41
Clinica Chimica Acta, 63 (1975) 41-47 0 Elsevier Scientific Publishing Company, Amsterdam -
Printed in The Netherlands
CCA 7216
SORBITOL AND OTHER POLYOLS IN LENS, ADIPOSE AND URINE IN DIABETES MELLITUS
TISSUE
D.J. HEAF and D.J. GALTON Diabetes
and Lipid
Research
Laboratory,
St Bartholomew’s
Hospital,
London
E.C.1
(U.K.)
(Received March 11, 1975)
Summary
Sugars and polyols (glucose, fructose, sorbitol, inositol and pentitols) have been measured in urine, adipose tissue and lens in groups of diabetic and nondiabetic patients. The daily excretions of glucose, sorbitol and inositol were raised in the diabetics. There was a linear relation between 24 hour urinary excretion of glucose and hexitols (r = +0.87, p < 0.001). In lens of diabetics there was an increase in glucose concentration, but not of fructose, sorbitol or inositol. Compounds readily detected in adipose tissue were inositol and glucose; in addition an unidentified carbohydrate was detected in adipose tissue that related to the concentration of tissue glucose. These results are discussed in relation to the possibility that tissue accumulation of polyols could be responsible for the secondary complications of diabetes such as cataracts.
Introduction
Apart from the disturbance of levels of blood glucose in diabetes mellitus it is now well established that there is an alteration in the rates of glucose metabolism in various tissues such as adipose tissue [l] and liver [2] . This is reflected by an alteration of levels of glucose metabolites; and for example in the diabetic erythrocyte there is accumulation of fructose diphosphate whereas in the diabetic adipocyte levels of glucose 6-phosphate fail to rise after an oral glucose load compared to non-diabetic controls [3]. It has also been proposed that unusual products of glucose metabolism accumulate in tissues of diabetics and are responsible for some of the secondary complications of this disease. Thus in alloxan-diabetic rats the development of cataracts has been postulated to depend on the accumulation of sorbitol in the lens which produces osmotic changes and lenticular damage [4]. Likewise the development of peripheral neuropathy in alloxan-diabetic rats has been attributed to the accumulation of sorbitol in peripheral nerves [ 51.
42
Although much work has been done on animal information on the levels of polyols and sugars in therefore reports measurements of glucose, fructose, tit& in urine, adipose tissue and tens to see if these the presence of diabetes melfitus.
models there is still little human tissues. This paper sorbitoi, inositol, and pencompounds are altered by
Materials and methods The following patients were investigated from the Diabetic, Lipid Clinic and Medical Wards of St Bartholomew’s Hospital. Twenty-six diabetics (mean age 60.5 + 3 years, weight 73 X!I3.3 kg, height 146 I 4.2 cm and blood sugar 16’7 it 22 mgJlO0 ml) and compared to twenty-three non-diabetics (mean age 53 F 5.2 years, weight 70.5 f 5.7 kg, height 142 L 4.4 cm). The diabetics ineluded juvenile and maturity-onset types and were under various stages of control by insulin or or& agents. Degree of control was estimated by the 24 hour urinary excretion of glucose. Preparation
for gas liquid chrmmtography
(GLC)
Urine 24 hour urine samples were collected from patients into bottles containing a few ml of toluene as preservative. The total urine volume was measured and an aliquot was stored in the deep freeze for assay of pentitols and hexitols. The urine sample (0.5 ml) was diluted with an equal volume of water and o-[U-‘“C] sorbitol was added (about 30 000 cpm) as intend standard. A small amount of urease powder (approx. 1 mg) was added and after mixing, the tube was placed in a water bath at 30°C. After 10 minutes incubation the pH was adjusted to 7 using dry IR 120 cation exchange resin (H’ form). The tube was further incubated, with repeated neutralization until no further rise in pH occurred, to remove ail urea. The urine was then deionised by further addition of cation exchange resin, and anion exchange resin Deacidite, (CO; form). The urine was then transferred, by decanting and washing (i.e. resin was left behind), to an hmicon Membrane Centrifuge Filter (‘Centriflo’) for removal of protein. The ultrafiltrate was evaporated to dryness under a stream of air in a 60°C waterbath. The residue was then transferred to a 1.5 mf screw top glass vial using several washes of methanol. Meth~oI was then blown off under a stream of dry nitrogen and 0.2 ml of oxime reagent fpyridine 10 ml, hydroxyl~~ne HCI 60 mg) was added to convert glucose to its oxime derivative by heating in a water bath at 6O*C for 0.5 hour. This had the advantage that multiple peaks of glucose isomers were reduced to one peak of glucose oxime on GLC. Silylation reagent (0.1 ml) was then added (Sweeley et al. [6] ) and the reaction carried out at room ~mpe~ture for 10 minutes. Pyridine and silylation reagents were blown off under a stream of oxygen-free nitrogen. Hexane (0.5 ml) containing silylation reagent (0.2 ml silyl reagent/l0 ml hexane) to protect the ethers from decomposition by atmospheric oxygen or water was added to each vial. Ahquots of hexane extracts (SO ~1) were counted in 10 ml of Kinards S~~nt~~~t in a Packard Scintillation Counter (model 2240) to determine recoveries of added D-[U-‘4C]sorbitol.
