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Long-term effects of eicosapentaenoic acid on diabetic peripheral neuropathy and serum lipids in patients with type II diabetes mellitus
Long-Term Effects of Eicosapentaenoic Acid on Diabetic Peripheral Neuropathy and Serum Lipids in Patients With Type II Diabetes Mellitus Yukichi Okuda, Masakazu Mizutani, Masashi Ogawa, Hirohito Sone, Michiko Asano, Yukari Asakura, Masaaki Osaka,Seiji Suzuki, Yasushi Kawakami, James B. Field, and Kamejiro Yamashita
ABSTRACT The present study was undertaken to investigate the efficacy of a new, highly purified (purity greater than 91%), ethyl esterification product from natural eicosapentaenoic acid (EPA-E, C20:5 03) in patients with type II diabetes mellitus (NIDDM). Wemodynamic changes were assessed at the level of the dorsalis pedis artery using an ultrasonic color Doppler duplex system before and after oral administration of EPA-E at a dose of 1800 mg/day for 48 weeks. The cross-sectional area of the dorsalis pedis artery increased significantly from 2.5 t 0.2 to 3.9 2 0.4 mm* (48 weeks, mean + SE, p < 0.05). Moreover, EPA-E improved the clinical symptom (coldness, numbness) as well as the
INTRODUCTION yperglycemia and the metabolic derangements of sorbitol and myo-inositol associated with it diabetic neuropathy.‘” On the other hand, there is strong evidence of
..-~. Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan, and Division of Endocrinology and Metabolism (J.B.F.) Department of Medicine, Diabetes Research Laboratory, St. Luke’s Episcopal Hospital, Baylor College of Medicine, Houston, Texas, USA. Reprint requests to be sent to: Dr. Yukichi Okuda, Department of Internal Medicine, Institute of CIinical Medicine, University of Tsukuba, Tennodai l-l-l, Tsukuba, Ibaraki 305, Japan. jooumal of Diabetes and Its Complications 20:280-287 0 Elsevier Science Inc., 1996 655 Avenue of the Americas, New York, NY 10010
vibration perception threshold sense of the lower extremities [from 32.1 ? 8.5 to 16.1 +- 4.8 (48 weeks) pm]. A significant decrease of serum triglycerides was also noted by EPA-E administration. Furthermore, significant decrease of the excretion of albumin in urine Ifrom 24.4 + 3.3 to 13.9 2 1.8 (48 weeks) mg&Cr, 77< 0.051.The results of this study suggest that EPA-E has significant beneficial effects on diabetic neuropathy and serum lipids as well as other diabetic complications such as nephropathy and macroangiopathy. (Journal of Diabetes and Its Complications 10; 5:28+287,1996.)
vascular implication in the pathogenesis of diabetic mononeuropathy and polyneuropathy.t6 Eicosapentaenoic acid (EPA, C20:5 w3), a polyunsaturated fatty acid of the o-3 series extracted from fish oil, has been reported to inhibit plateIet aggregation, improve serum lipid and have an antithrombotic action.7,* Recently, a highly pure EPA (purity greater than 91%) has been obtained by ethyl esterification of natural EPA (ethyl all-cis-5,8,11,14,17-eicosapentaenoate, EPA-E; EpadelB Manufacturer: Mochida Pharmaceutical Co., Ltd., Tokyo, Japan).9,‘0 The present study was undertaken to investigate the efficacy of EPA-E on diabetic peripheral neuropathy and its effects on serum lipids in patients with noninsulin-dependent (type II) diabetes mellitus (NIDDM). 1056-S727/96/$15.00 SSDI 1056.8727(95)00081-C
1 Did
Camp 1996; 20:280-287
EICOSAPENTAENOIC
TABLE 1. CLINICAL FEATURES OF 21 NON-INSULIN-DEPENDENT DIABETIC PATIENTS (N = 21) 13 8
40-29 50-59 60-69 70-79
2 3 12 4
IN NIDDM
281
-**I
(A)
Gender Male Female Age (year)
ACID
Body-mass index (kg/m*) G-19 20-22 23-25 26-29 a30
Duration
of diabetes (year)
s5 6-10
311 Duration
5 8 8
of nephropathy
I
I
4
12
24
48 wk
(year)
s2
5
34
11
z5
5
Treatment Diet OH-4 Insulin Hemoglobin Good
1
0
12
(B)
SCORE -**1 -**I 3
7 2
A,, (X,) 11
s7.0
Fair 7.1-8.9
6
Poor 29.0
4
Retinopathy C-1 Scott Ia IIa IIIa Nephropathy C-1 Early Overt AS0 C-1 (+) OHA, on?/ Izp~&m~ic
RESEARCH
8 7 5
1 5 16
I
I
I
I
0
12
24
48 wk
0 0
FIGURE 1 Chnnges in symptom score (A) Coldness, (B) Nmbness; Score: 3, severe; 2, moderate; I, sligirt; 0, nlxmf *“p c: 0.01.
