Organic Fluorocompounds in Human Plasma: Prevalence and

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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 2, 2016 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0028.ch007

Organic Fluorocompounds in Human Plasma: Prevalence and Characterization W. S. GUY Department of Basic Dental Sciences, University of Florida, Box J424, Gainsville, Fla. 32610 D. R. TAVES Department of Pharmacology and Toxicology, University of Rochester, Rochester, N.Y. 14642 W. S. BREY, JR. Department of Chemistry, University of Florida, Gainsville, Fla. 32611 Taves discovered that samples of his own blood serum con tained two distinct forms of fluoride (1-4). Only one of these was exchangeable with radioactive fluoride. The other, non -exchangeable form was detectable as fluoride only when sample preparation included ashing. This paper is concerned with three aspects of this newly discovered, non-exchangeable form: 1) its prevalence in human plasma, 2) how its presence in human plasma affects the validity of certain earlier conclusions about the metabolic handling of the exchangeable form of fluoride, and 3) its chemical nature. Preliminary work in this laboratory suggested that the non -exchangeable form was widespread in human plasma but did not exist in the plasma of other animals. Ashing increased the amount of fluoride an average of 1.6 ± 0.25 SDμM(range 0.4-3.0) in samples of plasma from 35 blood donors in Rochester, N.Y. (5). No such fluoride was detectable (above 0.3 μM) in blood serum from eleven different species of animal including horse, cow, guinea pig, chicken, rabbit, sheep, pig, turkey, mule and two types of monkey (6). Standard methods for analysis of exchangeable fluoride in serum have in the past included ashing as a step in sample preparation (7). Taves showed that the amount of fluoride in serum that would mix with radioactive fluoride was only about one-tenth the amount generally thought to be present based on analyses using these older methods (4). When plasma samples from individuals living in cities having between 0.15 and 2.5 ppm fluoride in their water supply were analysed by these older methods, no differences were found between the averages for the different cities. This led to the conclusion that "homeostasis of body fluid fluoride content results with intake of fluoride up to and including that obtained through the use of water with a fluoride content of 2.5 ppm" (8). If the non-exchangeable form of fluoride predominated in these samples, differences in the exchangeable fluoride concentration would probably not have been apparent, and it would be unnecessary to postulate such rigorous 117 Filler; Biochemistry Involving Carbon-Fluorine Bonds ACS Symposium Series; American Chemical Society: Washington, DC, 1976.


