The measurement of iron and iron-binding capacity in plasma containing deferoxamine

BRIEF

CLINICAL

LABORATORY

AND OBSERVATIONS

The measurement of iron and iron-binding capacity in plasma containing deferoxamine N o r m a n R. Gevirtz and Louis R. Wasserman With the technical assistance of Gertrude Lurinsky NEW

YORK,

N.

Y.

T H E PI~ASMA iron concentration is of importance in establishing the diagnosis and evaluating the course of acute iron poisoning. T h e method used for iron assay, therefore, must give data of clinical value even with specimens obtained during periods of therapy with iron chelating agents such as deferoxamine. The studies reported below demonstrate that deferoxamine interferes with some of the described methods for plasma iron determination. However, this interference may be useful clinically.

analytic procedures. (With experience the hydrosulfite can be estimated visually as the amount at the tip of a small spatula.) The optical density was read at the appropriate wave length 1-a in a Beckman B spectrophotometer. The patients studied were receiving deferoxamine for therapeutic purposes. RESULTS

T h e in vitro addition of deferoxamine to plasma, or the parenteral administration of

M A T E R I A L S AND M E T H O D S

Table I. Representative iron values in plasma with added deferoxamine (in vitro)

Plasma iron concentration was determined by the methods of Ramsey, 1, a Schade and associates, 2 and U I B C (unsaturated iron binding capacity) by the method of Schade and associates. 2 Deferoxamine "~ was added to plasma for the in vitro studies as indicated in Table I. Powdered sodium hydrosulfite (Na2S204, low in iron), 2 mg. per milliliter of plasma, was added to the reaction mixture after completion of the iron

Plasma control

Plasma with deferoxamine

Plasma with de/eroxamine, Na~S~O,, added

Ramsey1

228 162

135t 112t

225t 174t

Schade and associates2

228 196 9I

135t 49t 135

225t 204t 835

Ramseya

168 190

166" 186t

166" 195t

Method

From the Department of Hematology, The Mount Sinai Hospital. Aided in part by United States Public Itealth Service Grant A M 01063, the National Institute o[ Arthritis and Metabolic Diseases, Grant FR-71, Division of Research Facilities, and by the A'lbert A. List, Frederick Machlin, and Anna Ruth Lowenberg Research Funds.

The plasma iron concentration is apparently decreased when deferoxamine is added, and the addition of sodium hydrosulfite is ~ecessary to detect the total iro~ present when analysis is performed by tile methods of Ramsey1 or Schade and assoeiates. 2 ~0.1 mg. of deferoxamine per millillter of plasma. ~0.2 rag. of deferoxamine per milliliter of plasma. ++0.5 rag. of defero~amine per milliliter of plasma.

~Kindly supplied as Desferal methane-sulfonate by Dr. W. F. Westlin o[ Ciba Pharmaceutical Co., Summit, N. J.

802

Volume 68

Number 5

Brie[ clinical and laboratory observations

deferoxamine to patients, results in falsely low values for serum iron when the methods of R a m s e y ~ or S c h a d e a n d associates2 are used ( T a b l e s I a n d I I ) . T h e a d d i t i o n of a strong reducing agent, sodiunl hydrosulfite, is necessary for accurate determinations with these methods. N o modification of the 1957 m e t h o d of R a m s e y a is necessary to measure total iron in p l a s m a which contains deferoxamine. A n a p p a r e n t increase in the U I B C is noted in the p l a s m a of patients receiving deferoxamine (Table III). T h e a d d i t i o n of sodium hydrosulfite is n o t necessary w h e n d e t e r m i n i n g iron in aqueous solutions of ferrioxamine-deferoxamine. F u r t h e r m o r e , d e f e r o x a m i n e is quantitatively m e a s u r e d as an a p p a r e n t U I B C , e.g., an aqueous solution of 0.05 mg. of d e f e r o x a m i n e p e r milliliter os w a t e r gave an a p p a r e n t U I B C of 438 (predicted 425). DISCUSSION T h e q u a n t i t a t i v e colorimetric analysis of iron in p l a s m a is based u p o n the f o r m a t i o n

T a b l e II. P l a s m a iron values in patients receiving p a r e n t e r a l d e f e r o x a m i n e for t h e r a p y of iron o v e r l o a d i n g s y n d r o m e

Method Schade and associates2

Ramsey a

Plasma after deferoxamine administration 288 269 213 150

Plasma after deferoxamine, NaxS~O~ added 370 324 286 196

169 178

i80 188

80 3

of a colored c o m p o u n d from d i v a l e n t iron a n d a specific chromogenic agent. Since transferrin-bound iron is in the trivalent state, ~ the m e t h o d o l o g y requires b o t h a dissociation of iron from transferrin a n d a reduction f r o m the trivalent to the divalent state. D e f e r o x a m i n e has a stability constant for trivalent iron t h a t is greater t h a n t h a t of transferrin, ~ and, therefore, m a y chelate iron t h a t was b o u n d to transferrin. I n p l a s m a ( a n oxidizing system for ferrous cation) the m i l d reducing agents present in some serum iron analytic procedures 1' 2 a r e u n a b l e to reduce the ferric cation in f e r r i o x a m i n e to ferrous cation. A strong reducing agent in a d e q u a t e concentration, 2 mg. of sodium hydrosulfite per milliliter of plasma, is n e e d e d for this r e d u c t i o n to ensure detection of all iron present in the plasma. I n aqueous solutions, however, the Schade 2 reagents have sufficient reducing potential to p e r m i t detection of ferrioxa m i n e iron. T h e Ramsey a m e t h o d of 1957 has sufficient reducing potential to ensure detection of all iron in p l a s m a w i t h o u t modification for the presence of deferoxamine. D u r i n g the t r e a t m e n t of a p a t i e n t with acute iron toxicity the p l a s m a iron concentrations as m e a s u r e d by the m e t h o d of S c h a d e a n d associates z or the 1953 m e t h o d of R a m s e y 1 m a y be a m o r e useful guide for the evaluation of clinical toxicity t h a n is the total p l a s m a iron c o n c e n t r a t i o n determ i n e d after hydrosulfite t r e a t m e n t or by the 1957 m e t h o d of Ramsey. a These m e t h o d s d e t e r m i n e both transferrin-bound iron a n d the clinically toxic n o n c h e l a t e d iron2, 6 D e f e r o x a m i n e - b o u n d iron is nontoxic, a n d