21 qgerzts; ASO, arteriosclerosis obliterans.
DESIGN
AND
METHODS
Patients. Twenty-one patients with NIDDM and chronic peripheral diabetic neuropathy were entered into this study. The clinical characteristics of these patients are listed in Table 1. The mean age of the study population was 62.5 + 1.8 years, the mean duration of diabetes was 10.0 + 1.3 and the mean duration of neuropathy was 3.5 +- 0.4 years. They all had clinically evident bilateral peripheral somatic neuropathy in the
lower extremities. All of them had shown characteristic symptoms (e.g., burning pains, especially at night or while resting, numbness, coldness) and signs (e.g., reduced sensation, absent tendon reflexes) for at least 10 months. All patients had arteriosclerosis obliterans (ASO) as determined by Doppler echography, digital subtraction angiography, and absent foot pulses. None of them had severe cardiovascular or renal diseases, or had nephropathy due to alternative causes such as alcohol dependence or nerve entrapment syndrome were excluded. All patients gave their informed consent to the study, which was conducted according to the guidelines expressed in the Declaration of Helsinki.
TABLE 2. CHANGES
IN VIBRATORY PERCEPTION DURING TREATMENT Before -. 0 weeks
VPT Tibia (km) Ulna (km)
THRESHOLD (VPT) OF TIBIA WITH EPA-E During
32.1 2 8.5 13.3 r 1.4 _----... ..- -
..-
AND ULNA
_..
12 weeks
24 weeks
48 weeks
19.1 2 3.6* Y.5 2 0.4* ---
22.1 : 6.P+ Y.6 210.7
16.1 x 4.8** 9.8 - 2.y
EPA-E, eicosapmiaenoic acid. Mea11 L SEM. *p < 0.05, ** p < 0.07 us. before (0 zuerksi.
Study Design and Treatment. EPA-E (EpadelB, obtained from the pharmacy at the University of Tsukuba hospital, batch number 1017) was administered orally at a dose of 1800 mg; 21.8 kcal/day, in three divided doses, immediately after meals, over a period of 48 weeks. The calories of EPA-E (21.8 kcal/day) were added to existing diet of each patients. Blood pressure and pulse rate were measured before, and 12, 24, and 48 weeks after the administration of EPA-E. Prior to the examination, all subjects had to rest in the supine position for 30 min. The critical measurements were the cross-sectional area of the dorsalis pedis artery and the blood flow index, which were assessed noninvasively using a new ultrasonic color Doppler duplex system (Toshiba SSA-270A, Toshiba Co., Yokohama, Japan).” This system consists of a B-mode scanner and an ultrasonic range-gated pulsed Doppler echography. The blood flow index was obtained by multiplying the mean blood flow velocity by the cross-sectional area of the dorsalis pedis artery. The blood velocity and cross-sectional area of the artery were the mean of two or more measurements taken after allowing the patients to rest for 20 min in the supine position at room temperature (25”-27°C). The vibratory perception threshold (VET), as an indicator of sensory disturbance, was measured at both the medial malleolus of the tibia and styloid process of both ulnae using a vibratory sensation meter model TM-31 (Medic
TABLE
3. HEMODYNAMICS
International Co., Tokyo, Japan). Neurological subjective symptoms (coldness, numbness) were classified into four levels in increasing order of severity (0.1.2.3). At 0, 12, 24, and 48 weeks, venous blood was obtained from all patients before breakfast to determine fasting plasma glucose, hemoglobin (Hb) A,,, serum lipids (triglyceride, total cholesterol, high-density lipoprotein (HDL) cholesterol, apolipoproteins), blood urea nitrogen (BUN), creatinine (Cr), and the plasma composition of unsaturated fatty acids (not determined at 48 weeks). Urine samples were also collected to determine albuminuria. Data Analysis and Statistical Methods. All data in this paper are presented as the mean + SEM and were analyzed for statistical significance by the paired f test or Wilcoxon test. A p value of 0.05 or less was considered significant. RESULTS Effect on Clinical Symptoms. EPA-E significantly improved clinical symptoms such as coldness and numbness (p < 0.01, Wilcoxon test) (Figure 1). Measurement of the Vibratory Perception Threshold. VM was significantly improved at both arm and leg during treatment with EPA-E (Table 2). Improvement was detected by 12 weeks of treatment but showed no further progression thereafter.