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homeostatic c o n t r o l mechanisms f o r f l u o r i d e . In t h i s study plasma samples were c o l l e c t e d from a t o t a l of 106 i n d i v i d u a l s l i v i n g i n f i v e d i f f e r e n t c i t i e s with between 0.1 and 5.6 ppm f l u o r i d e i n t h e i r p u b l i c water supply. These were analyzed f o r both forms of f l u o r i d e . I n t h i s way the r e l a t i o n ship between exchangeable f l u o r i d e c o n c e n t r a t i o n i n the plasma and the consumption of f l u o r i d e through d r i n k i n g water was r e evaluated, and the prevalence of the non-exchangeable form was further studied. With respect to the chemical nature of the non-exchangeable form of f l u o r i d e s e v e r a l l i n e s of evidence suggested that i t was some s o r t of organic fluorocompound of intermediate p o l a r i t y , t i g h t l y bound to plasma albumin i n the blood. I t migrated with albumin during e l e c t r o p h o r e s i s of serum a t pH nine (3) and was not u l t r a f i l t e r a b l e from serum ( 2 ) . Attempts at d i r e c t e x t r a c t i o n from plasma with s o l v e n t s of low p o l a r i t y l i k e heptane, petroleum ether and e t h y l ether were g e n e r a l l y u n s u c c e s s f u l . Treatment of albumin s o l u t i o n (prepared by e l e c t r o p h o r e s i s of plasma) with c h a r c o a l a t pH three d i d remove the bound f l u o r i n e f r a c t i o n . And f i n a l l y , when plasma p r o t e i n s were p r e c i p i t a t e d with methanol a t low pH the f l u o r i n e f r a c t i o n o r i g i n a l l y bound to albumin appeared i n the methanol-water supernatant i n a form which s t i l l r e q u i r e d ashing to r e l e a s e f l u o r i n e as i n o r g a n i c f l u o r i d e (5) . Based on these c o n s i d e r a t i o n s the non-exchangeable form of f l u o r i d e i n human plasma i s r e f e r r e d to as "organic f l u o r i n e " throughout the r e s t of t h i s paper. In order to f u r t h e r c h a r a c t e r i z e the organic f l u o r i n e f r a c t i o n , i t was p u r i f i e d from 20 l i t e r s of pooled human plasma and c h a r a c t e r i z e d by f l u o r i n e nmr. M a t e r i a l s and Methods A n a l y t i c a l Methods. Values f o r organic f l u o r i n e were c a l c u l a t e d by taking the d i f f e r e n c e between the amount of i n o r g a n i c f l u o r i d e i n ashed and unashed p o r t i o n s of the same m a t e r i a l . The f o l l o w i n g procedure was used to prepare ashed samples: 1) samples (sample s i z e f o r plasma was 3 ml) were placed i n platinum c r u c i b l e s and mixed with 0.6 mmoles of low f l u o r i d e MgCl and 0.1 mmoles of NaOH, 2) these were d r i e d on a h o t p l a t e and then ashed (platinum l i d s i n place) f o r 2-4 h r a t 600° C i n a muffle furnace which had been modified so that the chamber received a flow of a i r from outside the b u i l d i n g (room a i r increased the blank and made i t more v a r i a b l e ) , and 3) ashed samples were d i s s o l v e d i n 2 ml of 2.5 Ν H S0^ and t r a n s f e r r e d t o polystyrene d i f f u s i o n dishes using 2 r i n s e s with 1.5 ml of water. The f o l l o w i n g procedure was used f o r s e p a r a t i o n of f l u o r i d e from both ashed and unashed samples: 1) samples (sample s i z e f o r unashed plasma was 2 ml) were placed i n d i f f u s i o n dishes (Organ C u l t u r e Dishes, Falcon P l a s t i c s , Oxnard, C a l i f . , absorbent 2

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Filler; Biochemistry Involving Carbon-Fluorine Bonds ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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removed, r i n s e d with water), a c i d i f i e d with 2 ml of 2.5 Ν H SO^, and a g i t a t e d with a g e n t l e s w i r l i n g a c t i o n on a l a b o r a t o r y shaker f o r 30 min to remove C0 ; 2) f o r each sample the trapping s o l u t i o n (0.5 ml, 0.01 Ν NaOH + p h e n o l t h a l e i n - p - n i t r o p h e n o l i n d i c a t o r ) was placed i n a small polystyrene cup i n the c e n t e r - w e l l of the d i f f u s i o n d i s h , 1 drop of 10% T r i t o n - X 100 was added to the sample to decrease surface t e n s i o n and make the d i f f u s i o n r a t e more uniform between samples c o n t a i n i n g plasma and those not, the l i d with a small hole made near i t s l a t e r a l margin was sealed i n t o p l a c e with petroleum j e l l y , 0.02 ml of 4% hexamethyldisiloxane (Dow Corning, F l u i d 200, 0.65 c s , Midland, Mich.) i n ethanol was i n j e c t e d through the hole i n the l i d i n t o the sample, and the hole was sealed Immediately with petroleum j e l l y and a s t r i p of p a r a f f i n f i l m ; and 3) samples were d i f f u s e d with g e n t l e s w i r l i n g f o r a t l e a s t 6 hr, d i f f u s i o n was terminated by breaking the s e a l , and trapping s o l u t i o n s were removed (the i n d i c a t o r c o l o r was checked a t t h i s p o i n t to i n s u r e that they were s t i l l a l k a l i n e ) and d r i e d i n a vacuum oven (60° C, 26 in-Hg vacuum, i n the presence of a NaOH d e s i c c a n t ) . 2


