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Automated enzymatic assay for measurement of lithium ions in human serum.

For decades, lithium carbonate has been one of the most effective agents for the treatment of bipolar disorder
This article is an expansion of a section entitled Treatment from within the main article: Bipolar disorder

Bipolar disorder has not currently been cured but it can be managed.
 (manic depressive psychosis). Lithium acts by altering intraneuronal metabolism of catecholamines Catecholamines
Family of neurotransmitters containing dopamine, norepinephrine and epinephrine, produced and secreted by cells of the adrenal medulla in the brain.
, inhibiting noradrenaline-sensitive adenylate cyclase, reducing synaptic transmission, and increasing neuronal excitability with modification of the central nervous system (CNS See Continuous net settlement.


See continuous net settlement (CNS).
) amine concentrations. Recent studies have shown that lithium holds promise against Alzheimer disease. Lithium has many side effects, however. Overdosage of lithium can cause acute [Li.sup.+] intoxication, which occurs quite often because of lithium's narrow therapeutic index. For example, serum [Li.sup.+] concentrations >1.5 mmol/L 12 h after a dose usually indicate a significant risk of intoxication. Therefore, the timely and accurate monitoring of serum concentrations of lithium after a therapeutic dose is critical (1-4).

The most commonly used methods to detect serum lithium are ion-selective electrodes (ISEs) and flame emission photometry photometry (fōtŏm`ətrē), branch of physics dealing with the measurement of the intensity of a source of light, such as an electric lamp, and with the intensity of light such a source may cast on a surface area. . ISE Ise (ē`sā), city (1990 pop. 104,164), Mie prefecture, S Honshu, Japan, on Ise Bay. It is one of the foremost religious centers of Shinto, the site of the shrines of Ise.  analyses rely on ion-specific electrodes. Ideally, each electrode possesses a unique ionselective property that allows it to respond to the desired ion. In practice, however, interference from other ions in the sample compromises the specificity of the detecting electrode, rendering the electrodes susceptible to false readings. The instrumentation for ISE analysis is relatively expensive, requires routine maintenance that is sometimes cumbersome and time-consuming, and demands that the operating technician have considerable skill and knowledge for accurate and consistent readings. Flame emission photometry relies on the principle that certain atoms, when energized by heat, become excited and emit a light of characteristic wavelength. Radiant energy produced by atoms in the flame is directly proportional to the number of atoms excited in the flames, which is directly proportional to the concentration of the substance of interest in the sample. Like ISE analysis, the instrumentation required for this method is complex and expensive. Moreover, flame emission photometry requires the use of combustible gas, necessitating expensive hazard prevention measures. More recently, a dye-based lithium assay has been developed and used in certain automated chemistry analyzers (5).

We recently developed an enzymatic lithium assay, which has been adapted to most automated clinical chemistry analyzers. In this assay, lithium is determined through a kinetic coupling system involving a lithium-sensitive enzyme, 3',5'-bisphosphate nucleotidase nucleotidase /nu·cleo·ti·dase/ (noo?kle-o-ti´das) an enzyme that catalyzes the cleavage of a nucleotide into a nucleoside and orthophosphate.

, from yeast. This enzyme is sensitive to lithium inhibition, with an [IC.sub.50] of 0.1 mmol/L. The enzyme is also sensitive to sodium inhibition, with a much higher [IC.sub.50] (>20 mmol/ L). The inhibitive effect of serum sodium ions is effectively masked by the sodium-specific binding reagent Kryptofix 221. Through enzymatic coupling, substrate adenosine 3',5'-bisphosphate (PAP) is converted to hypoxanthine hypoxanthine /hy·po·xan·thine/ (-zan´then) a purine base formed as an intermediate in the degradation of purines and purine nucleosides to uric acid and in the salvage of free purines. Complexed with ribose it is inosine.  by enzymatic reactions to generate uric acid and hydrogen peroxide ([H.sub.2][O.sub.2]). The generated [H.sub.2][O.sub.2] then reacts with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-m-toluidine (EHSPT) and 4-aminoantipyrine (4-AA) to form a quinone quinone

Any member of a class of cyclic organic compounds comprising a six-membered unsaturated ring (see saturation) to which two oxygen atoms are bonded as carbonyl groups (−C=O; see functional group).
 dye with an absorbance absorbance /ab·sor·bance/ (-sor´bans)
1. in analytical chemistry, a measure of the light that a solution does not transmit compared to a pure solution. Symbol .

 maximum at 556 nm. The rate of dye formation is inversely proportional to the lithium concentration in serum:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII ASCII or American Standard Code for Information Interchange, a set of codes used to represent letters, numbers, a few symbols, and control characters. Originally designed for teletype operations, it has found wide application in computers. ]

where PNP is purine nucleoside phosphorylase Purine nucleoside phosphorylase (also known as PNPase) is an enzyme (EC involved in purine metabolism. PNP metabolizes inosine into hypoxanthine and guanosine into guanine, in each case creating ribose phosphate. .