43
Adipose tissue Subcutaneous adipose tissue was obtained by needle biopsy [ 161 from patients and placed in 2 ml of deionised water containing tracer D -[ U-’ 4 C] sorbitol to correct for recoveries. Lipid was extracted with three washes with petroleum spirit (b.p. 40”--6O”C) in a glass homogeniser. The aqueous extract was deionised with a minimum quantity of cation and anion exchange resins until further addition of the cation exchange resin produced no further fall in pH. The solution was then transferred with several washes of the resin with deionised water to an Amicon Membrane Centriflo Filter. The ultrafiltrate was evaporated to dryness under a stream of N2 in a water bath at 60°C. Sugars and polyols were redissolved in methanol and transferred with two washes to a glass screw-top vial. Meth~ol was blown off under Nz and glucose was converted to its oxime using hydroxylamine-pyridine reagent (0.1 ml) at 60°C for 30 minutes. Polyols, sugars and glucose oxime were converted to trimethyl silyl ethers using a silylation reagent (50 ~1) after the method of Sweeley et al. [6]. Reagents were then blown off under a stream of oxygen-free nitrogen at room temperature. T~methylsilyl compounds were redissolved in hexane 50 ~1 which contained silylating reagent and 1 1.11was used for GLC analysis whereas 20 ~1 were used to estimate recoveries of added D-[ UJ-‘~C] sorbitol.
Lens Cataractous lenses from diabetics and non-diabetic patients were weighed and then extracted in methanol (5 ml) containing D-[U-“Cl sorbitol (to calculate recoveries) in a glass homogeniser. The suspension was transferred to a centrifuge tube and centrifuged until the supernatant was clear. The methanolic extract was evaporated to a small volume and then transferred to a screw-top vial for preparation of glucose oxime. Silylation reagent (0.1 ml) was then added and the vial left at room temperature for 10 minutes. Reagents and pyridine were blown off under a stream of oxygen-free nitrogen at room temperature. Hexane containing silylation reagent was added (0.5 ml) and the vial thoroughly mixed.
Gas liquid chromatography Trimethyl silyl ethers of polyols and sugar were separated by a Pye Series 104 Gas Chromatograph isothermally at 160°C with a detector temperature at 200°C. Column conditions were 7 feet by 0.25 inch glass tube containing Chromosorb W (100-200 mesh) coated with silicone gum SE. 30 (3%). Nitrogen carrier flow was 40 ml/min and the air flow was 675 ml/min. The compounds were identified by retention times compared with retention times of authentic standards. The TMS ethers emerged from the column in the order: erythritol, xylitol, arabitol, ribitol, fructose, sedoheptulose, mannitol, sorbitol, galactitol, glucose oxime and inositol. Arabitol and ribitol were coincident whereas xylitol, which could not be completely separated from the other pentitols emerged first. Pentitols were measured as ribitol at a retention time relative to inositol of 0.204. Under the column conditions inositol emerged after 21.5 minutes and no further compounds emerged after this. Standards of sugars and polyols were derivatised and silylated in the same