T a b l e I I I . T h e effect of p a r e n t e r a l l y a d m i n i s t e r e d d e f e r o x a m i n e on the m e a s u r e d U I B C in p l a s m a I II

Time UIBC

7A.M. 27

9 a.l'a. 27

10:10 A.M. 169

11A.lVt. 132

1P.W. 65

3 I'.M. 46

Time 7A.M. 8A.•. 9 A.~S. 9:20 A.~S. 9:40 A.~. 10:10 A.M. 11:50 A.M. UIBC 27 15 39 200 321 352 201 TIBC -364 391 -626 702 655 Deferoxamine, 2,0 gin:, was given intramuscularly at 9:04 A.~. fox"study I and 3.0 gin. was given intramuscularlyat 9:05 A.~. for study II. The marked increase in unsaturated iron binding capacity after deferoxamine administration is attributed at least in part to circulating deferoxamine.

80 4

Brief clinical and laboratory observations

contributes to the r a p i d amelioration of clinical symptomatology observed when it is used for the t h e r a p y of acute iron toxicity. 7' s

SUMMARY 1. Several of the widely used analytic procedures for p l a s m a iron do not measure total p l a s m a iron when deferoxamine is present. A modification which consists of a d d i n g a strong r e d u c i n g a g e n t , sodium hydrosulfite, at the end of the usual iron analytic m e t h o d is necessary. 2. T h e significance of p l a s m a iron conc e n t r a t i o n as directly m e a s u r e d (without hydrosulfite a d d i t i o n ) for evaluating the course of acute iron toxicity is discussed.

REFERENCES

May 1966

2. Schade, A. L., Oyama, J., Reinhart, R. W., and Millar, J. R.: Bound iron and unsaturated iron-binding capacity of serum; rapid and reliable quantitative determination, Proc. Soc. Exper. Biol. & Med. 87: 443, 1954. 3. Ramsey, W. N. M.: The determination of iron in blood plasma or serum, Clin. chirn. acta 2: 214, 1957. 4. Bothwell, T. H., and Finch, C. A.: Iron metabolism, Boston, 1962, Little, Brown & Company. 5. Keberle, H.: The biochemistry of desferrioxamine and its relation to iron metabolism, Ann. New York Acad. Sc. 119: 758, 1964. 6. Beutler, E., Fairbanks, V. F., and Fahey, J. L.: Clinical disorders of iron metabolism, New York, 1963, Grune & Stratton, Inc. 7. Henderson, F., Vietti, T. J., and Brown, E. B.: Desferrioxamine in the treatment of acute toxic reaction to ferrous gluconate, J. A. M. A. 186: 1139, 1963. 8. Gevirtz, N. R., and Rausen, A. R.: Personal observations.

I. Ramsey, W. N. M.: The determination of iron in blood serum or plasma, Biochem. J. 53: 227, 1953.

The effect of deferoxamine on the determination of serum iron and iron-binding capacity R a y E. Heifer, M.D., ~ a n d Denis O. Rodgerson, F.I.M.L.T. DENVER,

COLO,

THE INTRODUCTION of the new iron chelating agent, d e f e r o x a m i n e t ( D F ) , has offered a substantially i m p r o v e d m e t h o d for r e d u c t i o n of the m o r b i d i t y a n d m o r t a l i t y rates of acute iron poisoning. I t is a p p a r e n t f r o m reports in the l i t e r a t u r e t h a t m a n y workers are using the serum iron level to estimate the success of t h e r a p y ? -4 I t is the Supported in part by United States Public Health Service Grant MO1-FR-69. ~Address, Depart~'aent of Pediatrics, University of Colorado Medical Center, 4200 East Ninth Avenue, Denver, Colo. 00220. "~Deferoxamlne methane sulfonate was supplied by the C1BA Pharmaceutical Company~ Sgmmit~ N. J.

purpose of this p a p e r to d e m o n s t r a t e t h a t s t a n d a r d l a b o r a t o r y p r o c e d u r e s for the det e r m i n a t i o n of serum iron a r e u n r e l i a b l e after D F has been a d m i n i s t e r e d ? I t will be shown t h a t total serum iron in the presence of D F can be a c c u r a t e l y d e t e r m i n e d by a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y ; the c o n c e n t r a t i o n of b o u n d a n d u n b o u n d or "free" iron can also be d e t e r m i n e d . C o n v e n t i o n a l m e t h o d s for t h e d e t e r m i n a tion of serum iron d e p e n d on the f o r m a t i o n of a complex b y the chelation of iron with a c h r o m o g e n 2 I r o n is r e m o v e d f r o m transferrin b y acidification, reduction, a n d pre-