DURING
TREATMENT -
Before
Blood pressure Systolic (mm Hg) Diastolic (mm Hg) Pulse rate (beats/min) Area (mm*) Blood flow index
-
0 weeks
12 weeks
129 73 73 2.3 17
128 72 71 3.4 25
EPA-E, eicosapenfaenoic acid. Mean +- SEM. “p < 0.05, ““p < 0.01 ZIS.before (0 wwks).
‘+ 3 +- 3 ? 2 -c 0.2 -c 2 -
= -t ? ? -t
3 2 3 0.3** 4**
.
WITH EPA-E . During .--____ 24 weeks 128 72 69 3.3 34
z ? Ifi 2 k
3 2 2 0.3* 3**
-
. 48 weeks 126 68 74 3.9 46
rt z It t 2
2 2 2 0.4* 6”*
/ Did? Camp 7996; 10:280-287
EICOSAPENTAENOIC
TABLE 4. LABORATORY
DATA
BEFORE AND DURING
TREATMENT
WITH
Before No. of Patients Body weight Plasma glucose Hemoglobin A,,. Fructosamine Triglyceride 2 150 mg/dL < 150 mg/dL Total cholesterol 2 220 mg/dL < 220 mg/dL HDL cholesterol Apolipoprotein AI AI1 B CII CIII
(kg) (mg/dL) (‘L) (wol/L) (mg/W (WdL) (mg/dL) (mg/dL) I”$Y
0 weeks 58.0 138 7.3 326 182.8 263.0 84.6 196.6 235.0 183.8 50.6 141.2 31.3 103.7 5.8 12.9
21
21 21 21 21 11 10 21 6 15 21 21 21 21 21 21
(:g/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL)
0.74 5.9 -+ + 0.5 0.05
21
:/A:mg’dL)
2 2.1 F 10 F 0.3 + 13 -+ 33.8 2 48.9 + 13.4 2 6.7 +- 6.9 2 5.6 2 3.6 t 6.0 +- 1.6 -c 6.7 i 1.2 t 1.6
ACID
283
IN NIDDM
EPA-E
During 12 weeks 58.0 147 7.3 328 134.1 174.9 70.0 185.1 210.0 178.4 50.4 138.7 29.3 89.1 4.1 12.4 5.4 0.66
24 weeks
2 2.2 2 13 i- 0.3 f- 11 k 22.2* -t 30.2* -+ 9.5 t 5.9* t 15.3* 2 5.3 f 4.2 -t 5.7 -c 1.4* t- 4.6** i 0.5* i- 1.0 i 0.4 -c 0.04*
58.3 143 7.7 333 174.0 251.5 88.5 197.5 235.6 180.2 46.3 130.6 30.5 97.8 5.8 17.9 7.7 0.76 --~-~~~
48 weeks
5 2.1 k 16 z 0.5 2 15 2 33.6 i 45.1 +- 18.7 5 10.1 2 16.5 2 8.8 t- 5.4 2 7.0* 2 2.2 2 5.9 t 1.5 2 4.8 + 1.3 z 1.3
58.1 166 7.6 329 161.0 214.0 81.5 194.8 215.6 185.3 47.5 131.8 33.3 98.0 5.4 15.1 6.7 0.76 .._..._ ~_
rt t -’ ‘t i t ii 2 1 -’ 5 t it z z ?
2.2 18 0.4 16 35.3** 51.2** 18.2 8.9 17.3 9.6 6.9 7.9* 1.5 6.1 1.0 2.4 0.8 0.04
EPA-E, cicosapcrltncnoic ncid; HDL, hi,@-densit!/ lipoprotci~~. Mcarl t SEM. * P i 0.05, **p < 0.07 vs. bCf0l.Pf0 weeks).
TABLE
5. CHANGES
IN PLASMA DURING
COMPOSITION OF UNSATURATED TREATMENT WITH EPA-E Before
Unsaturated
0 weeks
Fatty Acids
Linoleic acid C18:2 w6 n-Linolenic acid C18:3 03 Dihomo-y-linolenic acid C20:3 w6 Arachidonic acid C20:4 w6 (AA) Eicosapentaenoic acid C20:5 03 (EPA) Docosapentaenoic acid C22:5 03 Docosahexaenoic acid C22:6 w3 Total of 18 acids C16:O w C24:l EPA/AA AEI’A ~_~Meari t SEM.
