Table 1.

EffeCt of Freezing on ALT and AST In Serum Mean difference, Activity range,
Method Sample Set I Test U/I
13-135

Fresh vs frozen
62 33 2

Fresh vs

refrlg
3 wks

2 wks

ALT IFCC IFCC (P-5-P-treated) ALT IFCC AST Sample Set 2 Pre-IFCC ALT Sample Set 3 ALT IFCC IFCC AST a Afterthree weeksat 4 #{176}C.

13-135
14-45

7-69 9-114
11-69

5
568

ceeded the upper limit of normal, the change in value upon dilution could still alter the clinical significance of the results. Because the use ofa 10-fold dilution could result in loss of sensitivity in the lower end of the measurement scale, we investigated the effects of reducing the volume of the sample and standards from 50 to 20 iL and using 25 arb. Units/mL instead of 50 arb. units/ mL as the highest-concentration standard. Decreasing the sample volume resulted in dilution linearity up to
25 arb. units/mL, clearly identiIring

86

12 7

In summary, whereas AST analysis with the IFCC reagents (in the absence of exogenous pyridoxal phosphate) is unaffected by freezing, the same is not true for ALT analysis. The only difference between the reagents used is the Tris buffer in the IFCC method, which suggests a possible change in the conformation of ALT in this buffer. References
1. Wroblewski F, LaPue JS. Serum glutamic pyruvic transanuinase (SGP-T) in hepatic disease: a Preliminary report. Ann Intern Med 1956;45:801. 2. Bergmeyer HU, Bowers Jr GN, Moss DW. Provisional recommendations on IFCC methods for the measurementof catalytic concentrations of enzymes. Clin Chem 1977;23:887-99. 3. Bergmeyer HU, Horder Jr M, Bowers Jr GN, Moss DW. Provisional recommendations on IFCC methods for the measurement of catalytic concentrations of enzymes. J Clin Chem Clin Biochem 1977; 15:719-20. 4. Bergmeyer HU, Bowers Jr GN, Horder M, Moss DW. IFCC methods for the measurement of catalytic concentration of en-

has a high for breast cancer. It is widely used to monitor therapy, to detect relapses, and to exclude bone metastases in conjunction with bone scintigraphy. Reports from various centers on the measurement of MCA in serum of patients with primary breast cancer and in serum of tumorfree patients after treatment of the primary cancer and benign breast diseases are in general agreement (2-4). So far, there are few reports on the utility ofMCA for monitoring patients with metastatic disease (5, 6). During a study ofthe change in MCA concentrations in serum in metastatic breast cancer, some sera showed a nonlinear response with dilution. Such results would lead to underestimations of MCA in serum. To ensure maximal sensitivity of the assay, we investigated the phenomenon and now propose a simple modification ofthe enzyme immunoassay test procedure used (Roche Diagnostics; 1), to obviate the observed
antibody specificity nonlinear response of rceults on dilu-

b12 (1). MCA

those sera that needed further dilution. We examined the effect of this change in procedure with 42 sera from patients with metastatic breast cancer whose first-measured values had been <50 arb. Units/roL, which would not have received special comment. The nominal median MCA concentration of these samples was 21.6 arb. Units/mL (range 10.1-44.7) when first measured; this increased to a median of 36 arb. units/ mL (range 10.5->250) when measured by the modified procedure (P <0.001). We therefore found this modified procedure suitable for quantifying MCA in metastatic disease. This change in procedure had no effect on MCA concentrations measured in sera from 25 patients with primary tumors, 20 with benign breast disease, nine each of tumor-free patients with or without adjuvant chemotherapy, and 10 with local recurrence. The cause of the nonlinearity with dilution of certain sera in the MCA test is not clear. It is interesting that this effect appears to be associated with metastatic breast cancer but is not seen in all patients. Moreover, it is not influenced by freezing and thawing.

zymes. J Clin 1980;18:521-34.

Chem Clin

Biochem

Rajani Prasad Kathy Firkins Jean Fiorello Quincy#{174} Laboratory Services 5100 E. 24th St. Kansas City, MO 64127

tion of samples. In a preliminary experiment, 40 of 45 sera from patients with metastatic breast cancer, chosen because of their unexpectedly low values, reached a plateau only upon dilution. For samples from patients with nonmetastatic breast cancer, the dilution-corrected values for MCA were not altered by
dilution.

