Labs of Lincoln. 555 South 70th St. Lincoln, NE 68510. Charles. L. Welch. Samuel E. Boon. HeterogeneIty of Calcitonln in. Patientswith Neopiasia. To the Editor:.
Table 1. PrecIsion Data for PhenylalanineDeterminations Serum pools Within-run (n = 10) Mean, mgfL 22 SD, mg/L 1.5 CV, % 6.8 Between-run (n = 5) Mean, mg/L 22 SD, mg/L 2.3 10.5 CV, %
41 1.0
73 1.5
2.4
2.1
42
69
3.2
5.4
7.6
7.8
We compared the results for phenylalanine obtained by our enzymic procedure (y) with those by the spectrofluorometric method (x) of McCanian and Robins (5). Linear regression analysis of 25 serum samples from phenylketonuric patients gave the following: y = 0.971x - 2.35; r = 0.990; 57 = 74 mg/L; = 79 mg/L. Phenylalanine concentrations ranged from 26 to 200 mg/L for the fluorometric procedure andfrom 19to 191 mgfLforour PALcatalyzed assay. Our adaptation of an enzymic determination of serum phenylalanune for use on a centrifugal analyzer provides a simple, rapid means for monitoring phenylalanune concentrations in phenylketonuric patients and for screening newborns for this disorder. References
1. Shen R-S, Abell CW. Phenylketonuria: A new method for the simultaneousdeterminationofplasma phenylalanine and tyrosine. Science 197,665-667 (1977). 2. Shen R-S,Richardson CJ, Rouse BM, Abell CW. An enzymatic assay of plasma phenylalanine
and tyrosine
forthe detec-
tion and management of phenylketonuria. Biochem Med 26,211-221 (1981). 3. McKnight RP, Willis JE, Shen R-S, Abell CW. Evaluation of the PaT Stat” kinetic uv test set for the determination of phenylalanine and tyrosine in serum or plasma. Gun Biochem 16, 157-162 (1983). 4. Jervis GA, Drejza EJ. Phenylketonuria: Blood levels of phenylpyruvic and orthohydroxyphenylacetic acids. Gun Chim Acta 13, 435-441 (1966). 5. McCaman MW, Robins E. Fluorometric methodfor the determinationof phenylalanine in serum. J Lab Clin Med 59, 885 (1962). Charles
Samuel
L. Welch
E. Boon
Clin. Labs of Lincoln 555 South 70th St. Lincoln,
forms larger than the 32-amino acid CT monomer (CT M) (1). Some of these species could be formed by noncovalent binding to plasma proteins; others probably result from disulfide bridge formation between CT monomers. The origin of this heterogeneity might be the secretion of multiple CT species that appear to be a feature of normal thyroid tissue (2). Increased concentrations of immunoreactive CT (iCT) have been found in the serum of patients with medullary thyroid carcinoma and with various nonthyroid tumors. However, the validity of serum iCT as a tumor marker is controversial (3-5). In a recent study to evaluate the CT content of nonthyroid human tumors, we found a high content of iCT in hepatic metastases from a patient with bronchial carcinoid (5). Further to analyze the nature of this iCT material, we fractionated the tissue extract by reversed-flow chromatography on a 2.5 x 65 cm column of Ultragel AcA-202 (Pharmacia, Uppsala, Sweden), eluting with phosphate buffer, pH 7.5. We measured iCT in the effluent by RIA (5). The goat antiserum we used, 03-377 (European PTH Study Group), recognized some sequence between amino acid residues 11 and 32, as shown in studies with synthetic fragments of CT’ (kindly provided by Dr. B. Argemi, Groupe Hospitalier de la Timone, Marseille, France). Material with immunoreactivity was eluted in a broad elution zone, from the void volume to beyond the position of the CT M (Figere 1). Nevertheless, RIA of the iinmunoreactive material in the tumor extract gave a dilution curve that was superimposable on the standard curve for CT M. The inimunochemical heterogeneity in this tumor extract differs from that found for sera from three patients with medullary thyroid carcinoma, in which CT exceeded 20 ng/mL (normal: a125 pg/mL). The distribution of the immunoreactivity in these sera after gel filtration, as described previously, showed essentially three peaks. One peak appeared at the void volume of the column (I), another peak at the position of the CT dimer (II), and the third peak corresponded to the CT M (ifi). The distribution of this profile was analogous in two cases: about 30, tive
121 P9I5
.102
ci
NE 68510
HeterogeneIty of Calcitonln
in
Patientswith Neopiasia To the Editor: Circulating calcitonin (CT) consists of as many as four or five immunoreac-
05
Ag. 1. Fractionationbygel filtrationof calcitonm immunoreactivityin hepatic metastasesof a patientwithbronchialcarcinoid Anvwsmark the elutton position of -Iabeled human calcitoninmonomerand the salt volume (1251)
786 CLINICALCHEMISTRY, Vol. 31, No. 5, 1985
20, and 45% of total immunoreactivity eluted in peaks 1,11,and ifi, respectively. However, in the third case studied, the distribution was about 30, 40, and 30%, respectively. Radioimmunoassayable CT in peaks I and III, but not in peak H, was superimpesable on the standard curve for CT M. Thus, we found the same chromatographic proffle but a heterogeneous distribution of serum iCT species among individual patients with medullary thyroid carcinoma. These results and those of others (68) suggest that at least some of the divergent findings among
different
groups who measured CT in tumor patients may be explained by the heterogeneity of iCT among different neoplasia and also among patients with the same kind of neoplasm. References 1. Snider RH, Silva OL, Moore ChF, Becker KL. Immunochemical heterogeneity of calcitonin in man: Effect of radioimmunoassay. Gun Chim Acta 76, 1-14 (1977). 2. WoloszczukW, Kovarik J. Heterogeneity of unimunoreactive calcitonin in extracts of normal human thyroid. Horm Metab Res 13, 460-463 (1981). 3. Coombes RC, Greenberg PB, Hillyard C, Maclntyre I. Plasma-immunoreactive-calcitonin inpatientswith non-thyroid tumours. Lancet i,1080-1083 (1974). 4. Body JJ, Kellermann G, Muquardt C, Borkowski A. Serum immunoreactive calcitonin: Useful as a general tumour marker? Horns Metab Res 15,624-625 (1983). 5. Babe M, Esbrit P, Rapado A. Estudio de la produciOn de calcitonina per tumores humanos. Rev Gun Esp 167, 313-316 (1982). 6. Franchimont
P, Heynen G. Parathormone and Galcitonin Radioimmunoassay in Various Medical and Osteoarticular Disorders, Masson Inc., Barcelona, 1976.
7. Roof BS, Weinstein R,Vujic I,Burdash NM. Immunoheterogeneity of the calcitonina of hypercalcemia, breast and lung cancers and medullary carcinoma of thyroid. Biomedicine 30,82-90 (1979). 8. RudeRK, Matsumoto C, Singer FR. Heterogeneity of immunoreactive calcitonin in extracts of medullary thyroid carcinoma. Mineral Electrolyte Metab 7, 292-297 (1982). Milagrosa Babe Pedro Esbrit Aureiio Rapado Unidad Metab#{243}lica Fundaci#{243}nJiimi nez DEar Avda. Reyes Gat#{243}licos 2 28040 Madrid, Spain
Differentiationof Mislabeled
Samples by LDH Isoenzyme Assay To the Editor:
The clinical laboratory is often faced with the problem of mislabeled speci-