(mol%)
(t&md ‘~Fi%; (mol%) WmL) (mol%) ‘yl%; (iq/mb (mol%) (y!:;;
0
‘“(“m’:5;i0 b.g/mL) (mol%) k/n-U -
27.80 797.2 0.94 33.0 0.90 27.6 4.30 124.0 3.02 84.7 0.88 29.0 3.94 128.4 100.00 2977.2 0.69 0.00 0.00
2 I.03 i 80.0 5 0.08 t 9.0 + 0.06 -c 2.3 -+ 0.27 -+ 5.5 i 0.47 +- 10.2 ? 0.05 + 2.5 t 0.26 i 11.2 1- 381.0 i- 0.08
FATTY ACIDS During
12 weeks 25.79 702.0 0.90 25.9 0.73 21.4 4.28 121.5 5.49 150.6 1.32 41.0 3.79 119.4 100.00 2754.8 1.30 2.46 65.8
24 weeks
f 0.75”* -c 49.5 t 0.07 +- 3.5 -+ 0.05*** +- 1.6”” + 0.25 -+ 5.9 t 0.50*** I 9.3*** i 0.07*** ? 2.7** -c 0.25 + 10.1 + + i t
197.2 0.10*** 0.34 9.1 -~~..
-.-
25.04 761.8 1.03 37.3 0.72 24.1 3.87 122.4 5.73 175.5 1.21 43.3 3.84 144.1 100.00 3166.1 1.47 2.52 86.4
t t ? t i 2 ? L ? t + 2 i t-
1.05* 86.2 0.08 8.9 0.05** 2.9% 0.24* 10.7 0.62*** 19.5*** O.OS** 5.4** 0.23 23.6
2 z 2 r
439.4 0.12*** 0.54 18.2
Changes in Fasting Plasma Glucose and HbA,,. Fast-
ing plasma glucose and the level of glycosylated globin A,, showed no significant ment with EPA-E (Table 4).
Y=-1.53X+1.20 r=-0.643 (p
0
n= 18 -301 -4
’ I 1 I 1 -2 0 2 4 6 EICOSAPENTAENOIC
I
I
1
8
10
12
ACID
mol%
09 Q 0.2 Q
u? .4 Y 0
t
o-
\
Y=-o.o5x-o.o5 r=-0.588 (p.eO.05)
l 00
n= 18
\ 0
l 0
0
5 -0.2 CL $ -0.4 -
0
g ,.,I -4
\
-2 0 2 4 6 EICOSAPENTAENOIC
r=0.557 (pcO.01)
l
I
I
I
8 10 12 ACID mol%
I
1
-2 0 2 4 6 EICOSAPENTAENOIC
I
I
8 10 12 ACID mol’%
FIGURE 2 Correlation hetzueen A eicosapentaenoic acid alzd A linoleic ucid (A), Adihomo-y-linolenic acid (B), and A docosapentaenoic mid CC).
Hemodynamic Effects of EPA-E. Hemodynamic changes are sumrnarizecl in Table 3. No significant changes were observed in blood pressure and pulse rate. However, both the cross-sectional area of the dorsalis pedis artery and blood flow index significantly increased. Changes in Body Weight. There was no significant changes in body weight of the patients (Table 4).