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F l u o r i d e was determined by potentiometry with the f l u o r i d e e l e c t r o d e . The system used c o n s i s t e d of a f l u o r i d e e l e c t r o d e o r i e n t e d i n an i n v e r t e d p o s i t i o n (model 9409A, Orion Research Inc. Cambridge, Mass.), a calomel reference e l e c t r o d e ( f i b e r t y p e ) , a p l a s t i c vapor s h i e l d which j u s t f i t t e d over the bodies of both e l e c t r o d e s forming an enclosed sample chamber i n which watersaturated t i s s u e paper was placed above the sample to prevent e v a p o r i z a t i o n of the sample, and a high impedence voltmeter (model 401, O r i o n ) . Samples were prepared and read i n the f o l l o w i n g way: 10 μΐ of 1 M HAc was drawn i n t o a polyethylene m i c r o p i p e t t e (Beckman Micro Sampling K i t , Spinco Div., Beckman I n s t . Co., Palo A l t o , C a l i f . ) and deposited i n t o the cup c o n t a i n i n g the r e s i d u e from the trapping s o l u t i o n a f t e r d r y i n g ; the f l e x i b l e t i p of the m i c r o p i p e t t e was used to wash down the w a l l s of the cup; and the s o l u t i o n was then t r a n s f e r r e d to the surface of the f l u o r i d e e l e c t r o d e and the reference e l e c t r o d e brought i n t o p o s i t i o n . Surfaces of the two e l e c t r o d e s were b l o t t e d dry between samples. Samples were read i n order of i n c r e a s i n g expected concentra t i o n and sets of samples were read between bracketing c a l i b r a t i o n standards. These standards were used i n two d i f f e r e n t ways during a run. F i r s t , they were flooded onto the e l e c t r o d e surfaces to e q u i l i b r a t e them to concentrations expected f o r samples and to make them uniform. This procedure permitted the a n a l y s t to take reasonably s t a b l e readings f o r samples w i t h i n one minute. Secondly, they were used i n 10 y l volumes f o r readings used i n preparing the standard curve. Values f o r i d e n t i c a l samples ( u s u a l l y t r i p l i c a t e s ) were averaged and the average blank was subtracted from sample means. These were then d i v i d e d by the average f r a c t i o n a l recovery of