manner as for lens extract. Silyl derivatives were redissolved in hexane (1 ml) containing silylating reagent and 1 1.11of each standard was used for calibration containing lo-70 mg of each compound. Calibration curves were linear over this range. Chromatography was carried out immediately after preparation of hexane solutions of standard and sample trimethylsilyl ethers to avoid any risk of error arising from evaporation or decomposition. The identity of peaks detected were confirmed by retention time measurements on S.E. 30 coated Chromosorb W at 160°C and neopentyl glycol succinate (NGS) coated Chromosorb W at 130°C. The recovery of sorbitol during preparation of derivatives was measured. The recovery of D-[U-‘4 C] sorbitol from lenses during the process of extraction was 78 + 4%. The coefficient of variation of 6 replicates for urinary inositol was 8.4%, for hexitols was 6.2% and for pentitols 2.3%. Results Urine The polyols found in urine are presented in Table I. Since it was found difficult to resolve the hexitols (sorbitol, galactitol, mannitol) from each other, as was the separation of pentitols (xylitol, ribitol, arabitol) the results are expressed as total hexitol and pentitol excretion per 24 hours. Pentitol excretion was not affected by the presence of diabetes even though the mean glucose excretion was 75-fold higher than in non-diabetic controls. However, urinary excretion of hexitols (including sorbitol) was increased 5-fold in the diabetic group. There was also a slight increase in excretion of inositol in diabetic urine. The degree of glycosuria also related to the urinary excretion of hexitols as observed in Fig. 1. Adipose tissue Inositol and glucose were readily detected in human adipose tissue. Their presence was confirmed using two other columns (2% NGS on Chromosorb W at 130°C and 1% polyethylene glycol adipate (PEGA) on Celite at 130°C). A peak having a retention time close to sorbitol and galactitol was detected in most extracts of adipose tissue. This peak emerged after the hexitols and by TABLE
I
POLYOL
EXCRETION
Urine
was
sugars
were measured
in
extracted
parentheses.
large
variation
Patient
group
Non-diabetics
IN for
URINE
pol~ols
by gas-liquid
Significances in urinary
(mg/24
t
13.9
(23) Diabetics P
3900 (23) co.01
and
DIABETICS
sugars
as described
chromatography differences
excretion
Glucose
52.3
of
OF
were
of glucose
h)
Inositol
49.9 67.8 (23) N.S.
tested
by
Trimethyl
are means
silyl
kS.E.M.
non-parametric
sign
ethers
with test
of polyols
number [17]
and
of patients
because
of
the
and polyols.
(mg124
* 10.5
(23) f 1950
in Methods.
and results
h)
Hexitol
27.1
(mg/24
5
5.2
(23) k 16.2
135 (23) 10.01
h)
Pentitol
29.1
(mg/24
k 3.1
(23) ?- 46
30.0 (23) N.S.
* 5.1
h)
45
THE EFFECT OF DlABETES ON EXCRETION OF URINARY HMITOLS 0 DIABETIC
*NON DIABETIC
1
1
1M
10
1.m
lO,fXO
IW,cm
GLUCOSE (mg124 hrrl
Fig. 1. Urinary gIucose and hexitols were extracted from 0.5 ml of a 24 hour urine collection as described in Methods. Trimethyl siIy1 ethers were prepared and measured by gas liquid chromatography. Points are individual patients comprising diabetics (0) under various degrees of control and non-diabetics (0).
TABLE
II
CONTENTS
OF SUGARS
AND POLYOLS
IN HUMAN
LENS
Lenses were removed at operation for cataracts and extracted in methanol as described in Methods. Trimethyl siIy1 ethers of polyols and sugars were measured by gas liquid chromatography. Results are presented for individual patients. Significances of differences were tested by Student’s ‘t’ test. Patient
Glucose
Diabetics F.D. F.L. D.B. S.M. z.c.* A.C. A.K. E.F.
913 88.5 671.6 128 698 336
Mean S.E.M. Non-diabetics G.H. M.F. M.H. P.R. L.D. T.R. Mean S.E.M. P
(ccglg)
-
Fructose
(clglg)
-
Sorbitol
(pg/g)
Inositol
-
19.2 70.2 15.4
81.4 84.4 87.3 140.9 (1243) 135 23.4 31.3
472 137
29.9 13.4
83.3 17.1
113 90.9 67.5 108 30.8 77.9
10.7 8.2 7.1 29.4 37.7 28.7
23.0 86.5 27.2 13.4 56.7 101.7
0.71 3.3 1.23 1.32 5.61 6.77
20.3 5.3 N.S.
51.4 14.8 N.S.
3.1 1.03 N.S.
