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Determination of monosaccharides and sugar alcoholsin tissues from diabetic rats by high-performance liquid chromatography with pulsed amperometric detection
ANALYTICALBIOCHEMISTRY 2 0 6 , 98-104 (1992)
Determination of Monosaccharides and Sugar Alcohols in Tissues from Diabetic Rats by High-Performance Liquid Chromatography with Pulsed Amperometric Detection N o b o r u T o m i y a , T a k e s h i Suzuki, Juichi Awaya, 1 K u n i h a r u Mizuno, Akira Matsubara, Kazumasa Nakano, and Masayasu Kurono Mie Research Laboratory, Sanwa Kagaku Kenkyusho, Co., Ltd., 363 Shiozaki, Hokusei-cho, Inabe-gun, Mie 511-04, Japan Received April 20, 1992
A sensitive and simple high-performance liquid chrom a t o g r a p h i c m e t h o d h a s b e e n d e v e l o p e d to d e t e r m i n e t h e c o n c e n t r a t i o n o f m o n o s a c c h a r i d e s a n d s u g a r alcohols in animal tissues. Five neutral monosaccharides ( D - g l u c o s e , D - g a l a c t o s e , D - m a n n o s e , D - f r u c t o s e , a n d Dribose) and three neutral sugar alcohols (myo-inositol, g l y c e r o l , a n d D-sorbitol) p r e d o m i n a t e i n t h e r e n a l cort i c e s a n d s c i a t i c n e r v e s o f rats. T h e s e m o n o s a c c h a r i d e s and sugar alcohols were extracted with distilled water, purified by deproteinization with ethanol, a Sep-Pak Cla c a r t r i d g e , a n d c o l u m n s o f D o w e x 5 0 W - X 8 a n d A m b e r l i t e C G - 4 0 0 , t h e n s e p a r a t e d o n C a 2÷ a n d P b 2÷ c a t i o n exchange columns, eluted with deionized distilled w a t e r at 8 0 ° C , a n d d e t e c t e d u s i n g i n t e g r a t e d p u l s e d amperometry. About 10 pmol of each sugar was detecta b l e w i t h a s i g n a l - t o - n o i s e r a t i o o f 1 0 : 1 . D - G l u c o s e , Dfructose, D-sorbitol, and D-mannose were higher in both t h e r e n a l a n d s c i a t i c t i s s u e s o f d i a b e t i c r a t s t h a n in those of normal animals. D-Ribose and glycerol were h i g h e r i n t h e r e n a l c o r t e x o f d i a b e t i c a n i m a l s . © 1992 Academic Press, Inc.
Gas chromatography (GC) is the standard method used to determine sugars and sugar alcohols in animal tissues (1). Although it is sensitive, it requires chemical modification of sample sugars and sugar alcohols prior to chromatography to produce volatile derivatives which can be detected. Recently, several high-performance liquid chromatographic (HPLC) methods using precolumn derivatization (2-5) have been developed and used for determination of sorbitol and myo-inositol.
1 To w h o m correspondence should be addressed.
98
Sensitive analytical methods for determination of individual monosaccharides, which do not require derivatization, use a combination of high-performance anionexchange chromatography (HPAEC) 2 and pulsed amperometoric detection (PAD) (6-8). However, no sensitive and simple method has been developed for the simultaneous determination of a mixture of monosaccharides and sugar alcohols. We examined the elution profiles of D-glucose, D-galactose, D-mannose, D-fructose, D-ribose, D-sorbitol, myo-inositol, and glycerol using several different H P L C columns and developed a new method which permits separation and detection of minute quantities of these carbohydrates, without forming derivatives, in a single chromatographic run. In this report we describe this method and an application of this method in studying the carbohydrate concentration in diabetic rats. Hyperglycemia often results in diabetic complications including n e p h r o p a t h y and neuropathy. D-Glucose, D-fructose, and D-sorbitol all increase and myo-inositol decreases in the renal cortices (9) and sciatic nerves (10,11) of diabetic animals. However, the effect of hyperglycemia on other carbohydrate metabolites derived from D-glucose is unknown. In a preliminary study, we found t hat D-galactose, D-mannose, D-ribose, and glycerol are also present in the extracts of these tissues in normal rats. We examined the levels of D-galactose, D-mannose, D-ribose, and glycerol as well as Dglucose, D-fructose, D-sorbitol, and myo-inositol in the renal cortices and sciatic nerves of diabetic animals using the chromatographic method described in this report. 2 Abbreviations used: H P A E C , high-performance anion-exchange chromatography; PAD, pulsed amperometoric detection; PED, pulsed electrochemical detector.
0003-2697/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
CHROMATOGRAPHIC
DETERMINATION
OF M O N O S A C C H A R I D E S
99
AND ALCOHOLS
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F I G . 1. E l u t i o n profile of s t a n d a r d m o n o s a c c h a r i d e s a n d s u g a r alcohols o n a S h o d e x S U G A R SC1011 c o l u m n e l u t e d w i t h w a t e r at 1.0 m l / m i n . D e t e c t i o n w a s by P E D u s i n g i n t e g r a t e d p u l s e d a m p e r o m e t r y mode. P e a k s : 1, D-glucose; 2, D-galactose; 3, D - m a n n o s e ; 4, D-fructose; 5, myo-inositol; 6, glycerol; 7, D-arabitol ( i n t e r n a l s t a n d a r d ) ; 8, D-ribose; 9, D-sorbitol (1 n m o l each). Details are described u n d e r M a t e rials a n d M e t h o d s .
MATERIALS AND METHODS Chemicals
The following materials were purchased from the sources indicated: D-glucose, D-galactose, D-fructose, Dribose, D-arabitol, D-sorbitol, myo-inositol, glycerol, and Glucose B-test Wako (Wako Pure Chemical Industries, Osaka, Japan); D-mannose (Katayama Chemical Industries, Osaka, Japan); streptozotocin (Sigma, St. Louis, MO). Dowex 50W-X8, 100-200 mesh (H + form) (Bio-Rad Laboratories, Richmond, CA); and Amberlite CG-400 (Type I) (Organo Co., Ltd., Tokyo, Japan). Animals
Male Sprague-Dawley rats (from SLC Japan, Shizuoka) weighing about 180 g were subjected to experiments after acclimatization for 1 week. Their body
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F I G . 3. E l u t i o n profile of s t a n d a r d m o n o s a c c h a r i d e s a n d s u g a r alcoh o l s on a D i o n e x C a r b o P a c P A l c o l u m n e l u t e d w i t h 16 mM N a O H at 0.8 m l / m i n . D e t e c t i o n w a s by P E D u s i n g i n t e g r a t e d p u l s e d a m p e r o m e t r y mode. T h e s u g a r s are t h e s a m e as t h o s e listed in t h e legend to Fig. 1.
weight at the beginning of the experiments averaged about 250 g. The animals were given food, CA-1 (Clea Japan, Inc., Tokyo, Japan), and water ad libitum. Diabetes was induced by intravenous injection of 60 mg/kg of streptozotocin dissolved in 50 mM citrate buffer, pH 4.5. Age-matched control animals (normal rats) were given the buffer by intravenous injection. Four to 7 days after injection of streptozotocin, blood was collected from the ocular vein with a heparinized capillary tube and plasma was prepared by centrifugation at 1700g for 10 min at 4°C. Plasma glucose concentration was determined by an enzymatic method using glucose oxidase and peroxidase (Glucose B-test Wako). Rats with glucose levels of 300 mg/dl or higher were selected for the study. Twenty eight days after the injection of streptozotocin, the diabetic (n = 5) and normal control rats (n = 5) were sacrificed and their sciatic nerves and kidneys were removed.