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f l u o r i d e (usually 90 to 95%) i n standards treated the same way as the sample s e t . P l a s t i c w a r e (Falcon P l a s t i c s ) was used f o r a l l a n a l y t i c a l procedures to avoid contamination by f l u o r i d e from g l a s s . Liquid volume measurements were made with 1, 5 and 10 ml p o l y s t y r e n e p i p e t t e s and a polycarbonate volumetric f l a s k (100 ml). Reagents were p u r i f i e d to i n s u r e uniformly low blanks. Water was r e d i s t i l l e d and d e i o n i z e d . A c e t i c a c i d and ammonia were r e d i s t i l l e d . F l u o r i d e contamination i n MgCl2 ( a n a l y t i c a l grade) was reduced by preparing a 1 M s o l u t i o n c o n t a i n i n g HC1 to pH 1 and scrubbing with hexamethyldisiloxane vapor i n a column through which the s o l u t i o n was continuously r e c y c l e d . Following scrubbing the s o l u t i o n was b o i l e d to one t h i r d volume to remove any r e s i d u a l v o l a t i l e s i l i c o n e s and then made j u s t b a s i c with NHi+OH. F l u o r i d e contamination i n I^SO^ was reduced by repeated e x t r a c t i o n s of a 6.7 Ν s o l u t i o n with hexamethyldisiloxane and then b o i l i n g to one t h i r d volume to remove the r e s i d u a l s i l i c o n e . Buffered c a l i b r a t i o n standards were made from the same NaOH and HAc stock s o l u t i o n s as f o r samples. The blanks f o r ashed samples ranged between 0.2 and 1.5 nmoles f l u o r i d e and were t y p i c a l l y about 0.5 nmoles. The blanks were smaller f o r unashed samples; these ranged between 0.05 and 0.2 nmoles f l u o r i d e and were t y p i c a l l y about 0.1 nmoles. Factors a f f e c t i n g recovery of f l u o r i d e during d i f f u s i o n were i n v e s t i g a t e d w i t h F~ t r a c e r . Recovery during d i f f u s i o n was 97% a f t e r 80 min from 5 ml containing 2 ml of plasma. Increasing the a c i d i t y of the sample up to 5 N, the volume of the sample up to 7.5 ml, the amount of c o l d F~ up to 1 pmole, the amount of f l u o r i d e complexors up to 1 ymole of ThiNC^)^ had no m a t e r i a l e f f e c t on the r a t e of f l u o r i d e d i f f u s i o n . The absence of both plasma and detergent i n the sample compartment markedly slowed the r a t e of d i f f u s i o n . Not shaking the sample a l s o slowed the r a t e of d i f f u s i o n . Increasing the a l k a l i n i t y of the trapping s o l u t i o n to 0.1 Ν increased the r a t e of d i f f u s i o n but the lower concentration, 0.01 N, was r e q u i r e d here to permit a lower i o n i c strength i n the sample reading s o l u t i o n . O v e r a l l recovery of added cold f l u o r i d e was measured. In samples containing n e i t h e r plasma nor detergent the recovery a f t e r 6 hr d i f f u s i o n averaged 93% and 95% f o r ashed and unashed samples, r e s p e c t i v e l y . In samples containing plasma the recovery was 95% a f t e r 3 hr d i f f u s i o n . The degree to which f l u o r i n e from organic fluorocompounds could be f i x e d as i n o r g a n i c f l u o r i d e by ashing v a r i e d from l e s s than 1% f o r v o l a t i l e compounds l i k e p - a m i n o b e n z o t r i f l u o r i d e , m-hydroxybenzotrifluoride, b e n z y l f l u o r i d e and benzotrifluoride to over 80% f o r l e s s v o l a t i l e compounds l i k e 5 - f l u o r o u r a c i l , f l u o r o a c e t a t e and p - f l u o r o p h e n y l a l a n i n e . Methods used here f o r s e p a r a t i o n of f l u o r i d e ( d i f f u s i o n at rm. temp.) (9) and i t s q u a n t i t a t i o n ( f l u o r i d e e l e c t r o d e ) (10) are considered to be q u i t e s p e c i f i c f o r f l u o r i d e . One p o t e n t i a l l y



