Reduction of pantethine in rabbit ocular lens homogenate

ELSEVIER

Journal of Pharmaceutical and Biomedical Analysis 15 (1997) 653-662

JOURNALOF PHARMACEUTICAL A N D BIOMEDICAL ANALYSIS

Reduction of pantethine in rabbit ocular lens homogenate Daniel H. Fisher *'1 Michael E. S z u l c 2 Oculon Corporation, Cambridge, MA, USA

Received 14 November 1995; accepted 17 May 1996

Abstract

In several animal models, preliminary studies have indicated that pantethine may inhibit cataract formation. Therefore, preclinical trials need to be conducted to study the pharmacology of pantethine in the ocular lens and to establish its efficacy. Since pantethine, which is a disulfide, can undergo a variety of chemical modifications such as reduction and formation of mixed disulfides, a detailed study was first conducted to determine the stability of pantethine in rabbit lens homogenate. A knowledge of the stability of pantethine in lens homogenate was necessary to establish if pantethine could be metabolized in the time it takes to harvest and homogenize a lens. The results of this study will be used to establish a protocol for harvesting and homogenizing lens samples. Pantethine (100/~ M) is completely reduced to pantetheine in rabbit lens homogenate in about 16 min. About 1.5% of the pantethine added to lens homogenate forms a mixed disulfide with lens proteins, and the remainder is found in the supernatant. The supernatant pantethine concentration decreases exponentially as a function of time, and the terminal half-life for this process is 3.3 rain. The free supernatant pantetheine concentration increases in pseudo first order manner as a function of time with a rate constant of 4.3 min. Pantethinase activity is not significant, because the free supernatant pantetheine concentration did not decrease. The exact mechanism of pantethine reduction in rabbit lens homogenate remains to be determined.

Keywords: Electrochemical detection; HPLC; Occular lens homogenate; Pantetheine; Pantethinase; Pantethine; Stability

1. I n t r o d u c t i o n

P r e l i m i n a r y w o r k by C l a r k a n d B e n e d e k [1] i n d i c a t e d t h a t p a n t e t h i n e (Fig. 1) m i g h t p r e v e n t the f o r m a t i o n o f c a t a r a c t s in several a n i m a l m o d els. This h y p o t h e s i s was tested f u r t h e r via preclin* Corresponding author. Tel.: (+ 1)913-864-5097; Fax: ( + 1)913-864-5071 ; e-mail:[email protected]

ical trials at O c u l o n C o r p o r a t i o n . T h e p u r p o s e o f the w o r k r e p o r t e d here was to s t u d y the stability o f p a n t e t h i n e in lens h o m o g e n a t e . T h e results will indicate if p a n t e t h i n e is chemically m o d i f i e d d u r ing the time r e q u i r e d to h a r v e s t the lens a n d p r e p a r e a lens h o m o g e n a t e . This i n f o r m a t i o n will be used to d e v e l o p a fully v a l i d a t e d p r o t o c o l for o c u l a r lens t o t a l p a n t e t h e i n e levels in a n i m a l s t r e a t e d with p a n t e t h i n e .

0731-7085/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved PH S0731-7085(96)01878-X

654

D.H. Fisher, M.E. Szulc / J. Pharm. Biomed. Anal. 15 (1997) 653 662

To conduct the proposed stability study, suitable analytical techniques were required. A review of the literature indicated previously published chromatographic methods for pantetheine, but none for pantethine. Fahey and co-workers derivatized thiols with monobromobianes [2] and applied their H P L C method to the determination of pantetheine in human red blood cells using ion-exchange [3] and reversedphase chromato-graphy [4]. A fluorescence method for total pantetheine using 4-aminosulphonyl-7-fluoro-2,1,3-benzoxadiazole was also developed at Oculon, but was not published. Because a method for pantethine had not been reported in the literature, glutathione was selected as a model compound for method selection. Many small, water-soluble thiols and disulfides can be quantitated using HPLC [5]. Thiols such as glutathione can be directly quantitated using HPLC with fluorescence detection (HPLC/FL), but the disulfides cannot be quantitated without a reduction step. Thiols and their corresponding disulfides can be directly quantitated using H P L C with electrochemical [6 11] or mass spectrometric detection [12]. Analysis of reduced and oxidized glutathione in the lens of the eye using H P L C with electrochemical detection (HPLC/EC) has been reported in the literature [13,14]. Therefore, H P L C / E C using a Hg/Au electrode was selected because pantethine could be detected without reduction and derivatization, pantetheine could be detected without derivatization, and an H P L C / E C method for analysis of glutathione in the lens of the eye had been reported [13,14]. Determing the stability of pantethine was important because harvesting the ocular lens and preparing the lens homogenate requires 10 rain. During this time period, pantethine could undergo a variety of chemical reactions (Fig. 2). It could be reduced to the free thiol, pantetheine, or it could form a mixed disulfide with protein or nonprotein moieties. Pantethine is rapidly degraded in plasma by pantethinase [15]. However, there are no reports of the stability of pantethine or pantethinase activity in ocular tissues.