Adopting the proposed procedure eliminates underestimating MCA values due to nonlinearity. In our experience with 150 patients with metastatic breast cancer, only 7.5% had values in excess of 250 arb. units/ mL. Four sera did not reach a plateau even after 100-fold dilution (>2500 arb. units/mL). The clinical significance of such increased concentrations of MCA remains uncertain.
virtually References 1. Stahli C, Caravatti M, Aeschbacher M, Kocyba C, Takacs B, Carmann H. Mucinlike carcinoma associated antigen defined by three monoclonal antibodies against

In a second experiment, Nonlinearity of Measurements of a Tumor Marker for Breast Cancer To the Editor: Mucin-like

a further

206 samples from patients with meta-

carcinoma-associated

antigen (MCA) is a glycoprotein antigen related to human milk-fat globulin defined by the murine monoclonal

static breast cancer, having values for apparent MCA between 14 and 50 arbitrary units/mL, were retested after being diluted 10-fold, to investigate the extent of this anomaly. Upon dilution, 113 of the 206 samples showed an MCA increase of 50%. Although the initial results had ex-

three different epitopes. Cancer Res 1988;48:6799-802 2. Cooper EH, Forbes MA, Hancock AK, Price JJ, Parker D. An evaluation of mucm-like carcinoma associated antigen

408 CLINICAL CHEMISTRY, Vol. 36, No. 2, 1990

(MCA) in breast cancer. Br J Cancer 1989;59:797-800. 3. Eskelinen K, Tikanoja S, Colan Y. Use

of tumour markers CA 15-3, MCA and

CEA in breast cancer diagnosis. J Tumor

Marker Oncol 1989;4:39-44. 4. Bombardieri E, Giion M, Mione R, Dittadi R, Bruscagnin G, Buraggi G. A mucinous-like carcinoma-associated antigen (MCA) in the tissue and blood of patients with primary breast cancer. Cancer 1989;63:490-5. 5. Rasoul-Rockenschaub S, Zielinski CC,
Kubista E, et al. Diagnostic value of mucinlike carcinoma associated antigen (MCA) in breast cancer. J Cancer Clin Oncol 1989;25:1067-72. 6. Bieglmayer C, Szepsi T, Neunteufel W. Follow-up of metastatic breast cancer pa-

of our data into a broader picture. First, a curve of pH vs the pseudofirst-order rate constants for the peroxidation of NAD(P)H demonstrates that, at the pH used by Jeyasingham
et al. in their study, pH 7.59, dinucleotide oxidation is negligible (5). This

portant contribution on heme proteins and hope they join me in recognizing that much has yet to be learned about regulatory activities surrounding this important group of proteins. References 1. Jeyasingham MD, Pratt DE, Rooprai HK. Interaction between pyridine nucleotide coenzymes and heme proteins as a possible source of error in assay of activities of coenzyme-linked enzymes. Clin

considerably strengthens their conclusions. Second, the reaction conditions that both groups used were somewhat similar. Ours were specifically as follows: [NAD(P)H] = 1.1 x i0 mol/L, [Fe(ll), MP-11, cyt ci = i0 mollL (hemoglobin at 1 g/L m 1.5 x 10 mol/L), and = 37 #{176}C.
Under these conditions (at pH 5.5 in

tients with mucin-like carcinoma associEH Cooper ated antigen: comparison to CA 15.3 and carcinoembryonic antigen. Cancer Lett 1988;42:199-206. M.A. Forbes Dept. Chem. Pat hol. Univ. of Leed.s. Leeds LS2 9NL, UJC. V. Laurence Cookridge Hosp. Radiotherapy Leeds LS16 6QB, UK. Centre