herno-
changes during treat-
Changes in Serum Lipids and the Plasma Composition of Unsaturated Fatty Acids. Compliance with EPA-E was confirmed with questionnaire which suggested full consumption of the prescribed regimen. Serum triglycerides (‘I’G) decreased significantly after treatment with EPA-E for 12 and 48 weeks. Total cholesterol (T-Chol) also showed a significant decrease after 12 weeks of treatment (p < 0.05). There were significant changes in TG among patients with a TG baseline value of greater than or equal to 150 mg/dL, and T-chol among those with a T-chol base line of greater than or equal to 220 mg/dL. HDI, cholesterol did not change significantly. While apolipoprotein (APO) CIII and Apo E, did not change significantly during EPA-E treatment; Apo AI, Apo AII, Apo B, and Apo CIl decreased significantly. The Apo B/APO Al ratio, which is an indicator of atherosclerosis also decreased significantly (p < 0.05) from 0.74 .+ 0.0s to 0.66 % 0.04 (12 weeks), but it returned to 0.76 +- 0.04 (48 weeks) (Table 4). Table 5 summarizes the changes in the plasma composition of unsaturated fatty acids during treatment with EPA-E. The molar ratio (mol%) of eicosapentaenoic and arachidonic acid (EPA/AA) increased significantly from 0.69 2 0.08 to 1.30 -C 0.10 (12 weeks) and 1.47 ? 0.12 (24 weeks) during EPA-E treatment (11< 0.001). While plasma Cl&3 w3 (a-linolenic acid), C22:6 w3 (docosahexaenoic acid) did not change significantly, Cl&2 w6 (linoleic acid), C20:3 w6 (dihomoy-linolenic acid), and C20:4 w6 (arachidonic acid) decreased significantly. On the other hand, C20:5 w3 (eicosapentaenoic acid) increased significantly from 3.02 z 0.47 to 5.49 -z 0.50 (12 weeks) and 5.73 ? 0.62 mol’% (24 weeks) (Table 5). There were significant correlations between A eicosapentaenoic acid and A linoleic acid (r = -O&43), A dihomo-y-linoleic acid (r = -0.588), and A docosapentaenoic acid (r = 0.557) (Figure 2A, B, and C). Renal Parameters. While serum BUN, creatinine, p2 microglobulin, and NAG (N-acetyl-P-D-glucosaminidase) did not change significantly during treatment with EPA-E, urinary albumin excretion rate, which is an indicator of early diabetic nephropathy decreased significantly (p < 0.01) (Table 6). DISCUSSION Bang and Dyerbery’? have suggested that ingestion of o-3 fatty acids may be responsible for the low incidence of cardiovascular disease, especially atherosclerosis, in a well-studied community of Greenland Eskimos. In fact, w-3 and w-6 fatty acids have been reported
/ Diab Cowlp 2996; 70:280-287
TABLE
6. CHANGES
EICOSAPENTAENOIC
IN RENAL
PARAMETERS
DURING
TREATMENT
Before Renal function
markers
Urinary components Albumin bg/gW Bz-Microglobulin (mg/gCr) NAG (U/gW Serum components BUN (mg/dL) Cr (mg/dL)
WITH
ACID
IN NIDDM
285
EPA-E
During
0 weeks
12 weeks
24 weeks
48 weeks
24.4 +- 3.3 0.26 -c 0.08 10.7 k 1.4
11.7 ? 1.0** 0.22 -+ 0.06 10.6 I 1.7
12.7 i 1.3** 0.23 5 0.04 9.7 -t 1.2
13.9 i 1.8* 0.21 -t 0.03
15.3 t 0.7 0.71 ? 0.03
14.7 ? 0.6 0.72 t 0.03
16.3 t 0.6 0.73 -+ 0.03
16.2 I 0.8 0.70 f 0.03
EPA-E, eicosapenfaenoic acid; NAG, N-acefyl-@-tqhcosanzinidase;
10.0 t 0.9
BUN, blood mea nifrogen; Cr, creafinine.
Mean + SEM. *p < 0.05, **p < 0.01 vs. before 10 weeks)
to have a wide variety of pharmacologic actions, including inhibition of platelet aggregation, reduction of thromboxane production, reduction of adhering capacity of whole blood, and increase of red cell deformability, thereby preventing microcirculation abnormalities.7JJ3 Various products containing fish-oil concentrates are now commercially available. However, the EPA used in previous studies were not highly purified (purity: less than 30%), and EPA was administered during a short-term period (less than 12 weeks).‘” Thus, these studies could not have elucidated the effects of EPA. In this study, we used a new EPA (EPA-E) for 48 weeks, which was highly purified (purity greater than 91%) by ethyl esterification. The results of this study indicated that treatment with EPA-E resulted in a clear improvement of diabetic neuropathy. This favorable effect of EPA-E is considered a consequence of the circulatory insufficiency of the lower extremities. Impaired blood rheological properties have been reported to be closely related to microcirculatory disturbances observed in patients with diabetes mellitus.‘2~‘h In this study, an increase of the crosssectional area of the dorsalis pedis artery was observed after EPA-E treatment. Thus, it is indicated that EPA-E caused the dilatation of peripheral arteries, which in turn resulted in a reduction of vascular resistance and an increase in blood flow. The biochemical mechanisms underlying these processes can be explained as follows: After EPA-E is incorporated into cell membrane in antagonism to arachidonic acid (AA), EPA is released from the membrane by the action of phospholipase Al. Then, EPA is oxygenated by cyclooxygenase and lipoxygenase in the same manner as AA. It also gives rise to prostanoids having one more double bond in the acyl side chain [i.e., prostaglandin I1 (PGI,), thromboxane A? (TXA1), PGDR, PGE?, and leukotrienes (LT) such as LT& Cs, D5, and Es], which decreases the relative amounts of eicosanoids that would otherwise be produced from AA. In addition, PG13produced from