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F I G . 2 . E l u t i o n profile of s t a n d a r d m o n o s a c e h a r i d e s a n d s u g a r alcohols o n a S h o d e x S U G A R SP0810 c o l u m n . S u g a r s were eluted, detected, a n d n u m b e r e d as described in t h e legend to Fig. 1.
0
5
10
15
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40
Elution Time (rain)
F I G . 4. E l u t i o n profile of s t a n d a r d m o n o s a c c h a r i d e s a n d s u g a r alcohols on two c o l u m n s in series: a S h o d e x S U G A R SC1011 c o l u m n followed by a S h o d e x S U G A R S P 0 8 1 0 c o l u m n . S u g a r s were eluted, detected, a n d n u m b e r e d as d e s c r i b e d in t h e legend to Fig. 1.
100
T O M I Y A E T AL.
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20 40 60 80 Injection Amount (pmol) - ~ - D-Glucose ~ - Glycerol
-D- D-Galactose - ~ - D-Arabitol
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Injection Amount (nmol) - ~ - D-Mannose ~ D-Ribose
~ D-Fructose - ~ - D-Sorbitol
-~- myo-lnositol
F I G . 5. Calibration curves for m o n o s a c c h a r i d e s a n d sugar alcohols. Aqueous solutions (25 #1 each) containing (a) 10-100 pmol or (b) 0.1-10 nmol of m o n o s a c c h a r i d e s and sugar alcohols were analyzed using a S U G A R SC1011 column a n d a S U G A R SP0810 column in series. Details are described u n d e r Materials a n d M e t h o d s .
HPLC
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F o u r different s e p a r a t i o n m e t h o d s were t e s t e d for resolution of m o n o s a c c h a r i d e s a n d sugar alcohols on a Dionex B i o - L C c h r o m a t o g r a p h : (1) a S h o d e x S U G A R SC1011 c o l u m n (8 X 250 m m ) ( S h o w a d e n k o , T o k y o , J a p a n ) e q u i p p e d with a S U G A R SC1011P g u a r d c o l u m n (8 X 25 m m ) ; (2) a S h o d e x S U G A R SP0810 c o l u m n (8 X 250 m m ) ( S h o w a d e n k o ) , e q u i p p e d with a S U G A R S P 0 8 1 0 P g u a r d c o l u m n (8 X 25 m m ) ; (3) the S U G A R SC1011 a n d S U G A R SP0810 c o l u m n s used in series with the SC1011P g u a r d column; a n d (4) a Dionex Carb o P a c P A l c o l u m n (4 X 250 m m ) (Dionex, S u n n y v a l e , CA) e q u i p p e d with a C a r b o P a c P A g u a r d c o l u m n (3 x 25 m m ) . T w e n t y - f i v e - m i c r o l i t e r s a m p l e s were u s e d in all four s e p a r a t i o n m e t h o d s . E l u t i o n in s e p a r a t i o n m e t h ods 1, 2, a n d 3 was with deionized, distilled w a t e r at 80°C a n d a flow r a t e of 1 m l / m i n . E l u t i o n in m e t h o d 4 was p e r f o r m e d with 16 mM N a O H at 25°C a n d a flow r a t e of 0.8 ml. D e t e c t i o n of m o n o s a c c h a r i d e s a n d s u g a r alcohols was carried out u s i n g Dionex p u l s e d e l e c t r o c h e m i c a l detector (PED) under integrated pulsed amperometry. N a O H at 0.5 M was a d d e d to the p o s t c o l u m n effluent with t h e D i o n e x anionic m i c r o m e m b r a n e s u p p r e s s o r ( A M M S - I I ) . T h e following pulse p o t e n t i a l s a n d d u r a tions were used: E1 = 0.10 V (tl = 0.50 s); E2 = 0.60 V (t2 = 0.08 s); E3 = - 0 . 6 0 V (t3 = 0.05 s). T h e signals w e r e
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F I G . 6. C h r o m a t o g r a m of m o n o s a c c h a r i d e s a n d sugar alcohols derived from t h e renal cortices o f n o r m a l (a) and diabetic (b) rats using a S U G A R SC1011 column a n d S U G A R SP0810 column in series. Sugars were eluted, detected, a n d n u m b e r e d as described in t h e legend to Fig. 1.
CHROMATOGRAPHIC DETERMINATION OF MONOSACCHARIDES AND ALCOHOLS integrated b e t w e e n 0.30 and 0.50 s and the response time of the P E D detector was set to 5 s.
Tissue Sample Preparation Neutral m o n o s a c c h a r i d e s and sugar alcohols were prepared by h o m o g e n i z i n g 20 mg of sciatic nerve tissue and 50 mg o f renal cortex in 1.15 ml of deionized, distilled water containing 100 or 500 n m o l of D-arabitol, respectively (used as an internal standard). Protein was precipitated and r e m o v e d from the h o m o g e n a t e by adding 2.8 ml of 99.5% ethanol and centrifuging the samples at 3500 rpm for 10 min at 4°C. T h e supernatant was concentrated to dryness in vacuo and resuspended in 1
101
ml o f deionized, distilled water. T h e s e samples were applied successively to a S e p - P a k Vac Ct8 cartridge (Millipore, Japan, T o k y o , Japan) and c o l u m n s of D o w e x 5 0 W - X 8 (H + form, 1 ml) and Amberlite CG-400 (CO~form, 1 ml). T h e s e were all w a s h e d with 6 ml of deionized, distilled water and the eluate was dried under a vacuum. T h e residue was dissolved with 1 ml of deionized, distilled water and was filtered (Ultrafree-C3GV, 0.22 #m, Millipore).