The assay is formulated into a lyophilized 2-reagent system with MES (Manufacturing Execution Software) Software that provides real time access to plant activities that include equipment, labor, orders and inventory. An MES integrates the data with enterprise resource planning (ERP) systems so that management has complete control of  buffer (pH 6.0). The assay analyzes nonhemolyzed serum samples on a variety of automated analyzers in as little as 10 min.

The precision of the Diazyme enzymatic lithium assay was evaluated on a Cobas Mira analyzer according to Clinical and Laboratory Standards Institute (formerly NCCLS NCCLS National Committee for Clinical Laboratory Standards ) guideline EP5-A. In the study, 2 specimens containing 1 and 2.3 mmol/L lithium, respectively, were tested in 2 runs per day with duplicates for a period of 20 working days. The results showed that within-run imprecision (CVs) was 4.7% for 1.0 mmol/L [Li.sup.+] and 3.3% for 2.3 mmol/L [Li.sup.+], respectively; and total imprecision (CVs) was 6.9% for 1 mmol/L and 5.5% for 2.3 mmol/L [Li.sup.+], respectively.

To demonstrate accuracy, the Diazyme lithium enzymatic assay was tested with individual serum samples and compared with both ISE and Thermo Trace colorimetric col·or·im·e·ter  
1. Any of various instruments used to determine or specify colors, as by comparison with spectroscopic or visual standards.

 methods. Three concentrations of serum calibrators containing 0 mmol/L (low), 1 mmol/L (medium), and 3 mmol/L lithium (high) were prepared by adding 1 mol/L lithium acetate stock solution to the pooled lithium-free serum. After verification with a Trace lithium assay, the calibrators were sent out to a certified laboratory for confirmation.

The pooled serum and the individual patient serum samples used for this study were from a certified commercial source and were accompanied by an Institutional Review Board certificate of approval for all protocols, including informed consent, used to collect samples. To ensure that the lithium concentrations were distributed across the reportable dynamic range, some lithium serum samples (especially at lithium concentrations >2.5 mmol/L) were supplemented to the targeted concentrations with a stock solution of 1.0 mol/L lithium.

The Diazyme enzymatic lithium assay and ISE were compared on Cobas Mira [n = 56; lithium concentration, 0-3 mmol/L; y = 1.008x + 0.10 mmol/L ([r.sup.2] = 0.953; [S.sub.y|x] = 0.042 mmol/L)], Hitachi 717 [n = 67; lithium concentration, 0-3 mmol/L; y = 0.987x + 0.072 mmol/L ([r.sup.2] 0.962; [S.sub.y|x] = 0.19 mmol/L)], and Synchron CX-7 [n = 38; lithium concentration, 0-3 mmol/L; y = 0.980x + 0.028 mmol/L ([r.sup.2] = 0.989; [S.sub.y|x] = 0.019 mmol/L)] analyzers. The Diazyme lithium enzymatic assay and Trace lithium reagent were also compared [n = 61; lithium concentration, 0-3 mmol/L; y = 1.083x - 0.071 mmol/L ([r.sup.2] = 0.950; [S.sub.y|x] = 0.20 mmol/L)].

We also determined the linearity of the Diazyme lithium enzymatic assay. A serum sample containing 0 mmol/L Li' was supplemented with lithium acetate stock solution to a concentration of 3.0 mmol/L [Li.sup.+]. The serum sample containing 3.0 mmol/L was then serially diluted with a serum sample containing 0 mmol/L lithium to obtain final lithium concentrations of 0, 0.5,1.0,1.5, 2.0, 2.5, and 3.0 mmol/L. The serum samples prepared as described were tested in triplicate with the Diazyme enzymatic lithium assay on the Cobas Mira. The result demonstrated that the assay was linear at least up to 3.0 mmol/L lithium (y = 0.9857x + 0.0548 mmol/L; [r.sup.2] = 0.9863).