81.3 12.3
Clinica Chimica Acta, 63 (1975) 41-47 0 Elsevier Scientific Publishing Company, Amsterdam -
Printed in The Netherlands
CCA 7216
SORBITOL AND OTHER POLYOLS IN LENS, ADIPOSE AND URINE IN DIABETES MELLITUS
TISSUE
D.J. HEAF and D.J. GALTON Diabetes
and Lipid
Research
Laboratory,
St Bartholomew’s
Hospital,
London
E.C.1
(U.K.)
(Received March 11, 1975)
Summary
Sugars and polyols (glucose, fructose, sorbitol, inositol and pentitols) have been measured in urine, adipose tissue and lens in groups of diabetic and nondiabetic patients. The daily excretions of glucose, sorbitol and inositol were raised in the diabetics. There was a linear relation between 24 hour urinary excretion of glucose and hexitols (r = +0.87, p < 0.001). In lens of diabetics there was an increase in glucose concentration, but not of fructose, sorbitol or inositol. Compounds readily detected in adipose tissue were inositol and glucose; in addition an unidentified carbohydrate was detected in adipose tissue that related to the concentration of tissue glucose. These results are discussed in relation to the possibility that tissue accumulation of polyols could be responsible for the secondary complications of diabetes such as cataracts.
Introduction
Apart from the disturbance of levels of blood glucose in diabetes mellitus it is now well established that there is an alteration in the rates of glucose metabolism in various tissues such as adipose tissue [l] and liver [2] . This is reflected by an alteration of levels of glucose metabolites; and for example in the diabetic erythrocyte there is accumulation of fructose diphosphate whereas in the diabetic adipocyte levels of glucose 6-phosphate fail to rise after an oral glucose load compared to non-diabetic controls [3]. It has also been proposed that unusual products of glucose metabolism accumulate in tissues of diabetics and are responsible for some of the secondary complications of this disease. Thus in alloxan-diabetic rats the development of cataracts has been postulated to depend on the accumulation of sorbitol in the lens which produces osmotic changes and lenticular damage [4]. Likewise the development of peripheral neuropathy in alloxan-diabetic rats has been attributed to the accumulation of sorbitol in peripheral nerves [ 51.
42
Although much work has been done on animal information on the levels of polyols and sugars in therefore reports measurements of glucose, fructose, tit& in urine, adipose tissue and tens to see if these the presence of diabetes melfitus.
models there is still little human tissues. This paper sorbitoi, inositol, and pencompounds are altered by
Materials and methods The following patients were investigated from the Diabetic, Lipid Clinic and Medical Wards of St Bartholomew’s Hospital. Twenty-six diabetics (mean age 60.5 + 3 years, weight 73 X!I3.3 kg, height 146 I 4.2 cm and blood sugar 16’7 it 22 mgJlO0 ml) and compared to twenty-three non-diabetics (mean age 53 F 5.2 years, weight 70.5 f 5.7 kg, height 142 L 4.4 cm). The diabetics ineluded juvenile and maturity-onset types and were under various stages of control by insulin or or& agents. Degree of control was estimated by the 24 hour urinary excretion of glucose. Preparation
for gas liquid chrmmtography
(GLC)
Urine 24 hour urine samples were collected from patients into bottles containing a few ml of toluene as preservative. The total urine volume was measured and an aliquot was stored in the deep freeze for assay of pentitols and hexitols. The urine sample (0.5 ml) was diluted with an equal volume of water and o-[U-‘“C] sorbitol was added (about 30 000 cpm) as intend standard. A small amount of urease powder (approx. 1 mg) was added and after mixing, the tube was placed in a water bath at 30°C. After 10 minutes incubation the pH was adjusted to 7 using dry IR 120 cation exchange resin (H’ form). The tube was further incubated, with repeated neutralization until no further rise in pH occurred, to remove ail urea. The urine was then deionised by further addition of cation exchange resin, and anion exchange resin Deacidite, (CO; form). The urine was then transferred, by decanting and washing (i.e. resin was left behind), to an hmicon Membrane Centrifuge Filter (‘Centriflo’) for removal of protein. The ultrafiltrate was evaporated to dryness under a stream of air in a 60°C waterbath. The residue was then transferred to a 1.5 mf screw top glass vial using several washes of methanol. Meth~oI was then blown off under a stream of dry nitrogen and 0.2 ml of oxime reagent fpyridine 10 ml, hydroxyl~~ne HCI 60 mg) was added to convert glucose to its oxime derivative by heating in a water bath at 6O*C for 0.5 hour. This had the advantage that multiple peaks of glucose isomers were reduced to one peak of glucose oxime on GLC. Silylation reagent (0.1 ml) was then added (Sweeley et al. [6] ) and the reaction carried out at room ~mpe~ture for 10 minutes. Pyridine and silylation reagents were blown off under a stream of oxygen-free nitrogen. Hexane (0.5 ml) containing silylation reagent (0.2 ml silyl reagent/l0 ml hexane) to protect the ethers from decomposition by atmospheric oxygen or water was added to each vial. Ahquots of hexane extracts (SO ~1) were counted in 10 ml of Kinards S~~nt~~~t in a Packard Scintillation Counter (model 2240) to determine recoveries of added D-[U-‘4C]sorbitol.