2. Experimental 2.1. Chemicals

Optima grade HPLC solvents were obtained from Fisher Scientific (Pittsburgh, PA). Sodium hydroxide (99.999 + %, electronic grade), chloroacetic acid (99 + %), redistilled 70% perchloric acid (99.999%), electronic grade mercury, redistilled nitric acid (99.999+%), and disodium ethylenediaminetetraacetic acid (EDTA, 99 + %) were supplied by Aldrich Chemical Company (Milwaukee, WI). Dithiothreitol (DTT, 99%) was supplied by Fluka Chemical (Ronkonkoma, NY). Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP, 98.8%) was purchased from Pierce Chemical (Rockford, IL). Pantethine (99.9%, Fig. 1) and bovine lens proteins were obtained from Sigma (St. Louis, MO). Pantetheine (Fig. 1) was synthesized, purified and characterized by Dr. Sham Patil at Oculon Corporation. The estimated purity of pantetheine using reversed-phase H P L C with UV detection at 230 nm is 92%, and the water content of the product is 7.7%. Pantetheine and pantethine were stored at - 2 0 ° C . Bis-(pantoyl-/~-aminoethyl) disulfide (Fig. 1), the internal standard for total pantetheine and pantethine determinations, was purified and characterized by Dr. Shashank Otiv at Oculon Corporation and was stored at - 2 0 ° C . Pantoylcysteamine (Fig. 1), the internal standard for supernatant-free pantetheine analysis, was synthesized by reducing bis-(pantoyl-fl-aminoethyl) disulfide with TCEP in acetate buffer. Purification was accomplished as described in Section 2.5.3. Pantoylcysteamine was stored at 4°C. 2.2. H P L C / E C equipment f o r pantetheine and pantethine assays

The chromatographic system consisted of a model 626 H P L C pump with associated column heater (Waters, Milford, MA), a model LC-44C electrochemical detector and flow cell (Bioanalytical Systems, West Lafayette, IN) and a model 9125 injector (Rheodyne, Cotati, CA). A 3.0 mm x 150 mm stainless-steel column packed with

655

D.H. Fisher, M.E. Szulc /' J. Pharm. Biomed. Anal. 15 (1997) 653-662

H H/,'~,

O

H~C3

H%~

OH H

_..

N

OH

H

_

O

H

:

N

H

H

OH

:"

O

H

SH

H~C~" H

pantetheine

.7

~.

OH

H

H

pantoylcysteamine

jo.

.o_ H

H

H

N -

H

H

N -

3

N

C

.

.~

H

-~--,,~/~

,,,.

H

H

H

H

=

H

N

~

H

OH

H3C

pantethine H.2-,, /OH

HO, 9.

OH

H

H

H

H

H

0

H

,,~"

~ . ~ H

OH

bis-(pantoyl-.p-aminoethyl) disulfide Fig. 1. Structures of pantetheine, pantethine, pantolycysteamine and bis-(pantoyl-/~-aminoethyl) disulfide.

Zorbax Stable Bond TM, 3.5 /~m, octyldecylsiloxane silica (Mac-Mod Analytical, Chadds Ford, PA) was prepared with titanium frits.

2.3. Preparation of the working electrode The following materials were purchased from Buehler (Lake Bluff, IL) for electrode polishing: 2.875 in, 15/Lm silicon carbide fibrpol PSA discs; 2.875 in, 9/Lm aluminum oxide fibrpol PSA discs;

2.875 in nylon polishing cloth PSA discs, 3 /~m Metadi II diamond polishing compound; 2.875 in microcloth polishing PSA discs; and Masterpolish. Spent amalgam was removed with 7.9 N nitric acid. The electrode was polished with a 15 /~m disc, 9/~m disc, 3 /~m polish on a nylon disc and alumina on a microcloth. The electrode was rinsed with water and methanol after the acid wash and polishing step. A drop of mercury ( ~ 5

656

D.H. Fisher, M.E. Szulc / J. Pharm. Biomed. Anal. 15 (1997) 653 662

HH~.