M.F. Beya J. de Chastonay F. Hoffmann-La

CH-4002

Roche

Basel, Switzerland

More on Heme Protein-Pyridine Nucleotide Interactions To the Editor: The recent report by Jeyasingham et al. (1) on the interaction of pyridine
nucleotides with hemoglobin and

acetate buffer) we monitored dinucleotide oxidation in both the presence and absence of H202. Although our interest was primarily in the kinetics of the peroxidation of NAD(P)H, we recognized that pyridine nucleotide binding to our catalytic species contributed to the overall reaction rate. Using difference spectra, we observed that binding ofsubstrate NAD(P)H to Fe(ll) could be monitored at -270 nm. Therefore, I am pleased to see that Jeyasingham et al. (1) were able to confirm our observation (5) that dinucleotide binding to Fe(ll) and heme proteins occurs, albeit in a more straightforward manner than our study permitted. We did, however, note that by monitoring the aforementioned spectral changes in the ultraviolet region we were able to determine that the binding reaction is not the rate-limiting step in the overall oxidation process. From a structural and mechanistic standpoint, tetra-coordinated Fe(II) as
it exists in heme has two available

Chem 1989;35:2129-33. 2. Bennun A, Needle MA, DeBari VA. Stimulation of the hexose monophosphate pathway in the human erythrocyte by Mn2: evidence for a Mn2-depondent NADPH peroxidase activity. Biochem Med 1985;33:17-21. 3. DeBari VA, Needle MA, Bennun A. Catalase as a manganese-dependent NADPH peroxidase [Communication]. Biochem Soc Trans 1985;13:125-7. 4. Kiebanoff SJ. The iron-H202---iodide cythtoxic system. J Exp Med 1982;156:12627. 5. Cruz R, Weinstein E, DeBari VA. Cornparison of enzyme and metal catalyzed dinucleotide peroxidations. Ann NY Aced Sci

1987;494:394-8.
6. Bennun A, Seidler N, DeBari VA, Divalent metals in the regulation of hemoglobin affinity for oxygen. Ann NY Acad Sci oxito NADP catalyzed by the heme-undecapeptide from cytochrorne C. Biochem Biophys Res Cominun 1982;113:710-6. dation of NADPH

1986;463:76-9. 7. Bodaness RB. The H202-mediated

Vincent

A. DeBari

Rheumatology Lab. St. Josephs Hosp. and Med. Ctr. School ofGrad. Med. Ethic. Seton Hall Univ. Paterson, NJ 07503

other heme proteins especially interested me, because of some work we reported several years ago and its bearing on this issue. Our approach to this subject came from a somewhat different orientation, i.e., from an interest in the enzymatic peroxidation of NADPH as a regulatory activity in erythrocyte metabolism (2, 3). Based on the description by Klebanoff (4) of an alternative (to the myeloperoxidase reaction) microbicidal system in neutrophils involving a classical Fenton-type reaction, we extended our study to Fe(ll) and reported (5) the results of a study of dinucleotide (NADH, NADPH) peroxidation by Fe(II), cytochrome c, and microperoxidase-11 (MP-11, an 11amino-acid fragment of cytochrome c). A set of fortuitous circumstances allows, to some extent, the integration

coordination sites, one, of course, for 02, the other being subject to physiological regulation by divalent cations (6). Binding of the adenine moiety of NAD(P)H at the regulatory coordination site, as suggested by the change in absorbance in the 260-270 nm region, could very nicely leave the nicetinamide moiety available for redox reactions at the catalytic site. This circumstance was also touched upon by the Jeyasingham group in their
final paragraph. I would be remiss if I did not point

LimItatIons of the Fructosamine Reaction To the Editor: Earlier we showed (1) that differences in spectral characteristics between standards and protein samples in the fructosamine reaction were related to the solubility of the diformazan product. Subsequently, Vogt et al. (2, 3) reported similar findings and also noted that uric acid is a potential interferant that can be removed by adding uricase to the initial reaction mixture. Most recently, Kallner (4) reported that addition of urate (0.5 mmol/L), in the presence of Triton x-100 surfactant (20 gIL), increases the value for serum fructosamine by
almost 10-fold. In the interim we have

out that in his study of the peroxidation of NADPH by MP-11, Bodaness (7) clearly recognized and reported heme protein-pyridine nucleotide binding. Apparently, neither he nor we recognized the potential application of our observation to the specific (and quite important) problem addressed by Jeyasingham et al. In conclusion, I congratulate Jeyasingham and co-workers for their im-

continued

to investigate

the general

assay methodology

and in particular

CLINICAL CHEMISTRY, Vol. 36, No. 2, 1990 409