy was admitted April 24, to th e University Medical Center for the Address reprint requests to Dr. Wolf, Department c * * M L OO -\c\na tr u J of Pathology AID 7200, University Hospital, 225 West first tlme o n November 28, He had Dickinson, San Diego, California been in good health until two and a half 73

2 74 HANCOCK, ONSTAD, AND WOLF A.J.C.P. Vol. 65 # FIG. 1. Peripheral blood, showing hypochromic, microcytic erythrocytes compared with a small lymphocyte. Wright's stain, x 100. years prior to admission, when abdominal pain, fever, episodic vomiting, and edema with ascites had developed. The diagnosis at the Social Security Hospital in Mexico had been pyelonephritis, and the patient had been treated with antibiotics, steroids, and diuretics. Initially, his condition had improved, with clearing of the ascites and edema, but over the subsequent two and a half years, the patient had experienced repeated episodes of edema and ascites, which usually responded to drug therapy. During one episode in Mexico a month prior to admission, stools had been grossly bloody, but a definite etiology had not been established. The child's first admission to the University Medical Center was necessitated by persistent edema, ascites, and episodic vomiting. Admission temperature was 99.8 F., pulse rate 110 per min., respiratory rate 24 per min., and blood pressure 120/72 mm. Hg. The patient was grossly edematous, with massive ascites and periorbital edema. The skin was pale and dry, with patchy regions of hyperkeratosis. The hair was dry and brittle. A grade I/VI systolic ejection murmur at the right sternal border was present. The lung fields were clear on auscultation. The liver and spleen were nonballottable. There was bilateral moderate pitting edema of the lower extremities to the level of the knees. The clinical impression was nephrotic syndrome. The significant laboratory values were: hemoglobin 8.0 Gm. per dl.; hematocrit 25%; erythrocyte count 3.26 million per ml., with mean cell volume 75 cu. fi, mean corpuscular hemoglobin 24 ^i fig. and mean corpuscular hemoglobin concentration 33%. The leukocyte count was 9,600 per ml., with 50 polymorphonuclears, 9 bands, 31 lymphocytes, 7 monocytes and 3 eosinophils. The platelet count was 630,000 per ml. The peripheral blood smear showed moderate microcytosis with hypochromia and moderate thrombocytosis (Fig. 1). The prothrombin time was 10.5 sec/11.4 sec; the partial thromboplastin time 30.4/28.0 sec. The reticulocyte count was 1.9% and the erythrocyte sedimentation rate 137 mm/min. (Westergren). Serum electrolytes were: sodium 139 meq. per 1.; C meq. per 1.; BUN 19 mg. per dl.; creatinine 0.5 mg. per dl.; calcium 7.2 mg. per dl.; total bilirubin 0.2 mg. per dl.; serum lactic dehydrogenase 138 I.U.; serum alkaline phosphatase 83 I.U. Total serum cholesterol was 465 mg. per dl. (normal mg. per dl.) and the serum triglyceride concentration was 505 mg. per dl. (normal mg. per dl.). Total serum protein was 3.3 Gm. per dl., with 0.6 Gm. per dl. albumin and 2.7 Gm. per dl. globulin. Serum protein electrophoresis performed on agarose gel showed a prominent increase in the alpha-2 fraction; the densitometric tracing is shown in Figure 2. The quantitative serum protein fractions were albumin 0.6 Gm. per dl., alpha Gm. per dl., alpha Gm.

3 January 1976 URINARY TRANSFERRIN LOSS per dl., beta 0.7 Gm. per dl., and gamma 0.2 Gm. per dl. The patient was oliguric, with a 24-hour urinary output averaging less than 50 ml. and a creatinine clearance of 10 ml. per min. A 24-hour urine obtained soon after admission showed a total protein of 1.22 Gm. The densitometric scan of the urinary protein electrophoresis is shown in Figure 3. The protein concentrations in the 24-hour urine were albumin 1,000 mg., alpha-1 7 mg., alpha-2 3 mg., beta 3 mg., and gamma 6 mg. Serum iron was 12 /Ag. per dl. and ironbinding capacity was 12 (xg. per dl. The urinary iron concentration was 64 fig. per dl. and the urinary iron-binding capacity was 366 ^ig. per dl. Serum immunoelectrophoresis (Fig. 4, top) showed decreased a l b u m i n, increased alpha-2 macroglobulin, and negligible transferrin. 75 Immunoelectrophoresis performed on undiluted urine (Fig. 4, middle) showed a prominent transferrin band. Immunoelectrophoresis performed with typespecific anti-transferrin antiserum showed a band identical to that of the normal serum control (Fig. 4, bottom). Other determinations were beta 1C 105 (normal ), Ch (normal ) and C'4 34 (normal 12-72). The antinuclear antibody test was negative, as was the test for Australia antigen. T h e patient was treated with albumin and diuretics, which resulted in a total weight loss of 10 pounds by the fifth hospital day. On the seventh hospital day, an open renal biopsy was performed. Light microscopy disclosed a moderate increase in the mesangial cells (Fig. 5). Silver stains showed increased mesangial FIG. 2 (left). Densitometric scan of the serum protein electrophoresis performed on agarose gel. There are decreased albumin, beta, and gamma fractions with a prominent increase in alpha-2 macroglobulin. FlG. 3 (right). Densitometric scan of unconcentrated 24-hour urine performed on agarose gel. There are significant increases in the albumin and beta globulin fractions.