OH O

H

~3H

O

H

H

H

H

H

pantetheine

t

HH//~. / O H O H3C H3C

H N

-L OH

RSH O I

---

H

h H / z . . ~OH .

H N

: H

SSR

.,c

_OH

HaC HC

o

H

o

,,.

--_-

I

OH

H

-

OH

H

i S--

~.l

H

H

H

RSH H S

pL

A

. . . .

-

I

H

/~

M

H

O

H

O

N

,,

H

H

pantetheine

:

H

H

I H A

A

H

I "

O

-

H

pantetheine mixed disulfide

H%

H

4-

It

H

O

H

~

HO.

""~"

N

I

H

-

=

-

H

H

3C HC

:

I

_

H

.,',?,

OH

3

pantethine /

HH','~

.I. Pantethinase (pantetheine is also a substrate)

OH

O

H

H

2 H3C .30

O

V

H N

-

I

OH

H

OH

~

H

H

H 4-

H

H/m,~..N

-

S--

H

H

N.,,~H

tJ

L~

H

H

H

cystamine

pantothenic acid Fig. 2. Some possible degradation pathways of pantethine in lens homogenates.

/xl) was applied to the electrode. After ~ 3 0 min, most of the mercury was removed from the surface with an index card. The electrode was conditioned in air overnight. The working electrode surface was renewed weekly.

frozen. The lens ( ~ 2 0 0 300 mg) was weighed, 0.9% saline (1:3 w/v) was added, and the lens was homogenized by sonication. Aliquots of the lens homogenate were used for the assays described below.

2.4. Lens harvesting and homogenizing

2.5. Total pellet, total supernatant and free supernatant pantetheine assays (Figs. 3A- 3C)

A rabbit eye was enucleated, frozen on dry ice and stored at - 7 0 ° C until ready for dissection. The lens was dissected while the eye was still

2.5.1. Chromatographic conditions The mobile phase was a solution of a citric acid

657

D.H. Fisher, M.E. Szule / J. Pharm. Biomed. Anal. 15 (1997) 653 662

Lens

/

Homogenate

I

J

I

[ ,ncubateat3VC

[

l

I B. Total Supernatant

A. Total Pellet Pantetheine

Dilute Supematant [ Add disulfide internal standard

Add disulfide internal standard

[ Reducedisulfides

1

C. Free Supernatant Pantetheine

Pantetheine

Wash and resuspend pellet

Reduce disulfides

Add Pantethine

Dilute Supematant

Dilute Supematant I

Add thiol internal standard

Add disulfide internal standard

[

[ Precipitate Proteins ]

.PLO,EOD

Filter

I

HPLC/ECD

[.

Pantethine / Concentration /

I=[Solid-Phase Extraction1~.

l

I

D. Supernatant Pantethine

I

1

Pantetheine / Concentration /

Fig. 3. Pantetheine and pantethine assay flow chart. buffer (50 mM, pH 2.6 with 0.2 m M EDTA) and acetonitrile (93%:7%, v/v). A single Hg/Au amalgam electrode (Waters Corporation, Milford, MA) was prepared as described in Section 2.3. The flow was set to 1.0 ml min-~ and the column was heated to 40°C. The detector was set to +0.150 V, 10 or 20 nA full scale and a time constant of 0.10 Hz.

2.5.2. Standards and quality control Pantethine stock standard and stock quality control solutions were prepared independently in

water. A 20/zl aliquot of each stock solution was diluted 1:10 with a 10% (w/v) solution of bovine lens protein in physiological saline. A 10 pl aliquot of 5.0 p M bis-(pantoyl-fl-aminoethyl) disulfide was added to each standard or quality control sample followed by 400 /~1 of reducing reagent (1.0 m M TCEP, 20 m M acetate, pH 4.8). For the free supernatant pantetheine assay, pantoyl cysteamine was used for the internal stan dard. The samples were heated at 37°C for 1 h. At the end of the reduction, 400 p l of precipitating reagent (0.6 M chforoacetic acid, 0.1 M perchloric