Statistical Calculations C o n c e n t r a t i o n s of m o n o s a c c h a r i d e s and sugar alcohols in rat tissues are expressed as the m e a n + SE and myo-lnosltol
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Dlabatelll
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FIG. 7. Monosaccharide and sugar alcohol concentrations in the renal cortices of normal and diabetic rats (n = 5). Bars and vertical lines indicate the mean _+ SE. +P < 0.05, ~+P < 0.01 vs normal.
102
T O M I Y A E T AL.
t h e d a t a were c o m p a r e d for significant difference using an u n p a i r e d S t u d e n t ' s t test. RESULTS AND DISCUSSION Five n e u t r a l m o n o s a c c h a r i d e s (D-glucose, D-galactose, D - m a n n o s e , D-fructose, a n d D-ribose) a n d t h r e e n e u t r a l s u g a r alcohols (myo-inositol, glycerol, a n d Dsorbitol) p r e d o m i n a t e in t h e renal cortices a n d sciatic n e r v e s of rats. A n u m b e r of m e t h o d s are available for s e p a r a t i o n a n d d e t e c t i o n of several of t h e s e c o m p o u n d s f r o m m i x t u r e s , b u t no p r e v i o u s m e t h o d provides simple, s i m u l t a n e o u s r e s o l u t i o n of all of t h e m . C a t i o n - e x c h a n g e c o l u m n s b e a r i n g N a +, Ca 2+, P b 2+, or Ag 2+ can be u s e d to s e p a r a t e v a r i o u s c a r b o h y d r a t e s t h r o u g h ligand exc h a n g e (12). M o n o s a c c h a r i d e s a n d s u g a r alcohols can be eluted f r o m Ca 2+ or P b 2+ resins with water, p e r m i t ting d e t e c t i o n of t h e s e c o m p o u n d s by P A D , a f t e r t h e effluent h a s b e e n alkaline. W e u s e d a c o m b i n a t i o n of c o l u m n s b e a r i n g Ca 2÷ a n d P b 2+ resins as well as P A D to s e p a r a t e t h e c o m p o u n d s m e n t i o n e d a b o v e in a single H P L C run.
Carbohydrate Separation on Single Columns W e studied t h e elution profiles of the m o n o s a c c h a rides a n d s u g a r alcohols m e n t i o n e d above using t h r e e different c o l u m n s . Figure 1 shows t h a t a Ca 2+ b e a r i n g S h o d e x S U G A R SC1011 c o l u m n resolved D-glucose, glycerol, a n d D-arabitol b u t was u n a b l e to s e p a r a t e Dgalactose f r o m D - m a n n o s e , D-fructose f r o m myo-inositol, or D-ribose f r o m D-sorbitol. T h e S h o d e x S U G A R SP0810 c o l u m n b e a r i n g P b 2+ resin resolved D-glucose, D-galactose, D-arabitol, D-ribose, a n d D-sorbitol b u t was u n a b l e to s e p a r a t e D - m a n n o s e f r o m D-fructose or m y o inositol f r o m glycerol (Fig. 2). H P A E C h a s b e e n r e p o r t e d to clearly s e p a r a t e s o m e m o n o s a c c h a r i d e s a n d s u g a r alcohols (6-8). W e f o u n d t h a t a D i o n e x C a r b o P a c P A l c o l u m n resolved D-glucose, D-galactose, D - m a n n o s e , D-fructose, a n d D-ribose as well as D-arabitol a n d D-sorbitol b u t was u n a b l e to s e p a r a t e glycerol a n d m y o - i n o s i t o l (Fig. 3).
Simple Carbohydrate Separation on Two Columns in Series D-Glucose, D-galactose, D - m a n n o s e , D-fructose, D-ribose, myo-inositol, glycerol, D-arabitol, a n d D-sorbitol were m i x e d t o g e t h e r a n d subjected to H P L C in a s y s t e m c o n t a i n i n g two c o l u m n s in series, a S h o d e x S U G A R SC1011 followed b y a S h o d e x S U G A R SP0810, a n d det e c t e d by P A D . T h e elution profile of this m i x t u r e of s t a n d a r d m o n o s a c c h a r i d e s a n d s u g a r alcohols is s h o w n in Fig. 4. Figure 5 shows t h a t the calibration curves for e a c h m o n o s a c c h a r i d e a n d s u g a r alcohol were a l m o s t linear up to 10 n m o l / i n j e c t i o n . T h e precision of m e a s u r e m e n t of elution t i m e s a n d p e a k a r e a s were t e s t e d b y
multiple injections a n d v a r i e d b y 0-0.1% (n = 5) a n d 0.4-1.6% (n = 5), respectively ( d a t a not shown). T h e limit of d e t e c t i o n of t h e s e c o m p o u n d s is a b o u t 10 p m o l with a signal-to-noise ratio of 10:1. T h i s value is c o m p a rable to or higher t h a n t h o s e o b t a i n e d using previously r e p o r t e d m e t h o d s (2-5). P r e v i o u s H P L C a n d GC m e t h o d s used to m e a s u r e sugars a n d s u g a r alcohols in tissue s a m p l e s h a v e dep e n d e d on d e r i v a t i z a t i o n of t h e s e c o m p o u n d for detection. T h i s o f t e n results in p e a k s which overlap t h o s e of t h e c o m p o u n d s of interest, m a k i n g good s e p a r a t i o n a n d m e a s u r e m e n t difficult. C h r o m a t o g r a m s of tissue s a m ples using the Ca 2+ a n d P b 2+ c o l u m n s in series clearly show t h a t no such i n t e r f e r i n g p e a k s are o b t a i n e d with this m e t h o d (Figs. 6 a n d 8).
Purification of Monosaccharides and Sugar Alcohols T h e r e c o v e r y of all the nine c a r b o h y d r a t e s was above 90%, w h e n t h e s t a n d a r d m i x t u r e c o n t a i n i n g 0.4-400 n m o l of each c a r b o h y d r a t e was t r e a t e d with ion-exchange resine a n d a S e p - P a k Cls cartridge as described u n d e r M a t e r i a l s a n d M e t h o d s . T h e recoveries of the int e r n a l s t a n d a r d , D-arabitol, t h r o u g h the whole purification p r o c e d u r e were 90.8 + 0.9 a n d 85.0 +_ 1.2% (renal cortices f r o m n o r m a l a n d diabetic rats, n = 5, respec-
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Eluti0n Time (min)
F I G . 8. C h r o m a t o g r a m of m o n o s a c c h a r i d e s and sugar alcohols derived from the sciatic nerves of n o r m a l (a) and diabetic (b) rats using a S U G A R SC1011 c o l u m n a n d S U G A R SP0810 c o l u m n in series. Sugars were eluted, detected, a n d n u m b e r e d as described in the legend to Fig. 1.
