Apr 7, 1988 - Glycated lysine. Furoslne. 1906. CLINICAL. CHEMISTRY,. Vol. 34, No. 9, 1988 tution indicate that some cyclosporine metabolites-partic- ularly.
tution indicate that some cyclosporine metabolites-particularly Ml, M13, M17, M18, and M21-possess immunosuppressive activity as demonstrated in vitro by use of lymphocyte-proliferation assays. Metabolites M17 and Ml in particular exhibit immunosuppressive activity in vitro approaching that produced by cyclosporine. Moreover, these metabolites appear to exhibit varying immunosuppressive potency depending upon the T-cell clones used in the test system (8,9). Therefore, the drug concentrations measured by the FPIA assay may more closely approximate the concentration of immunosuppressive drug present in the circulation. These differences between the two assay systems, coupled with the fact that there is greater imprecision with the RIA method, account for the observed correlation coefficient of 0.884 as well as the greater scatter observed at high values. There are two potential problems with the Thx plasma cyclosporine assay: (a) Assay linearity of 1000 1ig/L was not sufficient in our laboratory because about four to six samples in a tray of 20 needed to be diluted. (b) With prolonged use, the integrity of the kit may be compromised because, overtime, control values may exceed the high limit, possibly owing to some evaporation of the reagent. However, we observed no substantial evaporation during an analytical run: control values at the beginning and end of several assays were 247 ± 9.6 and 243 ± 9.4 mg/L (n = 24), respectively. The TDx method offers a simpler procedure with fewer handling steps and faster turnaround time. The instrunientation is automated and compact, allowing better use of
CLIN.
34/9, 1906-1908
CHEM.
Glycated
technologist time and laboratory space. Reagents and supplies present minimal storage and disposal problems and no radioactivity hazard. References
1. Sanghvi A. Impact of organ transplant program on the chemistry laboratory at the University of Pittsburgh. Arch Lab Med 1986;110:95-7. 2. Inununosuppressant Drug Assay: TDx Cyclosporine and olites. Package insert dated 4/13/87. North Chicago, IL:
clinical Pathol MetabAbbott
Laboratories. 3. Van den
Berg JW, Verhof ML, De Boes AJ, Schaim SW. Cyclosporin A assay: conditions for sampling and processing of blood. Clin Chini Acts 1985;147:291-7. 4. Cyclosporin RIA-K1T. Instructions for the determination of cyclosporin concentrations in blood, or plasma, or serum. 5th ed. (revised August 1986). Basle, Switzerland: Sandoz Ltd. 5 Popelka polarization surement
SR,
Miller
DM,
immunoassay of fluorescence
Holen
II analyzer polarization
JT,
Kelso DM. Fluorescence for the rapid, precise meawith use of disposable cu-
vettes. Clin Chem 198127:1198-201. 6. Blecka L.J, Jackson GtJ. Immunoassays in therapeutic drug monitoring. Clinics Lab Med 1987;7:357-69. 7. Jolley ME, Stroupe SD, Schwenzer CJ, et al. Fluorescence polarization immunoassay. ifi. An automated system for therapeutic drug determination. Clin Chem 1981;27:1575-9. 8. Zeevi A, Venkatramanan R, Burckart G, et al. Sensitivity of activated human lymphocytes to cyclosporine and its metabolites. Hum Immunol, in press. 9. Zeevi A, Erias G, Burckart G, et al. Immunosuppressive effect of cyclosporine
Transplant
metabolites
from
human
bile on alloreactive
T cells.
Proc, in press.
(1988)
Lysine Increases
in Serum Samples
Stored at
2000
Lauge SchSffer The glycated lysine in serum protein was determined by HPLC measurement of furosine in hydrolyzed serum, with synthetic NaBOCN#{128}(1 -deoxy-D-fructosyl)-L-lysine as the standard. The glycated lysine value increased with time of storage when the serum was kept at -20 #{176}C, possibly because of an ongoing reaction between glucose and the samino groups of lysine in the frozen serum. The problem was eliminated by using storage at -70 #{176}C or by acidifying the serum before storing it at -20 #{176}C. Additional glycated
Keyphrases: proteins
sample .
handling
.
variation,
source
diabetes
by determination of furosine in hydrolyzed serum (7, 8). All the methods require that samples be analyzed shortly after collection. Here I show why this is so, and propose storage conditions under which samples can validly be kept longer.
Materials
and Methods
Sample preparation. A mixture of 100 pL of serum and 500 1zL of 9.6 mol/L HC1 was hydrolyzed at 95#{176}C for 20 h in a 1.5-niL Eppendorf plastic vial. The hydrolysate was then partly neutralized with 800 pL of saturated sodium formate. NH2-CH
-NH-CH-CO-
-COOH
(CH2)4
Determination of glycated albumin or glycated serum protein as an index of diabetic control over the preceding two to three weeks has been proposed as a useful supplement to the determination of glycated hemoglobin, which reflects the control over the preceding four to six weeks (13). Glycated serum protein has been determined by various color reactions (1, 4,5), by affinity chromatography (6), and
(?H2)4
NH
NH
_________ 8 N HCI
12
CH2
c-o
95#{176}c, 20 h CHOH
ro
CHOH CHOH
Department of Clinical Biochemistry, Novo Research Novo Alle, DK-2880 Bagsvaerd, Denmark. Received March 29, 1988; accepted May 16, 1988. 1906
CLINICAL
CHEMISTRY,
Vol. 34, No. 9, 1988
Institute,
cH2OH
Glycated
lysine
Furoslne
400
untreated, #{149}-#{149} +glucoee 0-0
6-6
a-a 0-0
300
I-.
-20 ‘C 1 mg/mI, 2 tng/tnL
0.25
+glucone untreated, -70 ‘C +glucoee 2 mg/mLIdtrlc +cItrlc acId 10 ing/mL
-20 -20
C C
-J
acId
-20
10 mg/mL
0
-20
E E
‘C
0.20
200 0.15
op
p)0.05
0
p