658

D.H. Fisher, M.E. Szulc / J. Pharm. Biorned. Anal. 15 (1997) 653 662

acid and 1.0 mM EDTA, pH 2.8) was added and the samples were vortexed. 2.5.3. Solid-phase extraction The supernatants were filtered using a centrifugal filter at 10°C. Solid-phase extraction (SPE) tubes (ENVI-18, 100 mg, Supelco, Bellefonte, PA) were conditioned with 1.0 ml of methanol followed by 2.0 ml of a phosphate buffer (100 mM, pH 2.8 with 0.2 mM EDTA, conditioning solvent). The filtered supernatants were extracted under a vacuum of 5 in Hg at a rate of 2 - 3 drops per minute. The columns were dried by gradually increasing the vacuum to 15 in Hg and holding for 30 s. The compounds were eluted with 0.2 ml of acetonitrile. Residual solvent was reduced to about 10/tl in a centrifugal vacuum concentrator (Labconco, Kansas City, MO). The sample was reconstituted in 200/~1 of conditioning solvent. 2.5.4. Total pellet pantetheine assay (Fig. 3A) Lens homogenates were first brought to 37°C. Duplicate samples of 100 p M pantethine in lens homogenate were incubated at 37°C. A 20 /~1 aliquot was removed from each tube of lens homogenate at 5, 10, 15, 20, 25 and 30 rain, added to 180/tl of precipitating solution, vortexed, and frozen on dry ice. When all of the samples had been collected, they were thawed and centrifuged at 13 000g for 15 rain. The supernatants from the above samples were removed from the pellets. The pellets were washed three times with 200/tl of the acetate buffer, and resuspended in 190 ¢tl of acetate buffer with sonication. A 400 /~1 aliquot of reducing reagent was added followed by 10 ill of 5.0 /iM bis-(pantoyl-fl-aminoethyl) disulfide. The samples were incubated at 37°C for 60 min and vortexed every 10 min. A 400/zl aliquot of precipitating solution was added, and the samples were then vortexed. (If required, lens homogenate samples were diluted to reduce the actual pantetheine homogenate concentration to within the range of the standard curve.) The samples were then processed as described in Section 2.5.3. 2.5.5. Total supernatant pantetheine assay (Fig. 3B) This assay was identical to that outlined in

Section 2.5.4. except that following centrifugation a 20/zl aliquot of each supernatant was added to 180 Ill of the acetate buffer (20 mM, pH 4.8). The samples were then processed as described, starting with the reduction. 2.5.6. Free supernatant pantetheine assay (Fig. 3C) Lens homogenate samples containing 100 /zM of pantetheine were prepared as described in Section 2.5.4. A 20 /~1 aliquot of each homogenate was removed after 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105 and 120 min and added to 180 /tl of precipitating solution. Following centrifugation at 13 000g for 15 min, 20/tl of each supernatant was added to 10 /~1 of 5.0 /tM pantoylcysteamine, 580/tl of acetate buffer (20 mM, pH 4.8) and 400 /tl of precipitating solution in a 1.5 ml polypropylene microcentrifuge tube. Samples were then processed as described in Section 2.5.3. 2.6. Supernatant pantethine assay (Fig. 3D) 2.6.1. Chromatographic conditions The mobile phase consisted of a phosphate buffer (50 mM, pH 2.8 with 0.2 mM EDTA) and acetonitrile (86%:14%, v/v). The flow was set to 0.7 ml min -~. A dual Hg/Au working electrode (Bioanalytical Systems) was prepared as described in Section 2.3. The LC44-C detector was set to - 1 . 2 0 0 V (upstream electrode), + 0.150 V (downstream electrode), 50 nA and 0.10 Hz. 2.6.2. Standards and quality control Two stock solutions of pantethine in methanol were independently prepared and stored at 4°C. The standards and quality control sample were prepared before each use by diluting the stock solutions. A 40 /zl aliquot of each standard or quality control was added to 1.5 ml polypropylene microcentrifuge tubes followed by a 160 izl aliquot of precipitating reagent and 20 /~1 of 200 /zM bis-(pantoyl-fl-aminoethyl) disulfide. Samples were centrifuged at 13 000g for 15 min. The supernatant was filtered using a 0.2 /tm, 1.5 ml microcentrifuge filter for 5 min at 3000g.

D.H. Fisher, M.E. Szuh' / J. Pharm. Biomed, Anal. 15 (1997) 653 662

2.6.3. Supernatant pantethine assay (Fig. 3D) A 10 /tl aliquot of a 100 /~M solution of pantethine in water was added to 90 /tl of lens homogenate in duplicate. After 1 min, the samples were processed as described for the pantethine standards and quality control. This process was repeated for the following time points: 2, 4, 6, 8, 10, 15, 20, 25, and 30 min.