43
Adipose tissue Subcutaneous adipose tissue was obtained by needle biopsy [ 161 from patients and placed in 2 ml of deionised water containing tracer D -[ U-’ 4 C] sorbitol to correct for recoveries. Lipid was extracted with three washes with petroleum spirit (b.p. 40”--6O”C) in a glass homogeniser. The aqueous extract was deionised with a minimum quantity of cation and anion exchange resins until further addition of the cation exchange resin produced no further fall in pH. The solution was then transferred with several washes of the resin with deionised water to an Amicon Membrane Centriflo Filter. The ultrafiltrate was evaporated to dryness under a stream of N2 in a water bath at 60°C. Sugars and polyols were redissolved in methanol and transferred with two washes to a glass screw-top vial. Meth~ol was blown off under Nz and glucose was converted to its oxime using hydroxylamine-pyridine reagent (0.1 ml) at 60°C for 30 minutes. Polyols, sugars and glucose oxime were converted to trimethyl silyl ethers using a silylation reagent (50 ~1) after the method of Sweeley et al. [6]. Reagents were then blown off under a stream of oxygen-free nitrogen at room temperature. T~methylsilyl compounds were redissolved in hexane 50 ~1 which contained silylating reagent and 1 1.11was used for GLC analysis whereas 20 ~1 were used to estimate recoveries of added D-[ UJ-‘~C] sorbitol.
Lens Cataractous lenses from diabetics and non-diabetic patients were weighed and then extracted in methanol (5 ml) containing D-[U-“Cl sorbitol (to calculate recoveries) in a glass homogeniser. The suspension was transferred to a centrifuge tube and centrifuged until the supernatant was clear. The methanolic extract was evaporated to a small volume and then transferred to a screw-top vial for preparation of glucose oxime. Silylation reagent (0.1 ml) was then added and the vial left at room temperature for 10 minutes. Reagents and pyridine were blown off under a stream of oxygen-free nitrogen at room temperature. Hexane containing silylation reagent was added (0.5 ml) and the vial thoroughly mixed.
Gas liquid chromatography Trimethyl silyl ethers of polyols and sugar were separated by a Pye Series 104 Gas Chromatograph isothermally at 160°C with a detector temperature at 200°C. Column conditions were 7 feet by 0.25 inch glass tube containing Chromosorb W (100-200 mesh) coated with silicone gum SE. 30 (3%). Nitrogen carrier flow was 40 ml/min and the air flow was 675 ml/min. The compounds were identified by retention times compared with retention times of authentic standards. The TMS ethers emerged from the column in the order: erythritol, xylitol, arabitol, ribitol, fructose, sedoheptulose, mannitol, sorbitol, galactitol, glucose oxime and inositol. Arabitol and ribitol were coincident whereas xylitol, which could not be completely separated from the other pentitols emerged first. Pentitols were measured as ribitol at a retention time relative to inositol of 0.204. Under the column conditions inositol emerged after 21.5 minutes and no further compounds emerged after this. Standards of sugars and polyols were derivatised and silylated in the same