CHROMATOGRAPHIC DETERMINATION OF MONOSACCHARIDES AND ALCOHOLS tively), a n d 88.0 _ 2.3 a n d 80.0 + 0.6% (sciatic n e r v e s f r o m n o r m a l a n d d i a b e t i c rats, n = 5, r e s p e c t i v e l y ) .
Measurement of Monosaccharides and Sugar Alcohols in the Renal Cortices and Sciatic Nerves of Normal and Diabetic Rats D-Glucose, D-fructose, a n d D - s o r b i t o l all i n c r e a s e a n d m y o - i n o s i t o l d e c r e a s e s in t h e r e n a l c o r t i c e s (9) a n d sciatic n e r v e s (10,11) of d i a b e t i c a n i m a l s . H o w e v e r , t h e effect of h y p e r g l y c e m i a on o t h e r c a r b o h y d r a t e m e t a b o lites d e r i v e d f r o m D-glucose is u n k n o w n . M o n o s a c c h a r i d e s a n d s u g a r a l c o h o l s were e x t r a c t e d f r o m t h e r e n a l
Normal
c o r t i c e s a n d sciatic n e r v e s of n o r m a l r a t s a n d r a t s w i t h streptozotocin-induced diabetes. These samples were p u r i f i e d as d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s a n d t h e n s u b j e c t e d to H P L C u s i n g t h e t w o c o l u m n m e t h o d d e s c r i b e d above. F i g u r e s 6a a n d 6b s h o w t h a t p e a k s were o b s e r v e d for D-glucose, D - g a l a c t o s e , D - m a n n o s e , D-fructose, m y o - i n ositol, glycerol, D-ribose, a n d D - s o r b i t o l d e r i v e d f r o m r e n a l c o r t i c e s of n o r m a l a n d d i a b e t i c r a t s . T h e levels of t h e s e c o m p o u n d s in r e n a l c o r t e x a r e s h o w n in Fig. 7. S i g n i f i c a n t l y h i g h e r levels of D-glucose, D - m a n n o s e , Df r u c t o s e , D-ribose, D-sorbitol, a n d glycerol w e r e all f o u n d in t h e r e n a l c o r t i c e s of d i a b e t i c r a t s . T h e m e a n
Diabetes
,oOO5oo I D-,.no.o ®( / )
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0
(/)
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Normal
~-~
150
Dlabetell
D-Rlbose
D-Fructose
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1
0 Normal
103
10000
J_
5000
o
Normal
Normal
D-Sorbltol
myo°lnosltol
I Normal I
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##
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Normal
Diabetes
FIG. 9. Monosaccharide and sugar alcohol concentrations in the sciatic nerves of normal and diabetic rats (n = 5). Bars and vertical lines indicate the mean _ SE. $~P < 0.01 vs normal.
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TOMIYA ET AL.
level of D - g a l a c t o s e w a s a l s o e l e v a t e d in t h e d i a b e t i c a n i m a l s , h o w e v e r , m y o - i n o s i t o l was r e d u c e d to a b o u t 80% of t h e level f o u n d in n o r m a l rats. T h e e l u t i o n p r o f i l e s of m o n o s a c c h a r i d e s a n d s u g a r a l c o h o l s f r o m t h e sciatic n e r v e s of n o r m a l a n d d i a b e t i c r a t s a r e s h o w n in Figs. 8a a n d 8b. D-Glucose, D - m a n nose, D-fructose, m y o - i n o s i t o l , glycerol, D-ribose, a n d D - s o r b i t o l w e r e p r e s e n t in t h i s t i s s u e in b o t h n o r m a l a n d d i a b e t i c r a t s . T h e levels of t h e s e c o m p o u n d s a r e s h o w n in Fig. 9. S i g n i f i c a n t l y h i g h e r levels of D-glucose, Dm a n n o s e , D-fructose, a n d D - s o r b i t o l were all f o u n d in t h e sciatic n e r v e s of d i a b e t i c rats. T h e m e a n level of g l y c e r o l w a s also e l e v a t e d in t h e d i a b e t i c a n i m a l s . As in r e n a l t i s s u e m y o - i n o s i t o l w a s r e d u c e d to a b o u t 80% of t h e level s e e n in t h e n o r m a l a n i m a l s . W e h a v e d e v e l o p e d a s i m p l e , s e n s i t i v e m e t h o d of m e a s u r i n g all of t h e m o n o s a c c h a r i d e s a n d s u g a r alcoh o l s p r e s e n t in r e n a l c o r t i c e s a n d sciatic n e r v e tissue. U s i n g t h i s m e t h o d to c o m p a r e t h e levels of s i m p l e sug a r s in t h e s e t i s s u e s in d i a b e t i c a n d n o r m a l a n i m a l s , we h a v e d e m o n s t r a t e d a l t e r e d levels of D - m a n n o s e , glycerol, a n d D-ribose as well as t h e p r e v i o u s l y k n o w n c h a n g e s in t h e levels of D-glucose, D-fructose, a n d Ds o r b i t o l . T h e s e r e s u l t s i n d i c a t e t h a t i m b a l a n c e s in c a r b o h y d r a t e m e t a b o l i s m o c c u r n o t o n l y in p o l y o l p a t h w a y i n t e r m e d i a t e s , b u t also in o t h e r m o n o s a c c h a r i d e s a n d
s u g a r a l c o h o l s . T h e s e c h a n g e s m a y be i m p o r t a n t in det e r m i n i n g h o w d i a b e t i c c o m p l i c a t i o n s d e v e l o p in animals. REFERENCES 1. Sweely, C. C., Bentley, R., Makita, M., and Wells, W. W. (1963)d. Am. Chem. Soc. 85, 2497-2507. 2. Dethy, J-M., Callaert-Deveen, B., Janssens, M., and Lenaers, A. (1984) Anal. Biochem. 143,119-124. 3. Lloyd, P., and Crabbe, M. J. C. (1985) d. Chromatogr. 343,402406. 4. Akanuma, H., Yamanouchi, T., Ono, H., Nomura, K., and Akanuma, Y. (1985) d. Biochem. 97,579-588. 5. Miwa, I., Kanbara, M., Wakazono, H., and Okuda, d. (1988) Anal. Biochem. 173, 39-44. 6. Rocklin, R. D., and Pohl, C. A. (1983) J. Liq. Chromatogr. 6, 1577-1590. 7. Hardy, M. R., Townsend, R. R., and Lee, Y. C. (1988) Anal. Biochem. 170, 54-62. 8. Peelen, G. O. H., De Jong, J. G. N., and Wevers, R. A. (1991) Anal. Biochem. 198, 334-341. 9. Poulsom, R., and Heath, H. (1983) Biochem. Pharmacol. 32, 1495-1499. 10. Clements, R. S., and Stockard, C. R. (1980) Diabetes 29,227-235. 11. Finegold, D., Lattimer, S. A., Nolle, S., Bernstein, M., and Greene, D. A. (1983) Diabetes 32,988-992. 12. Goulding, R. W. (1975) J. Chromatogr. 103,229-239.