2. 7. Calculations and data analysis A standard curve was prepared by plotting the peak height ratio vs. concentration. The slope (m), standard error of the slope (se,,,), intercept (b), standard error of the intercept (sg), and correlation coefficient (r) of the standard curve were determined using linear regression and analysis of variance. If the origin was not statistically different from zero, a linear regression through the origin was used. S-Plus was used for nonlinear-least-squares analysis of the data (StatSci, Seattle, WA).

3. Results

A chromatographic method was developed for pantetheine to measure total pellet pantetheine, total supernatant pantetheine and free supernatant pantetheine (Figs. 4A 4C). The reduction of pantethine and bis-(pantoyl-fl-aminoethyl) disulfide using T C E P in acetate buffer resulted in 100% conversion to pantetheine and pantoyl cysteamine. The standard curve for pantetheine was linear from 0.07-4.0 /iM and for a typical standard curve r = 0.999, m = 1.99, Sem = 0.02, and b ~ 0. The expected concentrations for each of the three quality control samples were 0.10, 0.40 and 1.6 /~M. The interassay average concentrations and standard deviations for three quality control samples were 0.12 /LM_+0.01 /~M ( n = 5 ) , 0.41 tiM +0.01 /tM ( n = 7 ) , and 1.58 itM ± 0 . 0 4 /lM (n = 7). Another chromatographic method was developed for pantethine (Fig. 4D) to measure free supernatant pantethine. The standard curve was linear from 0.5 125 tiM ( r = 0 . 9 9 9 , m =0.0401, Sem = 0.0004 and b ~_ 0). The interassay average

659

concentration for the 125 /tM quality control sample was 128 /lM + 5 /~M (standard deviation, n =4). The majority of the added pantethine (98.5%) is found in the supernatant and the supernatant total pantetheine concentration is not a function of time under the conditions reported here (Fig. 5). The supernatant total pantetheine concentration (Fig. 3B) is the sum of the free pantetheine concentration plus pantetheine resulting from the reduction of pantethine and mixed disulfides of pantetheine. The average supernatant total pantetheine concentration is 191 /lM _+ 4 /~ M (standard deviation, n = 12) for all the samples (six time points in duplicate). The concentration of supernatant pantethine decreases exponentially with time (Eq. (1)). The rate constant is 0.25 min ~ with a standard error of 0.01 min ~. The calculated initial value was 103 /~M with a standard error of 3 I~M. Cpantethine

=

103e

(1)

0.25,

The concentration of supernatant pantetheine (Fig. 5) increases in a pseudo first order manner (Eq. (2)). Supernatant-free pantetheine concentration (Fig. 3C) was determined without reduction and so pantoylcysteamine (Fig. 1), a thiol, was used as the internal standard. The rate constant is 0.16 min ~ with a standard error of 0.02 rain t. The concentration of pantetheine plateaus at 208 /zM with a standard error of 8 /tM. The free supernatant pantetheine concentration in lens homogenate does not change in the time period from 30 120 rain. Cpantethein e =

208(1 -

e

o.16t)

(2)

Total pellet pantetheine concentration is basically 1.5% of the total pantethine concentration added to the lens homogenate and constant as a function of time (Fig. 5). Reduction of the resuspended pellet yielded the thiols of the internal standard and protein mixed disulfides of pantetheine (Fig. 3A). The average pellet total pantetheine concentration is 2.9 tiM + 0.4 /LM (standard deviation, n = 12) for all the samples (six time points in duplicate).

660

D.H. Fisher, M.E. Szulc

J. Pharm. Biomed. Anal. 15 (1997)653

662

73

il

]

'\

5oo-~

o~ 1 1 i ; ! 1 1 ; ]

I ,

,. ~ ~ ~ , 3.0

210

1.0

~ ~, i ~ 4.0

i l l ; l l

6.0

7.0

500~

\

oJ ....

£o ......

3!o'

'

~'~

~:o

-~-' . . . ~.o . .

.... ~]o

~ i,~i~[i~l~,~,

7,0

zJ

500----

//

c

\

O~

t/' ' \

\ j

I 1.0

2.0

3.0

4.0

~' ~ '5!0 ~ ~ I . . . . 6!0

7.0

I i! ! ,j'

iI

', il

500 4 -I

0q i i

,,,--~--]-T,----~].r.--i.:q,T~,TF~ 1 2 3

ri,T=.:..rT,[i 4

......... 5

!-i ~ 6

7

8

9

10

11

Fig. 4. Representative HPLC/EC chromatograms: (A) total pantetheine in the pellet; (B) total supernatant pantethine; (C) free supernatant pantetheine; (D) supernatant pantethine.