manner as for lens extract. Silyl derivatives were redissolved in hexane (1 ml) containing silylating reagent and 1 1.11of each standard was used for calibration containing lo-70 mg of each compound. Calibration curves were linear over this range. Chromatography was carried out immediately after preparation of hexane solutions of standard and sample trimethylsilyl ethers to avoid any risk of error arising from evaporation or decomposition. The identity of peaks detected were confirmed by retention time measurements on S.E. 30 coated Chromosorb W at 160°C and neopentyl glycol succinate (NGS) coated Chromosorb W at 130°C. The recovery of sorbitol during preparation of derivatives was measured. The recovery of D-[U-‘4 C] sorbitol from lenses during the process of extraction was 78 + 4%. The coefficient of variation of 6 replicates for urinary inositol was 8.4%, for hexitols was 6.2% and for pentitols 2.3%. Results Urine The polyols found in urine are presented in Table I. Since it was found difficult to resolve the hexitols (sorbitol, galactitol, mannitol) from each other, as was the separation of pentitols (xylitol, ribitol, arabitol) the results are expressed as total hexitol and pentitol excretion per 24 hours. Pentitol excretion was not affected by the presence of diabetes even though the mean glucose excretion was 75-fold higher than in non-diabetic controls. However, urinary excretion of hexitols (including sorbitol) was increased 5-fold in the diabetic group. There was also a slight increase in excretion of inositol in diabetic urine. The degree of glycosuria also related to the urinary excretion of hexitols as observed in Fig. 1. Adipose tissue Inositol and glucose were readily detected in human adipose tissue. Their presence was confirmed using two other columns (2% NGS on Chromosorb W at 130°C and 1% polyethylene glycol adipate (PEGA) on Celite at 130°C). A peak having a retention time close to sorbitol and galactitol was detected in most extracts of adipose tissue. This peak emerged after the hexitols and by TABLE
I
POLYOL
EXCRETION
Urine
was
sugars
were measured
in
extracted
parentheses.
large
variation
Patient
group
Non-diabetics
IN for
URINE
pol~ols
by gas-liquid
Significances in urinary
(mg/24
t
13.9
(23) Diabetics P
3900 (23) co.01
and
DIABETICS
sugars
as described
chromatography differences
excretion
Glucose
52.3
of
OF
were
of glucose
h)
Inositol
49.9 67.8 (23) N.S.
tested
by
Trimethyl
are means
silyl
kS.E.M.
non-parametric
sign
ethers
with test
of polyols
number [17]
and
of patients
because
of
the
and polyols.
(mg124
* 10.5
(23) f 1950
in Methods.
and results
h)
Hexitol
27.1
(mg/24
5
5.2
(23) k 16.2
135 (23) 10.01
h)
Pentitol
29.1
(mg/24
k 3.1
(23) ?- 46
30.0 (23) N.S.
* 5.1
h)
45
THE EFFECT OF DlABETES ON EXCRETION OF URINARY HMITOLS 0 DIABETIC
*NON DIABETIC
1
1
1M
10
1.m
lO,fXO
IW,cm
GLUCOSE (mg124 hrrl
Fig. 1. Urinary gIucose and hexitols were extracted from 0.5 ml of a 24 hour urine collection as described in Methods. Trimethyl siIy1 ethers were prepared and measured by gas liquid chromatography. Points are individual patients comprising diabetics (0) under various degrees of control and non-diabetics (0).
TABLE
II
CONTENTS
OF SUGARS
AND POLYOLS
IN HUMAN
LENS
Lenses were removed at operation for cataracts and extracted in methanol as described in Methods. Trimethyl siIy1 ethers of polyols and sugars were measured by gas liquid chromatography. Results are presented for individual patients. Significances of differences were tested by Student’s ‘t’ test. Patient
Glucose
Diabetics F.D. F.L. D.B. S.M. z.c.* A.C. A.K. E.F.
913 88.5 671.6 128 698 336
Mean S.E.M. Non-diabetics G.H. M.F. M.H. P.R. L.D. T.R. Mean S.E.M. P
(ccglg)
-
Fructose
(clglg)
-
Sorbitol
(pg/g)
Inositol
-
19.2 70.2 15.4
81.4 84.4 87.3 140.9 (1243) 135 23.4 31.3
472 137
29.9 13.4
83.3 17.1
113 90.9 67.5 108 30.8 77.9
10.7 8.2 7.1 29.4 37.7 28.7
23.0 86.5 27.2 13.4 56.7 101.7
0.71 3.3 1.23 1.32 5.61 6.77
20.3 5.3 N.S.
51.4 14.8 N.S.
3.1 1.03 N.S.
81.3 12.3