4 HANCOCK, ONSTAD, AND WOLF A.J.C.P. Vol. 65 Cathode Cathode Cathode FIG Serum Immunoelectrophoresis performed with polyvalent antisera normal control. 2. Patient's serum, showing decreased albumin and negligible transferrin band. 3. Normal serum control polyvalent antisera. 4. Patient's unconcentrated urine, showing increased albumin and heavy transferrin band equivalent to that of the control serum. 5. Serum control using type-specific anti-transferrin antiserum. 6. Patient's unconcentrated urine using type-specific anti-transferrin antiserum. fibrils; there was no evidence of focal sclerosis. The proximal convoluted tubules showed cytoplasmic vacuolization. Direct immunofluorescence study showed segmental, granular deposits of IgG accompanied by C'3 along portions of the glomerular basement membrane. Electron microscopy showed focal subendothelial immune deposits and fusion of foot processes of the visceral epithelial cells (podocytes). The final impression was mesangio-proliferative glomerulonephritis. Work-up for the intermittently Hematest-positive loose stools revealed a circumferential anal ulcer on proctoscopic examination. The patient was discharged on prednisone 15 mg. q.i.d., in addition to salt and fluid restriction. He was admitted for the second time to the University Medical Center on January 8, 1975, for ascites, hypertension and bilateral otitis media. He was Cushingoid, with pitting edema and ascites. Blood pressure was 120/86 mm. Hg. There was mild otitis media. The significant changes in the laboratory values since the first admission were an increase in the hematocrit to 35%, hemoglobin 10.8 Gm. per dl., erythrocyte count 4.18 million per ml., and reticulocyte count 4.0%. The erythrocytes continued to show microcytosis and hypochromia. Serum proteins were increased to a total of 5.5 Gm. per dl., with albumin 3.2 Gm. per dl. and globulins 2.3 Gm. per dl. The patient was again treated with diuretics and prednisone. Cyclophosphamide was also started. He was discharged to a child care center in San Diego for further supervision.

5 January 1976 URINARY TRANSFERRIN LOSS 77 Materials and Methods Protein electrophoresis of serum and urine was performed on agarose gel using the method of Elevitch and associates. 4 Immunoelectrophoresis of serum and urine was performed according to the method of Cawley. 3 Iron concentrations and total iron-binding capacities on serum and urine were determined using the colorimetric method of Giovaniello and colleagues. 6 Discussion The nephrotic syndrome is generally defined as a clinical entity characterized by massive proteinuria, lipidemia and lipiduria. It is secondary to multiple causes, with the common denominator of increased glomerular membrane permeability to serum proteins. One of the important serum proteins that may be lost in the urine of patients with the nephrotic syndrome is transferrin; this loss may lead to significant hypochromic, microcytic anemia secondary to altered iron transport. Iron in plasma is bound to a glycoprotein called "transferrin" (siderophilin), present in a concentration of about 200 mg. per dl. plasma. 1 Normally, less than 4 mg. of transferrin are excreted in the urine per 24 hours. 2 Transferrin has a molecular weight of 88,000 to 90,000 and an isolectric point of ph 5.9.' On electrophoresis, it moves as a beta-1-globulin. It is a specific transport protein for iron and is necessary for movement of iron from one site to another in the body. Transferrin has the capacity to bind mg. of ferric iron per Gm. of protein; this corresponds to the binding of two iron atoms per mole. 1 In adults the average iron-binding capacity of plasma is approximately 30 ng. per dl. with the transferrin binding capacity usually about one-third saturated with iron. 7 Transferrin normally leaves the plasma with a half-time of between 6.7 and 10.4 days. 10 It has been found that when transferrin is saturated, FlG. 5. Glomerulus from open renal biopsy in which there is proliferation of mesangial cells. There is vacuolization of the proximal convoluted tubules. Hematoxylin and eosin. x 160. the iron that is absorbed from the diet is deposited mainly in the liver, with little reaching the erythropoietic tissue of the bone marrow. 5 A similar phenomenon was observed in a case of congenital atransferrinemia, 8 and it would therefore appear that the major physiologic role of transferrin is transport of iron to the bone marrow. Low plasma levels of transferrin may be secondary to either.decreased production or accelerated loss, as in the patient in this case report. Three cases of congenital atransferrinemia have been reported. Two of the cases probably represent an acquired deficiency of the protein. Riegel and Thomas 12 described one patient with absence of the beta-globulin fraction just prior to death, while Hitzig and associates 9 described a child with erythroleukemia with absent transferrin on immunoelectrophoresis shortly before