Determination of Monosaccharides and Sugar Alcohols in Tissues from Diabetic Rats by High-Performance Liquid Chromatography with Pulsed Amperometric Detection N o b o r u T o m i y a , T a k e s h i Suzuki, Juichi Awaya, 1 K u n i h a r u Mizuno, Akira Matsubara, Kazumasa Nakano, and Masayasu Kurono Mie Research Laboratory, Sanwa Kagaku Kenkyusho, Co., Ltd., 363 Shiozaki, Hokusei-cho, Inabe-gun, Mie 511-04, Japan Received April 20, 1992
A sensitive and simple high-performance liquid chrom a t o g r a p h i c m e t h o d h a s b e e n d e v e l o p e d to d e t e r m i n e t h e c o n c e n t r a t i o n o f m o n o s a c c h a r i d e s a n d s u g a r alcohols in animal tissues. Five neutral monosaccharides ( D - g l u c o s e , D - g a l a c t o s e , D - m a n n o s e , D - f r u c t o s e , a n d Dribose) and three neutral sugar alcohols (myo-inositol, g l y c e r o l , a n d D-sorbitol) p r e d o m i n a t e i n t h e r e n a l cort i c e s a n d s c i a t i c n e r v e s o f rats. T h e s e m o n o s a c c h a r i d e s and sugar alcohols were extracted with distilled water, purified by deproteinization with ethanol, a Sep-Pak Cla c a r t r i d g e , a n d c o l u m n s o f D o w e x 5 0 W - X 8 a n d A m b e r l i t e C G - 4 0 0 , t h e n s e p a r a t e d o n C a 2÷ a n d P b 2÷ c a t i o n exchange columns, eluted with deionized distilled w a t e r at 8 0 ° C , a n d d e t e c t e d u s i n g i n t e g r a t e d p u l s e d amperometry. About 10 pmol of each sugar was detecta b l e w i t h a s i g n a l - t o - n o i s e r a t i o o f 1 0 : 1 . D - G l u c o s e , Dfructose, D-sorbitol, and D-mannose were higher in both t h e r e n a l a n d s c i a t i c t i s s u e s o f d i a b e t i c r a t s t h a n in those of normal animals. D-Ribose and glycerol were h i g h e r i n t h e r e n a l c o r t e x o f d i a b e t i c a n i m a l s . © 1992 Academic Press, Inc.
Gas chromatography (GC) is the standard method used to determine sugars and sugar alcohols in animal tissues (1). Although it is sensitive, it requires chemical modification of sample sugars and sugar alcohols prior to chromatography to produce volatile derivatives which can be detected. Recently, several high-performance liquid chromatographic (HPLC) methods using precolumn derivatization (2-5) have been developed and used for determination of sorbitol and myo-inositol.
1 To w h o m correspondence should be addressed.
98
Sensitive analytical methods for determination of individual monosaccharides, which do not require derivatization, use a combination of high-performance anionexchange chromatography (HPAEC) 2 and pulsed amperometoric detection (PAD) (6-8). However, no sensitive and simple method has been developed for the simultaneous determination of a mixture of monosaccharides and sugar alcohols. We examined the elution profiles of D-glucose, D-galactose, D-mannose, D-fructose, D-ribose, D-sorbitol, myo-inositol, and glycerol using several different H P L C columns and developed a new method which permits separation and detection of minute quantities of these carbohydrates, without forming derivatives, in a single chromatographic run. In this report we describe this method and an application of this method in studying the carbohydrate concentration in diabetic rats. Hyperglycemia often results in diabetic complications including n e p h r o p a t h y and neuropathy. D-Glucose, D-fructose, and D-sorbitol all increase and myo-inositol decreases in the renal cortices (9) and sciatic nerves (10,11) of diabetic animals. However, the effect of hyperglycemia on other carbohydrate metabolites derived from D-glucose is unknown. In a preliminary study, we found t hat D-galactose, D-mannose, D-ribose, and glycerol are also present in the extracts of these tissues in normal rats. We examined the levels of D-galactose, D-mannose, D-ribose, and glycerol as well as Dglucose, D-fructose, D-sorbitol, and myo-inositol in the renal cortices and sciatic nerves of diabetic animals using the chromatographic method described in this report. 2 Abbreviations used: H P A E C , high-performance anion-exchange chromatography; PAD, pulsed amperometoric detection; PED, pulsed electrochemical detector.
0003-2697/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
CHROMATOGRAPHIC
DETERMINATION
OF M O N O S A C C H A R I D E S
99
AND ALCOHOLS
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F I G . 1. E l u t i o n profile of s t a n d a r d m o n o s a c c h a r i d e s a n d s u g a r alcohols o n a S h o d e x S U G A R SC1011 c o l u m n e l u t e d w i t h w a t e r at 1.0 m l / m i n . D e t e c t i o n w a s by P E D u s i n g i n t e g r a t e d p u l s e d a m p e r o m e t r y mode. P e a k s : 1, D-glucose; 2, D-galactose; 3, D - m a n n o s e ; 4, D-fructose; 5, myo-inositol; 6, glycerol; 7, D-arabitol ( i n t e r n a l s t a n d a r d ) ; 8, D-ribose; 9, D-sorbitol (1 n m o l each). Details are described u n d e r M a t e rials a n d M e t h o d s .
MATERIALS AND METHODS Chemicals
The following materials were purchased from the sources indicated: D-glucose, D-galactose, D-fructose, Dribose, D-arabitol, D-sorbitol, myo-inositol, glycerol, and Glucose B-test Wako (Wako Pure Chemical Industries, Osaka, Japan); D-mannose (Katayama Chemical Industries, Osaka, Japan); streptozotocin (Sigma, St. Louis, MO). Dowex 50W-X8, 100-200 mesh (H + form) (Bio-Rad Laboratories, Richmond, CA); and Amberlite CG-400 (Type I) (Organo Co., Ltd., Tokyo, Japan). Animals
Male Sprague-Dawley rats (from SLC Japan, Shizuoka) weighing about 180 g were subjected to experiments after acclimatization for 1 week. Their body
I
5
I
25
I
10 15 Elution Time (min)
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20
F I G . 3. E l u t i o n profile of s t a n d a r d m o n o s a c c h a r i d e s a n d s u g a r alcoh o l s on a D i o n e x C a r b o P a c P A l c o l u m n e l u t e d w i t h 16 mM N a O H at 0.8 m l / m i n . D e t e c t i o n w a s by P E D u s i n g i n t e g r a t e d p u l s e d a m p e r o m e t r y mode. T h e s u g a r s are t h e s a m e as t h o s e listed in t h e legend to Fig. 1.