EPA is reported to have a more potent relaxant action on vascular smooth muscles than PGIZ17(Figure 3). Fish oils have a dose-dependent blood-pressure lowering effect in healthy individuals and in patients with essenIn this study, diastolic blood prestial hypertension. ‘8~19 sure was slightly decreased after 48 weeks of treatment. EPA-E induced inhibition of platelet function resulted in an improved blood rheology and vasodilatation with subsequent improvement of small neural blood vessels. This improvement might in turn contribute to a noticeable decrease of the vibratory perception threshold, a parameter that reflects the status of deep sensory function. In addition, we have already demonstrated that EPA-E prevents the inhibition of myo-inositol uptake
Gluco\r
.. I ,Na
._ ---&
r--EPA I=Fibrmectm
t
+LNa+ ,y
AA, PYC‘t
Na*~K*ATPa\e
1
1
I
t
FIGURE 3 Hypothetical mechanisms ofEPA on endothelial cells and peripheral neurons (Ml, myo-inositol; PIP, phosphatidylinositol-4-phosphate; PIPL, phosphatidylinositol-4,5-bisphosphate; IPi, inositol-1,4,5triphosphate; DG, diacylglycerol; PKC, protein kinase C; PLA?, phospholipase A,: ------*, inhibition).
286
OKIJI>A
F’I‘ AI
induced by a high glucose concentration in cultured human endothelial cells (ECS) (20). Moreover, since EPA-E potentiated Na’-K’ ATPase activity of ECS, the mechanism of increment of myo-inositol uptake indicates that EPA-E might affect the influx of myo-inositol through the action of Na’-K’ ATPase. Thus, it is suggested that EPA-E might ameliorate diabetic neuropathy by increasing the content of myo-inositol. On the other hand, long chain polyunsaturated fatty acids also form key structural components of the neural membrane21 23and influence membrane functions (including fluidity,‘.% the behavior of several membrane-associated enzymes and receptors) and the properties of myelin.2“,2i In addition, we have demonstrated that EPA inhibits fibronectin production induced by high glucose2h(Figure 3). The molar ratio (mol %) of eicosapentaenoic acid and arachidonic acid (EPA/AA) increased significantly in patients with NIDDM treated with EPA-E. In addition, the level of docosapentaenoic acid increased in response to EPA-E treatment (Table 5). Thus, compliance was considered to be good. As shown in Figure 2, there were significant inverse correlations between A eicosapentaenoic acid and A linoleic and (Y = -0.643), and A dihomo-y-linolenic acid (Y = -0.588). Linoleic acid is metabolized along a variety of pathways, one of which is its conversion to y-linolenic acid by A-6-desaturase. y-Linoleic acid is rapidly elongated to dihomo-y-linoleic acid which is subsequently desaturated by A-5-desaturase to arachidonic acid. It has been demonstrated that EPA strongly interferes with the metabolism of linoleic acid and dihomo-y-linolenic acid. The A-5-desaturase step converting dihomoy-linoleic acid to arachidonic acid is a rate-limiting step. Previous reports have shown that w-3 fatty acids inhibit the metabolism of o-6 acids, especially at the desaturation step.?7,28 A significant lowering of serum triglycerides was also noted during EPA-E administration. Similar results have been reported by several investigators.28”’ Although the exact mechanisms of the hypolipidemic effects of EPA have not been determined yet, a reduced rate of hepatic synthesis of very-low-density lipoproteins (VLDL) and the inhibition of the synthesis of VLDL-TG and VLDL apoprotein B have been thought to be the most likely causes8 EPA-E may prevent vascular disorders by reducing platelet aggregation and counteract thrombosis by virtue of its vasodilating action and its effect on lipids. Treatment with EPA-E was also followed by a reduction of urinary excretion of albumin, an index of early diabetic nephropathy, which suggests that EPA-E might also be effective in diabetic nephropathy, another manifestation of diabetic microangiopathy. In summary, the results of this study suggest that EPA-E might save as a useful treatment for diabetic
neuropathy and altered serum lipids composition well as other diabetic complications.
as
ACKNOWLEDGMENT The authors wish to thank Miss Y’oko Yokokura for technical assistance. This study was supported in part by a grant from the JapaneseMinistry of Education, Science and Culture, and another from the University
of Tsukuba Project Research.