6 78 HANCOCK, ONSTAD, AND WOLF A.J.C.P. Vol. 65 death. Heilmeyer and colleagues 8 described a 7-year-old girl with severe hypochromic anemia, absence of marrow iron, and hypoferremia associated with iron overloading of the liver, heart, and kidneys. Other conditions in which decreased iron-binding capacities are frequently found include nephrosis, uremia, malignancy, and protein malnutrition. 7 Rifkind and co-workers 13 reported two cases of significant urinary loss of transferrin associated with the nephrotic syndrome. Associated with the urinary loss of transferrin were low serum iron concentrations and increased urinary losses of iron, ranging in one case to 1,000 micrograms per day. Milliez and associates 11 demonstrated transferrin qualitatively in the urines of all of 30 patients with the nephrotic syndrome, regardless of etiology. Wiltink and colleagues 14 studied two patients with the nephrotic syndrome who lost appreciable amounts of iron in the urine. However, they found the total amount of iron excretion did not exceed 0.5 mg. per day, and concluded that the anemia observed could not be explained by this iron loss unless there was a reduction in iron stores. Our patient manifested the usual findings of the nephrotic syndrome associated with low serum iron and iron-binding capacity. Although the chronic anal ulcer was contributory to the total iron loss, the observed hypochromic, microcytic anemia was predominantly a manifestation of the significant urinary loss of transferrin and transferrin-bound iron, as demonstrated by urine immunoelectrophoresis and colorimetric determination of iron and total iron-binding capacity. The patient's chronic renal disease may also be partly responsible for the observed hypochromic, microcytic anemia, since this type of anemia may be seen with chronic disease. It is hoped that treatment with steroids and cytotoxic agents will control the proteinuria and the secondary irondeficiency anemia in this child. References 1. Awai M, Brown EB: Studies of the metabolism of I 131 -labeled human transferrin. J Lab Clin Med 61:363, Boyce WH, Garvey FK, Norfleet CM Jr: Proteins and other bicolloids of urine in health and in calculous disease. I. Electrophoretic studies at ph 4.5 and 8.6 of those components soluble in molar sodium chloride. J Clin Invest 33: , Cawley LP: Electrophoresis and Immunoelectrophoresis. Boston, Little, Brown, 1969, p Elevitch FR, Aronson SB, Feichtmeir TV, et al: Thin gel electrophoresis in agarose. Am J Clin Pathol 46: , Fawwaz RA, Winchell HS, Pollycove M, et al: Hepatic iron deposition in humans. I. Firstpass hepatic deposition of intestinally absorbed iron in patients with low plasma latent iron-binding capacity. Blood 30: , Giovaniello TJ, DiBenedetto G, Palmer DN, et al: Fully automated method for die determination of serum iron and total ironbinding capacity, Automation in Analytical Chemistry, Technicon Symposia 1967, volume 1. White Plains, New York, Mediad, Inc., 1968, pp Harris JW, Kellermeyer RW: The Red Cell. Cambridge, Harvard University Press, 1970, p82 8. Heilmeyer L, Keller W, Vivell O, et al: Die Kongenitale Atransferrinamie. Schweiz Med Wochenschr 91:1203, Hitzig WH, Schmid M, Betke K, Rothschild M: Erythroleukamie mit Hamoglobinpathie und Eisenstoff wechelstorung. Helv Paediatr Acta 15:203, Katz' JH: Iron and protein kinetics studied by means of doubly labeled human crystalline transferrin. J Clin Invest 40: , Milliez P, Hartmann L, Lagrue G: Immune and starch gel electrophoresis of serum and urine of subjects with nephrotic syndromes. J Urol 65: , Riegel C, Thomas D: Absence of beta globulin fraction in the serum protein of a patient with unexplained anemia. N Engl J Med 255:434, Rifkind D,' Kravetz HM, Knight V, Schade AL: Urinary excretion of iron-binding protein in the nephrotic syndrome. N Engl J Med 265: , Wiltink WF, van Eijk HG, Bobeck-Rutsaert MM, et al: Urinary iron excretion in the nephrotic syndrome. Acta Haematol 47: , 1972