weight at the beginning of the experiments averaged about 250 g. The animals were given food, CA-1 (Clea Japan, Inc., Tokyo, Japan), and water ad libitum. Diabetes was induced by intravenous injection of 60 mg/kg of streptozotocin dissolved in 50 mM citrate buffer, pH 4.5. Age-matched control animals (normal rats) were given the buffer by intravenous injection. Four to 7 days after injection of streptozotocin, blood was collected from the ocular vein with a heparinized capillary tube and plasma was prepared by centrifugation at 1700g for 10 min at 4°C. Plasma glucose concentration was determined by an enzymatic method using glucose oxidase and peroxidase (Glucose B-test Wako). Rats with glucose levels of 300 mg/dl or higher were selected for the study. Twenty eight days after the injection of streptozotocin, the diabetic (n = 5) and normal control rats (n = 5) were sacrificed and their sciatic nerves and kidneys were removed.
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15 I
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10 Elution Time (rain)
20
F I G . 2 . E l u t i o n profile of s t a n d a r d m o n o s a c e h a r i d e s a n d s u g a r alcohols o n a S h o d e x S U G A R SP0810 c o l u m n . S u g a r s were eluted, detected, a n d n u m b e r e d as described in t h e legend to Fig. 1.
0
5
10
15
20
-1
I
40
Elution Time (rain)
F I G . 4. E l u t i o n profile of s t a n d a r d m o n o s a c c h a r i d e s a n d s u g a r alcohols on two c o l u m n s in series: a S h o d e x S U G A R SC1011 c o l u m n followed by a S h o d e x S U G A R S P 0 8 1 0 c o l u m n . S u g a r s were eluted, detected, a n d n u m b e r e d as d e s c r i b e d in t h e legend to Fig. 1.
100
T O M I Y A E T AL.
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8
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-~- myo-lnositol
F I G . 5. Calibration curves for m o n o s a c c h a r i d e s a n d sugar alcohols. Aqueous solutions (25 #1 each) containing (a) 10-100 pmol or (b) 0.1-10 nmol of m o n o s a c c h a r i d e s and sugar alcohols were analyzed using a S U G A R SC1011 column a n d a S U G A R SP0810 column in series. Details are described u n d e r Materials a n d M e t h o d s .
HPLC
1.0 l a
F o u r different s e p a r a t i o n m e t h o d s were t e s t e d for resolution of m o n o s a c c h a r i d e s a n d sugar alcohols on a Dionex B i o - L C c h r o m a t o g r a p h : (1) a S h o d e x S U G A R SC1011 c o l u m n (8 X 250 m m ) ( S h o w a d e n k o , T o k y o , J a p a n ) e q u i p p e d with a S U G A R SC1011P g u a r d c o l u m n (8 X 25 m m ) ; (2) a S h o d e x S U G A R SP0810 c o l u m n (8 X 250 m m ) ( S h o w a d e n k o ) , e q u i p p e d with a S U G A R S P 0 8 1 0 P g u a r d c o l u m n (8 X 25 m m ) ; (3) the S U G A R SC1011 a n d S U G A R SP0810 c o l u m n s used in series with the SC1011P g u a r d column; a n d (4) a Dionex Carb o P a c P A l c o l u m n (4 X 250 m m ) (Dionex, S u n n y v a l e , CA) e q u i p p e d with a C a r b o P a c P A g u a r d c o l u m n (3 x 25 m m ) . T w e n t y - f i v e - m i c r o l i t e r s a m p l e s were u s e d in all four s e p a r a t i o n m e t h o d s . E l u t i o n in s e p a r a t i o n m e t h ods 1, 2, a n d 3 was with deionized, distilled w a t e r at 80°C a n d a flow r a t e of 1 m l / m i n . E l u t i o n in m e t h o d 4 was p e r f o r m e d with 16 mM N a O H at 25°C a n d a flow r a t e of 0.8 ml. D e t e c t i o n of m o n o s a c c h a r i d e s a n d s u g a r alcohols was carried out u s i n g Dionex p u l s e d e l e c t r o c h e m i c a l detector (PED) under integrated pulsed amperometry. N a O H at 0.5 M was a d d e d to the p o s t c o l u m n effluent with t h e D i o n e x anionic m i c r o m e m b r a n e s u p p r e s s o r ( A M M S - I I ) . T h e following pulse p o t e n t i a l s a n d d u r a tions were used: E1 = 0.10 V (tl = 0.50 s); E2 = 0.60 V (t2 = 0.08 s); E3 = - 0 . 6 0 V (t3 = 0.05 s). T h e signals w e r e
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F I G . 6. C h r o m a t o g r a m of m o n o s a c c h a r i d e s a n d sugar alcohols derived from t h e renal cortices o f n o r m a l (a) and diabetic (b) rats using a S U G A R SC1011 column a n d S U G A R SP0810 column in series. Sugars were eluted, detected, a n d n u m b e r e d as described in t h e legend to Fig. 1.
CHROMATOGRAPHIC DETERMINATION OF MONOSACCHARIDES AND ALCOHOLS integrated b e t w e e n 0.30 and 0.50 s and the response time of the P E D detector was set to 5 s.
Tissue Sample Preparation Neutral m o n o s a c c h a r i d e s and sugar alcohols were prepared by h o m o g e n i z i n g 20 mg of sciatic nerve tissue and 50 mg o f renal cortex in 1.15 ml of deionized, distilled water containing 100 or 500 n m o l of D-arabitol, respectively (used as an internal standard). Protein was precipitated and r e m o v e d from the h o m o g e n a t e by adding 2.8 ml of 99.5% ethanol and centrifuging the samples at 3500 rpm for 10 min at 4°C. T h e supernatant was concentrated to dryness in vacuo and resuspended in 1
101
ml o f deionized, distilled water. T h e s e samples were applied successively to a S e p - P a k Vac Ct8 cartridge (Millipore, Japan, T o k y o , Japan) and c o l u m n s of D o w e x 5 0 W - X 8 (H + form, 1 ml) and Amberlite CG-400 (CO~form, 1 ml). T h e s e were all w a s h e d with 6 ml of deionized, distilled water and the eluate was dried under a vacuum. T h e residue was dissolved with 1 ml of deionized, distilled water and was filtered (Ultrafree-C3GV, 0.22 #m, Millipore).