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Malasanos TH, Stacpoole PW: Biological effects of w-3 fatty acids in diabetes mellitus. Diabetes Care 14:116@1179, 19Yl.
EICOSAPENTAENOIC
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Cohen MI’, Urdanivia E, Surma M, Ciborowski J: Nonenzymatic glycosylation of basement membranes. In vitro studies. Diabetes 30:367-371, 1981.
16.
Barnes AJ, Locke I?, Scudder PR, Dormandy JA, Slack J: Is hyperviscosity a treatable component of diabetic microcirculatory disease? Lancef ii:789-791, 1977.
17.
Shibata N: Clinical application of EPA preparations. lschemic heart disease. Biomed Tker 25:79-88, 1991.
18.
Mortensen JZ, Schmidt EB, Nielsen Arl, Dyerberg J: The effect of N-6 and N-3 polyunsaturated fatty acids on hemostasis, blood lipids and blood pressure. Thromb Haernost 50543-546, 1983.
19.
Bonaa KH, Bjerve KS, Straume B, Gram IT, Thelle D: Effect of eicosapentaenoic and docosahexaenoic acids on blood pressure in hypertension: A population-based intervention trial from the Tromso study. N Engl ] Med 322:795-801, 1990.
20.
Okuda Y, Mizutani M, Tsukahara K, Yamashita K: Eicosapentaenoic acid prevents the inhibition of myo-inosito1 uptake induced by high glucose concentration in cultured human endotheliae cells. Horm Metab Res 25:127-128, 1993.
21.
Holman RT: Essential fatty acids in nutritional Ckcrn lnd 5:704-709, 1981.
22.
Sinclair HM: Low phosphate arachidonic acid values in diabetic platelets [Letter]. BMJ 286:648, 1983.
23.
Lin CJ, Peterson T, Eichberg J: The fatty acid composition of glycerolipids in nerve, brain and other tissues of the streptozotocin diabetic rat. Neuvockem Res 10: 1453-1465, 1985.
ACID
IN NIDDM
287
24.
Elliasson SG: Lipid synthesis in the peripheral nerves from alloxan diabetic rats. Lipids 1:237-240, 1966.
25.
Tilvis RS, Miettinen TA: Fatty acid compositions of serum lipids, red cells and platelets in insulin-dependent diabetic women. J Clin Endocrinol Metab 61:741-745,1985.
26.
Mizutani M, Okuda Y, Yamashita K: High glucose causes dysfunction in human vascular endothelial cells and preventive effects of eicosapentaenoic acids, in Yasugi T, Nakamura H, Soma M (ed), Advances in Polyunsaturated Fatty Acid Research. New York, Elsevier, 1993, pp. 201-202.
27. Juan H, Sametz W: Dihomo-y-Linolenic acid increases the metabolism of eicosapentaenoic acid in perfused vascular tissue. Prost Leuk Med 19:79-86, 1985. 28.
Nassar BA, Huang YS, Manku MS, Das UN, Morse N, Horrobin H: The influence of dietary manipulation with n-3 and n-6 fatty acids on liver and plasma phospholipid fatty acids in rats. Lipids 21:652-656, 1986.
29.
Illingworth DR, Connor WE, Harris WS, Goodnight SH: The influence of dietary w-3 fatty acids on plasma lipids and lipoproteins in human: Efficacy and mechanism. Reading Univevsify Conference on N3 Fatty acids. London, 1984, pp. 63.
30.
Harris WS, Connor WE, Inkeles SB, Illingworth DR: Dietary o-3 fatty acids prevent carbohydrate-induced hypertriglyceridemia. Metabolism 33:1016-1019, 1984.
31.
Simons LA, Hickie JB, Balasubramaniam S: On the effects of dietary n-3 fatty acids (MaxEPA) on plasma lipids and lipoproteins in patients with hyperlipidaemia. Atherosclerosis 54:75-78, 1985.
disease.