D.H. Fisher, M.E. Szulc J. Pharm. Biomed. Anal. 15 (1997) 653-662

661

220.00 210.00

. . . . . .

208

200.00 ................

190.00

+

180.00

~.~

+ .-

~-

----+ ...........

:-.:_-.-.=~-_ . . . . . .

2:~-

+

191

•

170.00

,/'~-

150.00 140.00

•

~" 13o.oo

J

"-~ 120.00

/

.= 110.00 /

100.00

8

90.00

u

80.00

9"

•

160.00

~e

/

/

/

/

/

Cpaotetheine= 208(1-e -°16t)

/

~

• Supematant

.......

+ Total 8upematant Pantetheine

.........

x Total Pellet P a n t e t h e i n e

/ •

/!

'i -- -'Free SuperoatantPantet,eio

70.00 60.00 50.00 40.00 30.00

Pantethine

t

/•

f/~ ~ - - / "~

Cpantethine = ] 0 3 e °25t

20.00 10.00 0.00 -10.00

i

i

5

10

15 Time

k

i

20

25

30

(min)

Fig. 5. Total pellet pantetheine, total supernatant pantetheine, free supernatant pantetheine and supernatant pantethine concentrations in lens homogenates plotted as a function of time.

4. Discussion For this study, H P L C / E C provided a suitable method for studying the stability of pantethine in rabbit lens homogenates. Although the initial goal was to develop one isocratic method for pantetheine and pantethine, two isocratic chromatographic methods were developed because the capacity factor for pantethine is very large under mobile phase conditions that permit adequate resolution of pantetheine and its internal standard. Each method is accurate, precise and linear over two orders of magnitude. The experimental design was such that pantethine was used as the standard for all three pantetheine assays. The primary reasons for this choice were that very pure preparations of pantethine are commercially available while pantetheine must be synthesized and the product is not as

pure. Secondly, pantetheine is more unstable than its corresponding disulfide, pantethine. Thirdly, a measure of reduced pantethine was required for all three assays. However, using pantethine as the standard for free supernatant pantetheine required that the standards be treated in a manner slightly different from the samples. The pantethine in the standards was completely reduced by TCEP, while the pantethine added to the samples was reduced to varying degrees in the lens homogenate to pantetheine. (The lens homogenate sample were not reduced with TCEP.) For both standards and samples, pantoylcysteamine was used as the internal standard. The net result was that thiols were still being measured. The decrease in pantethine concentration in lens homogenates is not due to pantethinase activity. Rabbit plasma pantethinase activity is known

662

D.H. Fisher, M.E. Szuh" / J. Pharm. Biomed. Anal. 15 (1997) 653 662

to be very high, yielding a rapid terminal haiti life for p a n t e t h i n e in plasma. However, pantethinase cleaves one equivalent o f p a n t e t h i n e to yield two equivalents o f p a n t o t h e n i c acid a n d one equivalent of cystamine (Fig. 2) b u t n o t two equivalents of pantetheine. In fact, p a n t e t h e i n e is also a substrate for p a n t e t h i n a s e (Fig. 2). Free p a n t e t h e i n e is stable in lens h o m o g e n a t e for at least 2 h. Therefore, d e g r a d a t i o n due to p a n t e t h einase is n o t significant. The most plausible hypothesis is that p a n t e t h i n e is being rapidly a n d completely reduced to p a n t e t h e i n e u n d e r the c o n d i t i o n s reported, as d e m o n s t r a t e d by the essentially complete conversion of p a n t e t h i n e to p a n t e t h eine. The exact m e c h a n i s m of this reduction rem a i n s to be elucidated. This m e a n s that a b o u t 87.5% ( ~ t h r e e half-lives) of the p a n t e t h i n e present in the lens is converted to p a n t e t h e i n e by the time the lens is thawed, h o m o g e n i z e d a n d refrozen.

Acknowledgements T h e a u t h o r s t h a n k Michelle Salt G r i m m for expertly harvesting a n d h o m o g e n i z i n g the rabbit lenses, J a y a n t i P a n d e a n d N a n c y H a r m o n y for their c o m m e n t s , a n d Chester D u d a a n d Pamela White o f Bioanalytical Systems for their technical assistance.

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