Statistical Calculations C o n c e n t r a t i o n s of m o n o s a c c h a r i d e s and sugar alcohols in rat tissues are expressed as the m e a n + SE and myo-lnosltol
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FIG. 7. Monosaccharide and sugar alcohol concentrations in the renal cortices of normal and diabetic rats (n = 5). Bars and vertical lines indicate the mean _+ SE. +P < 0.05, ~+P < 0.01 vs normal.
102
T O M I Y A E T AL.
t h e d a t a were c o m p a r e d for significant difference using an u n p a i r e d S t u d e n t ' s t test. RESULTS AND DISCUSSION Five n e u t r a l m o n o s a c c h a r i d e s (D-glucose, D-galactose, D - m a n n o s e , D-fructose, a n d D-ribose) a n d t h r e e n e u t r a l s u g a r alcohols (myo-inositol, glycerol, a n d Dsorbitol) p r e d o m i n a t e in t h e renal cortices a n d sciatic n e r v e s of rats. A n u m b e r of m e t h o d s are available for s e p a r a t i o n a n d d e t e c t i o n of several of t h e s e c o m p o u n d s f r o m m i x t u r e s , b u t no p r e v i o u s m e t h o d provides simple, s i m u l t a n e o u s r e s o l u t i o n of all of t h e m . C a t i o n - e x c h a n g e c o l u m n s b e a r i n g N a +, Ca 2+, P b 2+, or Ag 2+ can be u s e d to s e p a r a t e v a r i o u s c a r b o h y d r a t e s t h r o u g h ligand exc h a n g e (12). M o n o s a c c h a r i d e s a n d s u g a r alcohols can be eluted f r o m Ca 2+ or P b 2+ resins with water, p e r m i t ting d e t e c t i o n of t h e s e c o m p o u n d s by P A D , a f t e r t h e effluent h a s b e e n alkaline. W e u s e d a c o m b i n a t i o n of c o l u m n s b e a r i n g Ca 2÷ a n d P b 2+ resins as well as P A D to s e p a r a t e t h e c o m p o u n d s m e n t i o n e d a b o v e in a single H P L C run.
Carbohydrate Separation on Single Columns W e studied t h e elution profiles of the m o n o s a c c h a rides a n d s u g a r alcohols m e n t i o n e d above using t h r e e different c o l u m n s . Figure 1 shows t h a t a Ca 2+ b e a r i n g S h o d e x S U G A R SC1011 c o l u m n resolved D-glucose, glycerol, a n d D-arabitol b u t was u n a b l e to s e p a r a t e Dgalactose f r o m D - m a n n o s e , D-fructose f r o m myo-inositol, or D-ribose f r o m D-sorbitol. T h e S h o d e x S U G A R SP0810 c o l u m n b e a r i n g P b 2+ resin resolved D-glucose, D-galactose, D-arabitol, D-ribose, a n d D-sorbitol b u t was u n a b l e to s e p a r a t e D - m a n n o s e f r o m D-fructose or m y o inositol f r o m glycerol (Fig. 2). H P A E C h a s b e e n r e p o r t e d to clearly s e p a r a t e s o m e m o n o s a c c h a r i d e s a n d s u g a r alcohols (6-8). W e f o u n d t h a t a D i o n e x C a r b o P a c P A l c o l u m n resolved D-glucose, D-galactose, D - m a n n o s e , D-fructose, a n d D-ribose as well as D-arabitol a n d D-sorbitol b u t was u n a b l e to s e p a r a t e glycerol a n d m y o - i n o s i t o l (Fig. 3).
Simple Carbohydrate Separation on Two Columns in Series D-Glucose, D-galactose, D - m a n n o s e , D-fructose, D-ribose, myo-inositol, glycerol, D-arabitol, a n d D-sorbitol were m i x e d t o g e t h e r a n d subjected to H P L C in a s y s t e m c o n t a i n i n g two c o l u m n s in series, a S h o d e x S U G A R SC1011 followed b y a S h o d e x S U G A R SP0810, a n d det e c t e d by P A D . T h e elution profile of this m i x t u r e of s t a n d a r d m o n o s a c c h a r i d e s a n d s u g a r alcohols is s h o w n in Fig. 4. Figure 5 shows t h a t the calibration curves for e a c h m o n o s a c c h a r i d e a n d s u g a r alcohol were a l m o s t linear up to 10 n m o l / i n j e c t i o n . T h e precision of m e a s u r e m e n t of elution t i m e s a n d p e a k a r e a s were t e s t e d b y
multiple injections a n d v a r i e d b y 0-0.1% (n = 5) a n d 0.4-1.6% (n = 5), respectively ( d a t a not shown). T h e limit of d e t e c t i o n of t h e s e c o m p o u n d s is a b o u t 10 p m o l with a signal-to-noise ratio of 10:1. T h i s value is c o m p a rable to or higher t h a n t h o s e o b t a i n e d using previously r e p o r t e d m e t h o d s (2-5). P r e v i o u s H P L C a n d GC m e t h o d s used to m e a s u r e sugars a n d s u g a r alcohols in tissue s a m p l e s h a v e dep e n d e d on d e r i v a t i z a t i o n of t h e s e c o m p o u n d for detection. T h i s o f t e n results in p e a k s which overlap t h o s e of t h e c o m p o u n d s of interest, m a k i n g good s e p a r a t i o n a n d m e a s u r e m e n t difficult. C h r o m a t o g r a m s of tissue s a m ples using the Ca 2+ a n d P b 2+ c o l u m n s in series clearly show t h a t no such i n t e r f e r i n g p e a k s are o b t a i n e d with this m e t h o d (Figs. 6 a n d 8).
Purification of Monosaccharides and Sugar Alcohols T h e r e c o v e r y of all the nine c a r b o h y d r a t e s was above 90%, w h e n t h e s t a n d a r d m i x t u r e c o n t a i n i n g 0.4-400 n m o l of each c a r b o h y d r a t e was t r e a t e d with ion-exchange resine a n d a S e p - P a k Cls cartridge as described u n d e r M a t e r i a l s a n d M e t h o d s . T h e recoveries of the int e r n a l s t a n d a r d , D-arabitol, t h r o u g h the whole purification p r o c e d u r e were 90.8 + 0.9 a n d 85.0 +_ 1.2% (renal cortices f r o m n o r m a l a n d diabetic rats, n = 5, respec-
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F I G . 8. C h r o m a t o g r a m of m o n o s a c c h a r i d e s and sugar alcohols derived from the sciatic nerves of n o r m a l (a) and diabetic (b) rats using a S U G A R SC1011 c o l u m n a n d S U G A R SP0810 c o l u m n in series. Sugars were eluted, detected, a n d n u m b e r e d as described in the legend to Fig. 1.