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important i n t e r f e r e n c e , however, was c o d i f f u s a b l e organic a c i d s which might p a r t i a l l y n e u t r a l i z e the trapping s o l u t i o n and thus lower the pH of the b u f f e r e d reading s o l u t i o n . Indeed, i t was found that samples c o n t a i n i n g r e l a t i v e l y l a r g e concentrations of a c e t i c acid (e.g., f r a c t i o n s 2, 3 and 4 from step 4 i n the p u r i f i c a t i o n system) completely n e u t r a l i z e d the trap w i t h i n a few hours. The s i g n i f i c a n c e of t h i s problem i n the a n a l y s i s of f l u o r i d e i n blood plasma was i n v e s t i g a t e d i n two ways. F i r s t , four samples of human plasma were allowed to d i f f u s e f o r three weeks, and no change i n the c o l o r of the phenolphthalein i n d i c a tor i n the trapping s o l u t i o n was observed. Secondly, samples c o n t a i n i n g the same plasma were d i f f u s e d f o r d i f f e r e n t periods up to 158 hr and the apparent f l u o r i d e was determined. No changes were observed between samples which c o r r e l a t e d with d i f f u s i o n time. The s e n s i t i v i t y of the a n a l y t i c a l method was l i m i t e d by the blank r a t h e r than the s e n s i t i v i t y of the instruments used. R e p r o d u c i b i l i t y v a r i e d with the amount of f l u o r i d e being measured. The c o e f f i c i e n t of v a r i a t i o n averaged 55% i n the low range (samples c o n t a i n i n g 0.25 to 0.75 nmoles F ) and 6.6% i n the high range (10-12 nmoles F " ) . Blood Plasma. Human plasma was obtained from blood banks i n f i v e c i t i e s . According to p u b l i c records these c i t i e s had not changed the f l u o r i d e c o n c e n t r a t i o n of t h e i r p u b l i c water supply f o r a t l e a s t s i x years p r i o r to o b t a i n i n g the samples. Samples were r e c e i v e d i n i n d i v i d u a l polyethylene bags which were p a r t of the Fenwall ACD blood c o l l e c t i o n system. In blood c o l l e c t i o n using t h i s system 450 ml of blood i s drawn i n t o a bag containing 67.5 ml of anticoagulant a c i d c i t r a t e dextrose (ACD) s o l u t i o n . When the c e l l s are removed the ACD s o l u t i o n remains i n the plasma. Because of t h i s d i l u t i o n of plasma a c o r r e c t i o n f a c t o r of 1.3 was a p p l i e d to values obtained here f o r the c o n c e n t r a t i o n of f l u o r i d e . The p o t e n t i a l e r r o r i n t h i s f a c t o r was ± 0 . 1 because of v a r i a t i o n between standard l i m i t s f o r hematocrit and minimum volume of the blood donation. Bovine blood was obtained a t slaughter and mixed immediately with ACD s o l u t i o n i n 1 l i t e r polyethylene b o t t l e s . E l e c t r o p h o r e s i s . A continuous flow e l e c t r o p h o r e t i c separator (model FF-3, Brinkman I n s t . , Inc., Westbury, N.Y.) was employed. Sample flow r a t e was 2.3 ml/hr, b u f f e r flow r a t e was 72 ml/hr, v o l t a g e was 0.67 kv, and current was 140 mamp. Separa t i o n took 19 hr. P l a t e s e p a r a t i o n was 1 mm and operating tempera ture was between 2 and 4° C. The b u f f e r was 0.12% (NHi ) C0 , made by bubbling C 0 from dry i c e i n t o r e d i s t i l l e d ΝΗίψΟΗ u n t i l the pH reached 9.0. +

