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14. Lee VW, Willis C. Activity of human and nonhuman amylases ondifferent substrates used in enzymatic kinetic assay methods-a

16. Howe L, Elmslie RG. Stability patients with pancreatitis. Aust J Exp

of aniylase in serum

from

Biol

Med

Sci

197 1;49:513-5.

pitfall in interlaboratory 1982;77:290-6.

quality control. Am J Cliii Pathol

17. Tieta Saunders

NW. Clinical guide Co., 1983:54-5.

to

laboratory

tests.

Philadelphia:

WB

15. O’DonnellMD, McGeeney KF. Suitability of in the differential inhibition assay for human salivary aniylase. Clin Chem 1983;29:510.-2.

control materials

pancreatic

and

18. Eberhagen VD, !ssmer A, Brandmaier B.

Ausscheidung im Urin Biochem 1970;8:284-7.

bei

gesunden

Personen.

Uber die Amylase- Z Kim Chem Kim

CLIN. CHEM. 34/4, 757-760

(1988)

Factors InfluencingFluorescenceSpectra of Free Porphyrins

C#{233F.sProlo, AlbertoL Frisardl, Ernesto R. Resnlk, Angel E. M.

Schoua, and Alcira M. del C. fl$1

We recorded fluorescence excitation and emission spectra of

near-ultraviolet.

This useful property

is widely applied in

uro- and coproporphyrin tions, to see how these based on measurement

under different experimental condi- conditions influence quantifications of fluorescence intensity. We found

that, for bands

a and j3of the emission spectra

and the main

peak of the excitation spectra, fluorescence

depends on pH

and is minimal near pH 5 and near pH 7-7.5 for copro- and uroporphyrin, respectively. For band yof the emission spec- tra there was a constant decrease of fluorescense with increasing alkalinity of the solution. The intensity of porphyrin fluorescence also depends on ionic strength, reaching sharp maxima at 0.1 mol/L (for uroporphynn) and 1 mol/L (for coproporphyrin). The organic mixture ethyl acetate:acetic acid (4:1 by vol), commonly used to extract porphyrins from biological samples, markedly diminishes the fluorescence of both porphyrins as compared with the same concentration of each porphyrin in aqueous acidic solvent. Furthermore, when we measured different ratios of uro:copro mixture at three distinct pHs and buffers, we found that at pH 10.5 (in carbonate buffer) the measured units of fluorescence depend only on total porphyrin concentration and not on the composi- tion of the mixture.

clinical

laboratories to quantify

tion

being

the

relatively

high

porphyrins,

its

major limita-

concentration

necessary for

accurate spectrophotometric

measurement.

Another

characteristic

free

porphyrins

and

their

and most important esters is the intens

property of e red fluores-

cence emitted 400 nm, which

on radiation allows their

with light detection

of in

wavelength near concentrations of

about iO

moltL. Because of their

sensitivity

and specific-

ity, fluorometric

tetrapyrroles.

In

methods are our laboratory

now this

often used to

quantify

is the method

of choice

for samples investigation.

both from patients and from any other related Therefore, we undertook a study using differ-

ent experimental ionic strength,

conditions to determine and solvent composition on

the effect of

pH,

measurement

of

porphyrin fluorescence. MaterIals and Methods

Chemicals Coproporphyrin

ifi

methyl

ester was from

Porphyrin

Products, Legan, UT. Uroporphyrin

ifi was prepared from

turaco (Turacus sp.) feathers

(10),

a gift from C. Rimington.

Free porphyrins

were obtained from their

respective methyl

AdditIonal Keyphrases:

diagnosis of porphyrias

of porphyrins

in biological samples

determination

esters by acid hydrolysis

and

at room

temperature).

(HC1 6.85 mol/L,

Stock

solutions

24 h, were

in darkness

prepared

in

HC1 at concentrations

of 0.1 mol/L (for coproporphyrin)

or

0.5 moIJL (for uroporphyrin),

measured spectrophotometri-

The porphyrias are common fundamental

a group of diseases abnormalities in the

that have in heme biosyn-

cally. All

Both other

stocks were then diluted as indicated. chemicals used were reagent grade.

thetic pathway (1-5).

Diagnosis

characteristic rin excretion

history and physical or accumulation in

demands a combination of signs plus excess porphy- the body, so measurement

of

these

tetrapyrroles

acquires

clinical

significance

(6,

7).

There are quantifying

several methods for separating,

porphyrins,

metalloporphyrins,

identifying, and and their deriv-

atives, extensively reviewed by Falk (8) and Smith (9). The presence of a conjugated double-bond system in the tetrapyr-

role nucleus is the structural feature

responsible for

strong

and

characteristic

absorption

bands

in

the

visible

the and

Solutions

Buffers. To study the effect of pH, we used buffers covering

the pH adjusted Ionic

range from 1 to 11 (Table 1). The pH was carefully

at room strength.

temperature

Because

(25 #{17to wi,thin

KC1

dissociates

±0.02 unit.

completely

in

solution, we into account

used it to adjust solution ionic strength. the presence of HC1 and knowing the

Taking concen-

tration solution

of the ionic species, the ionic strength was calculated by using the equation a

()

of the

#{18 C1Z2,

where

C

is

the

concentration

and

Z

the

charge.

Centro

de Investigaciones sobre Porfirinas y Porfirias (CIPYP),

Apparatus

Ciudad

Universit.aria-Pabell#{243U-2do

Piso,

1428

Buenos

Aires,

Reptiblica Argentina. ‘Address correspondence to this author, at Viamonte 1881-1O

A, 1056 Buenos Aires, Argentina.

Dedicated to Prof. Dr. Claude Rimington FRS, on the occasion of his 85th birthday.

Received

July

24,

1987; accepted

January

22,

1988.

We used a Model RF-510 spectrofluorophotometer (Ski- madzu Co., Kyoto, Japan) with an optional Shimadzu R928

photomultiplier nm) and with

(wavelength measuring

an

Omnigraphic

2000

range,

220 to 900

recorder

(Houston

Instruments, Austin, ments we used a DB

TX). For spectrophotometric

measure-

Model

35

(Beckman

Instruments

Inc.,

CLINICAL CHEMISTRY, Vol. 34, No. 4, 1988 757

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