CHROMATOGRAPHIC DETERMINATION OF MONOSACCHARIDES AND ALCOHOLS tively), a n d 88.0 _ 2.3 a n d 80.0 + 0.6% (sciatic n e r v e s f r o m n o r m a l a n d d i a b e t i c rats, n = 5, r e s p e c t i v e l y ) .
Measurement of Monosaccharides and Sugar Alcohols in the Renal Cortices and Sciatic Nerves of Normal and Diabetic Rats D-Glucose, D-fructose, a n d D - s o r b i t o l all i n c r e a s e a n d m y o - i n o s i t o l d e c r e a s e s in t h e r e n a l c o r t i c e s (9) a n d sciatic n e r v e s (10,11) of d i a b e t i c a n i m a l s . H o w e v e r , t h e effect of h y p e r g l y c e m i a on o t h e r c a r b o h y d r a t e m e t a b o lites d e r i v e d f r o m D-glucose is u n k n o w n . M o n o s a c c h a r i d e s a n d s u g a r a l c o h o l s were e x t r a c t e d f r o m t h e r e n a l
Normal
c o r t i c e s a n d sciatic n e r v e s of n o r m a l r a t s a n d r a t s w i t h streptozotocin-induced diabetes. These samples were p u r i f i e d as d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s a n d t h e n s u b j e c t e d to H P L C u s i n g t h e t w o c o l u m n m e t h o d d e s c r i b e d above. F i g u r e s 6a a n d 6b s h o w t h a t p e a k s were o b s e r v e d for D-glucose, D - g a l a c t o s e , D - m a n n o s e , D-fructose, m y o - i n ositol, glycerol, D-ribose, a n d D - s o r b i t o l d e r i v e d f r o m r e n a l c o r t i c e s of n o r m a l a n d d i a b e t i c r a t s . T h e levels of t h e s e c o m p o u n d s in r e n a l c o r t e x a r e s h o w n in Fig. 7. S i g n i f i c a n t l y h i g h e r levels of D-glucose, D - m a n n o s e , Df r u c t o s e , D-ribose, D-sorbitol, a n d glycerol w e r e all f o u n d in t h e r e n a l c o r t i c e s of d i a b e t i c r a t s . T h e m e a n
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FIG. 9. Monosaccharide and sugar alcohol concentrations in the sciatic nerves of normal and diabetic rats (n = 5). Bars and vertical lines indicate the mean _ SE. $~P < 0.01 vs normal.
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TOMIYA ET AL.
level of D - g a l a c t o s e w a s a l s o e l e v a t e d in t h e d i a b e t i c a n i m a l s , h o w e v e r , m y o - i n o s i t o l was r e d u c e d to a b o u t 80% of t h e level f o u n d in n o r m a l rats. T h e e l u t i o n p r o f i l e s of m o n o s a c c h a r i d e s a n d s u g a r a l c o h o l s f r o m t h e sciatic n e r v e s of n o r m a l a n d d i a b e t i c r a t s a r e s h o w n in Figs. 8a a n d 8b. D-Glucose, D - m a n nose, D-fructose, m y o - i n o s i t o l , glycerol, D-ribose, a n d D - s o r b i t o l w e r e p r e s e n t in t h i s t i s s u e in b o t h n o r m a l a n d d i a b e t i c r a t s . T h e levels of t h e s e c o m p o u n d s a r e s h o w n in Fig. 9. S i g n i f i c a n t l y h i g h e r levels of D-glucose, Dm a n n o s e , D-fructose, a n d D - s o r b i t o l were all f o u n d in t h e sciatic n e r v e s of d i a b e t i c rats. T h e m e a n level of g l y c e r o l w a s also e l e v a t e d in t h e d i a b e t i c a n i m a l s . As in r e n a l t i s s u e m y o - i n o s i t o l w a s r e d u c e d to a b o u t 80% of t h e level s e e n in t h e n o r m a l a n i m a l s . W e h a v e d e v e l o p e d a s i m p l e , s e n s i t i v e m e t h o d of m e a s u r i n g all of t h e m o n o s a c c h a r i d e s a n d s u g a r alcoh o l s p r e s e n t in r e n a l c o r t i c e s a n d sciatic n e r v e tissue. U s i n g t h i s m e t h o d to c o m p a r e t h e levels of s i m p l e sug a r s in t h e s e t i s s u e s in d i a b e t i c a n d n o r m a l a n i m a l s , we h a v e d e m o n s t r a t e d a l t e r e d levels of D - m a n n o s e , glycerol, a n d D-ribose as well as t h e p r e v i o u s l y k n o w n c h a n g e s in t h e levels of D-glucose, D-fructose, a n d Ds o r b i t o l . T h e s e r e s u l t s i n d i c a t e t h a t i m b a l a n c e s in c a r b o h y d r a t e m e t a b o l i s m o c c u r n o t o n l y in p o l y o l p a t h w a y i n t e r m e d i a t e s , b u t also in o t h e r m o n o s a c c h a r i d e s a n d
s u g a r a l c o h o l s . T h e s e c h a n g e s m a y be i m p o r t a n t in det e r m i n i n g h o w d i a b e t i c c o m p l i c a t i o n s d e v e l o p in animals. REFERENCES 1. Sweely, C. C., Bentley, R., Makita, M., and Wells, W. W. (1963)d. Am. Chem. Soc. 85, 2497-2507. 2. Dethy, J-M., Callaert-Deveen, B., Janssens, M., and Lenaers, A. (1984) Anal. Biochem. 143,119-124. 3. Lloyd, P., and Crabbe, M. J. C. (1985) d. Chromatogr. 343,402406. 4. Akanuma, H., Yamanouchi, T., Ono, H., Nomura, K., and Akanuma, Y. (1985) d. Biochem. 97,579-588. 5. Miwa, I., Kanbara, M., Wakazono, H., and Okuda, d. (1988) Anal. Biochem. 173, 39-44. 6. Rocklin, R. D., and Pohl, C. A. (1983) J. Liq. Chromatogr. 6, 1577-1590. 7. Hardy, M. R., Townsend, R. R., and Lee, Y. C. (1988) Anal. Biochem. 170, 54-62. 8. Peelen, G. O. H., De Jong, J. G. N., and Wevers, R. A. (1991) Anal. Biochem. 198, 334-341. 9. Poulsom, R., and Heath, H. (1983) Biochem. Pharmacol. 32, 1495-1499. 10. Clements, R. S., and Stockard, C. R. (1980) Diabetes 29,227-235. 11. Finegold, D., Lattimer, S. A., Nolle, S., Bernstein, M., and Greene, D. A. (1983) Diabetes 32,988-992. 12. Goulding, R. W. (1975) J. Chromatogr. 103,229-239.