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P u r i f i c a t i o n System. Steps i n the p u r i f i c a t i o n system are summarized i n t a b l e I. In the f i r s t step one l i t e r of plasma (pooled from 5-6 i n d i v i d u a l s ) was d i a l y s e d i n seamless c e l l u l o s e tubing (1 i n . diameter) against 20 l i t e r s of water at 4° C. The d i a l y s a t e was changed twice at 24 hr i n t e r v a l s . In the second step d i a l y s e d plasma was f r e e z e d r i e d . In the t h i r d step the d r i e d powder from e l e c t r o p h o r e s i s was extracted with methanol i n a soxhelet e x t r a c t i o n apparatus (model 6810 G, Ace Glass, Inc., Vineland, N.J.). C e l l u l o s e e x t r a c t i o n thimbles (model 6812 G, Ace Glass) were soaked overnight i n methanol. Operating c o n d i t i o n s were 25° to 30° C under a vacuum of 24 in-Hg. Coolant f o r the condenser was 80% ethanol; i n l e t temperature was -10° to -20° C and o u t l e t tempera ture was -10° to 0°. Two l i t e r s of methanol were r e f l u x e d through the apparatus f o r a period of 4 hr and approximately 400 ml were l o s t to evaporation during that p e r i o d . Glass beads were placed i n the f l a s k to prevent bumping. In the f o u r t h step the r e s i d u e from the methanol e x t r a c t was f r a c t i o n a t e d according to the method described by Siakotos and Rouser (11) f o r separating l i p i d and n o n - l i p i d components. The method i s based on l i q u i d - l i q u i d p a r t i t i o n i n a column containing a dextran g e l (Sephadex G-25, coarse, beaded, Pharmacia F i n e Chemicals, Inc., N.Y.). Four eluents are used: 1) 500 ml chloroform/methanol, 19/1, saturated with water, 2) 1000 ml of a mixture of 5 p a r t s of chloroform/methanol, 19/1, and 1 part of g l a c i a l a c e t i c a c i d , saturated with water, 3) 500 ml of a mixture of 5 p a r t s chloroform/methanol, 19/1, and 1 part g l a c i a l a c e t i c a c i d , saturated with water, and 4) 1000 ml of methanol/water, 1/1. T h e i r method was modified f o r use here by i n c r e a s i n g the column length to that a t t a i n e d by using a f u l l 100 grams of dextran beads. Sample s i z e corresponded to that from 2.5 l i t e r s of the o r i g i n a l plasma. In the f i f t h step the residues from eluents 2 and 3 from two runs of step four were combined, a p p l i e d to a s i l i c i c a c i d column, and e l u t e d by reverse flow with an exponential gradient of i n c r e a s i n g amounts of methanol i n chloroform. The column (model SR 25/45, 2.5 cm i . d . χ 45 cm, Pharmacia) was f i l l e d to a height of 30 cm with s i l i c i c a c i d ( U n i s i l , 100-200 mesh, Clarkson Chem. Co., Inc., W i l l i a m s p o r t , Pa., heat a c t i v a t e d at 110° C f o r 2 days) and was washed with a complete set of e l u t i o n s o l v e n t s before use. The gradient maker (model 5858, set 4, Ace Glass Co.) was f i l l e d with 1 l i t e r of methanol i n the upper chamber and 2 l i t e r s of chloroform i n the lower. The flow r a t e was adjusted by the height of the s o l v e n t r e s e r v o i r s to an average of 3 ml/min f o r the f i r s t l i t e r of eluent. The sample had to be t r a n s f e r r e d to the column by repeated washings with chloroform because of i t s low s o l u b i l i t y i n t h i s s o l v e n t . This u s u a l l y r e q u i r e d about 30 ml of chloroform t o t a l . Dead volume for the system as 90 ml. F r a c t i o n s of 15 ml volume were c o l l e c t e d i n c a r e f u l l y cleaned g l a s s tubes.


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Table I PROCEDURE FOR PURIFICATION OF FLUOROCOMPOUNDS FROM BLOOD PLASMA Fraction Treated


blood plasma

Treatment step 1: exhaustive d i a l y s i s against d i s t i l l e d water

Fraction Removed smaller, waters o l u b l e components

plasma p r o t e i n s & protein-bound substances i n water s o l u t i o n

step 2:

plasma p r o t e i n s & protein-bound substances

step 3: methanol extraction—soxh e l e t , 25°C, 24 in-Hg vacuum

plasma p r o t e i n s

plasma l i p i d s

step 4: column chromatography— liquid-liquid p a r t i t i o n on Sephadex

l i p i d s of low p o l a r i t y and residual polar contaminants

polar l i p i d s

step 5: column chromatography— adsorption on s i l i c i c acid

lyophilization

water

unknown: severa1 yellow f r a c t i o n s

Figure 1. Relationship between the concentration of fluoride in human plasma and the concentration of fluoride in the drinking water



BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E

Figure 2. Relationship between the concentration of organic fluorine in human pfosma and the concentration of fluoride in the drinking water

TUBE NO. Figure 3. Separation of fluoride and organic fluorine in human plasma by electrophoresis. A sample (about 45 ml) of human phsma was electrophoresed in pH 9 buffer and fractions between the sampling port (near tube 72) and the positive pole (near tube 1) were analyzed for the fluoride content of both ashed and unashed aliquots. Relative concentrations of proteins were estimated by absorbance at 280 nm.