To determine the extent of interference from other cations and substances typically present in serum, we supplemented 1 mL of serum containing 1.0 mmol/L lithium with various concentrations of substances, and then assayed the sera (6 replicates) with the Diazyme enzymatic lithium assay on the Hitachi 917. The results are shown in Table 1.

In summary, we have developed an enzymatic coupling assay for quantitative measurement of lithium in nonhemolyzed human sera and have developed applications for commonly used automated chemistry analyzers. The assay uses 2 reagents, and applications have been developed for testing human serum specimens on the Cobas Mira, Synchron CX-7, and Hitachi 717. The within-run CV was <4.7%, and the total CV was <6.9%. The study testing human sera with lithium concentrations ranging from 0 to 3 mmol/L demonstrated good correlation with both a commercially available ISE method and a colorimetric method on various automated analyzers. The assay was linear up to 3.0 mmol/L. No interference was detected from the following substances at the indicated concentrations: sodium, 200 mmol/L; ammonium, 0.5 mmol/L; calcium, 4.0 mmol/L; magnesium, 2.0 mmol/L; ascorbic acid, 5.0 mmol/L; zinc, 0.25 mmol/L; iron, 0.25 mmol/L; copper, 0.25 mmol/L; potassium, 10 mmol/L; triglycerides, 2.82 mmol/L (250 mg/dL); and bilirubin Bilirubin

The predominant orange pigment of bile. It is the major metabolic breakdown product of heme, the prosthetic group of hemoglobin in red blood cells, and other chromoproteins such as myoglobin, cytochrome, and catalase.
, 770 [micro]mol/L (45 mg/dL).


(1.) Murguia JR, Belles JM, Serrano R. A salt-sensitive 3'(2'),5'-bis phosphate nuclectidase involved in sulfate activation. Science 1995;267:232-4.

(2.) Moyer TP, Pippenger CE. Therapeutic drug monitoring therapeutic drug monitoring Clinical pharmacology The regular measurement of serum levels of drugs requiring close 'titration' of doses in order to ensure that there are sufficient levels in the blood to be therapeutically effective, while avoiding potentially . In: Burtis CA, Ashwood ER, eds. Tietz textbook of clinical chemistry, 2nd ed. Philadelphia: WB Saunders, 1994:1094-154.

(3.) Amdisen A. Serum lithium determinations for clinical use. Scand J Clin Lab Invest 1967;20:104-8.

(4.) Wachtel MS, Paulson R, Plese C. Creation and verification of reference intervals. Lab Med 1995;26:593-7.

(5.) Rumbelow B, Peake M. Performance of a novel spectrophotometric lithium assay on a routine biochemistry analyzer. Ann Clin Biochem 2001;38: 684-6.

DOI : 10.1373/clinchem.2005.053439

Chao Don, Olga Aleshin, Abhijit Datta, and Chong Yuan * (Diazyme Laboratories Division, General Atomics, 3550 General Atomics Ct., San Diego, CA 92121; * author for correspondence: fax 858455-4754, e-mail
Table 1. Interference by cations and substances typically
found in serum.

Interferent              Concentration         Deviation, %

N[H.sub.4.sup.+]    0.5 mmol/L                     +0.8
[P.sub.I]           1.5 mmol/L                     -1.6
[Ca.sup.2+]         5.0 mmol/L                     +4.7
[Mg.sup.2+]         2.0 mmol/L                     +6.2
[Na.sup.+]          200 mmol/L                     +3.9
[K.sup.+]           10 mmol/L                      -0.8
[Cu.sup.2+]         0.25 mmol/L                    -0.8
[Fe.sup.3+]         0.25 mmol/L                    +0.8
[Zn.sup.2+]         0.25 mmol/L                    +0.8
Triglycerides       2.82 mmol/L (250 mg/dL)        -1.2
Ascorbic acid       5.0 mmol/L                     +0.8
Bilirubin           770  mol/L (45 mg/dL)          +6.5

(a) The interference study was carried out by adding various
concentrations of the substances examined to individual patient
serum samples and then testing with the Diazyme enzymatic lithium
assay (6 replicates) on a Hitachi 917 analyzer. The cations and
substances listed produced a <10% deviation from the result for
1.0 mmol/L lithium at the concentrations given.
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Title Annotation:Abstracts of Oak Ridge Posters
Author:Don, Chao; Aleshin, Olga; Datta, Abhijit; Yuan, Chong
Publication:Clinical Chemistry
Date:Oct 1, 2005
Previous Article:Development of a novel enzymatic cycling assay for total homocysteine.
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