BONDS

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Tubing and f i t t i n g s to the columns were p o l y t e t r a f l u o r o e t h y lene (supplied l a r g e l y by Chromatronix, Inc., Berkeley, C a l i f . ) . A l l solvents were r e d i s t i l l e d . Methanol and chloroform were ACS c e r t i f i e d ( F i s h e r S c i e n t i f i c Co.) and a c e t i c a c i d was a n a l y t i c a l reagent grade, U.S.P. ( M a l l i n c k r o d t Chemical Works, St. L o u i s ) . Solvents were removed from samples i n a f l a s h evaporator. NMR. The nmr spectrum was obtained on a V a r i a n XL-100 spectrometer with N i c o l e t Technology F o u r i e r Transform accessory. The sample was d i s s o l v e d i n an approximately 1/1 mixture of CH OH and CDCI3 and s p e c t r a were run i n a 5 mm tube. External r e f e r e n c i n g to CFCI3 was used f o r the chemical s h i f t s , and these are expressed with p o s i t i v e numbers to lower f i e l d ( i . e . , higher frequency). E x t e r n a l l o c k was used. T y p i c a l c o n d i t i o n s were a pulse length of 15 microseconds, a delay time between pulse c y c l e s of 2.5 sec, and a time constant of -1 sec f o r exponential processing. 3

Results Values f o r i n o r g a n i c f l u o r i d e (F~) and organic f l u o r i n e (R-F) i n 106 plasma samples from humans l i v i n g i n f i v e c i t i e s are shown i n t a b l e I I . These data show that the average f l u o r i d e c o n c e n t r a t i o n i n plasma i s d i r e c t l y r e l a t e d to the f l u o r i d e c o n c e n t r a t i o n i n the water supply, and that the average organic f l u o r i n e concentration i n plasma i s not. No r e l a t i o n s h i p between f l u o r i d e i n plasma and organic f l u o r i n e i n plasma was apparent by i n s p e c t i o n of values f o r i n d i v i d u a l samples. The d i s t r i b u t i o n s of the values w i t h i n c i t i e s are shown i n f i g u r e s 1 and 2. In both cases the d i s t r i b u t i o n s appear to be l o g normally d i s t r i b u t e d with only 3 or 4 i n d i v i d u a l s s u r p r i s i n g l y deviant. In the cases of the two i n d i v i d u a l s with l i t t l e or no apparent organic f l u o r i n e ( f i g u r e 2, Andrews group, l e f t margin), the i n o r g a n i c f l u o r i d e l e v e l s were both i n excess of 7 μΜ, making the d i f f e r e n c e measure ment f o r organic f l u o r i n e d i f f i c u l t . The o v e r a l l mean value f o r organic f l u o r i n e was 1.35 ± 0.85 SD μΜ. Plasma was electrophoresed i n an attempt to reproduce the f i n d i n g s of Taves (3) using plasma from another i n d i v i d u a l . Results shown i n f i g u r e 3 c l o s e l y match those found e a r l i e r i n that a predominant form of organic f l u o r i n e appeared to migrate with albumin at pH 9, and i n that organic and i n o r g a n i c forms were c l e a r l y separated. The recovery, mass balance and p u r i f i c a t i o n f a c t o r s f o r steps i n the p u r i f i c a t i o n system l i s t e d i n t a b l e I are recorded i n t a b l e I I I . These data show that about o n e - t h i r d of the o r i g i n a l amount of organic f l u o r i n e i n plasma i s recovered i n the major peak from s i l i c i c a c i d chromatography. Another t h i r d i s accounted for i n other f r a c t i o n s and the r e s t i s not accounted f o r , presumably because of adsorption to surfaces of containers i n


126

BIOCHEMISTRY

INVOLVING CARBON-FLUORINE

BONDS

Table I I CONCENTRATION OF FLUORIDE (F~) AND ORGANIC FLUORINE (R-F) IN BLOOD PLASMA SAMPLES FROM FIVE CITIES HAVING DIFFERENT FLUORIDE CONCENTRATIONS IN THEIR WATER SUPPLY

3

a


[F"] i n Plasma , uM C i t y ([F~] i n Water, ppm)

Mean± SD(n)

Albany, N.Y. (

Organic Fluorocompounds in Human Plasma: Prevalence and

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