ABSTRACT The present study was undertaken to investigate the efficacy of a new, highly purified (purity greater than 91%), ethyl esterification product from natural eicosapentaenoic acid (EPA-E, C20:5 03) in patients with type II diabetes mellitus (NIDDM). Wemodynamic changes were assessed at the level of the dorsalis pedis artery using an ultrasonic color Doppler duplex system before and after oral administration of EPA-E at a dose of 1800 mg/day for 48 weeks. The cross-sectional area of the dorsalis pedis artery increased significantly from 2.5 t 0.2 to 3.9 2 0.4 mm* (48 weeks, mean + SE, p < 0.05). Moreover, EPA-E improved the clinical symptom (coldness, numbness) as well as the
INTRODUCTION yperglycemia and the metabolic derangements of sorbitol and myo-inositol associated with it diabetic neuropathy.‘” On the other hand, there is strong evidence of
..-~. Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan, and Division of Endocrinology and Metabolism (J.B.F.) Department of Medicine, Diabetes Research Laboratory, St. Luke’s Episcopal Hospital, Baylor College of Medicine, Houston, Texas, USA. Reprint requests to be sent to: Dr. Yukichi Okuda, Department of Internal Medicine, Institute of CIinical Medicine, University of Tsukuba, Tennodai l-l-l, Tsukuba, Ibaraki 305, Japan. jooumal of Diabetes and Its Complications 20:280-287 0 Elsevier Science Inc., 1996 655 Avenue of the Americas, New York, NY 10010
vibration perception threshold sense of the lower extremities [from 32.1 ? 8.5 to 16.1 +- 4.8 (48 weeks) pm]. A significant decrease of serum triglycerides was also noted by EPA-E administration. Furthermore, significant decrease of the excretion of albumin in urine Ifrom 24.4 + 3.3 to 13.9 2 1.8 (48 weeks) mg&Cr, 77< 0.051.The results of this study suggest that EPA-E has significant beneficial effects on diabetic neuropathy and serum lipids as well as other diabetic complications such as nephropathy and macroangiopathy. (Journal of Diabetes and Its Complications 10; 5:28+287,1996.)
vascular implication in the pathogenesis of diabetic mononeuropathy and polyneuropathy.t6 Eicosapentaenoic acid (EPA, C20:5 w3), a polyunsaturated fatty acid of the o-3 series extracted from fish oil, has been reported to inhibit plateIet aggregation, improve serum lipid and have an antithrombotic action.7,* Recently, a highly pure EPA (purity greater than 91%) has been obtained by ethyl esterification of natural EPA (ethyl all-cis-5,8,11,14,17-eicosapentaenoate, EPA-E; EpadelB Manufacturer: Mochida Pharmaceutical Co., Ltd., Tokyo, Japan).9,‘0 The present study was undertaken to investigate the efficacy of EPA-E on diabetic peripheral neuropathy and its effects on serum lipids in patients with noninsulin-dependent (type II) diabetes mellitus (NIDDM). 1056-S727/96/$15.00 SSDI 1056.8727(95)00081-C
1 Did
Camp 1996; 20:280-287
EICOSAPENTAENOIC
TABLE 1. CLINICAL FEATURES OF 21 NON-INSULIN-DEPENDENT DIABETIC PATIENTS (N = 21) 13 8
40-29 50-59 60-69 70-79
2 3 12 4
IN NIDDM
281
-**I
(A)
Gender Male Female Age (year)
ACID
Body-mass index (kg/m*) G-19 20-22 23-25 26-29 a30
Duration
of diabetes (year)
s5 6-10
311 Duration
5 8 8
of nephropathy
I
I
4
12
24
48 wk
(year)
s2
5
34
11
z5
5
Treatment Diet OH-4 Insulin Hemoglobin Good
1
0
12
(B)
SCORE -**1 -**I 3
7 2
A,, (X,) 11
s7.0
Fair 7.1-8.9
6
Poor 29.0
4
Retinopathy C-1 Scott Ia IIa IIIa Nephropathy C-1 Early Overt AS0 C-1 (+) OHA, on?/ Izp~&m~ic
RESEARCH
8 7 5
1 5 16
I
I
I
I
0
12
24
48 wk
0 0
FIGURE 1 Chnnges in symptom score (A) Coldness, (B) Nmbness; Score: 3, severe; 2, moderate; I, sligirt; 0, nlxmf *“p c: 0.01.
21 qgerzts; ASO, arteriosclerosis obliterans.
DESIGN
AND
METHODS
Patients. Twenty-one patients with NIDDM and chronic peripheral diabetic neuropathy were entered into this study. The clinical characteristics of these patients are listed in Table 1. The mean age of the study population was 62.5 + 1.8 years, the mean duration of diabetes was 10.0 + 1.3 and the mean duration of neuropathy was 3.5 +- 0.4 years. They all had clinically evident bilateral peripheral somatic neuropathy in the
lower extremities. All of them had shown characteristic symptoms (e.g., burning pains, especially at night or while resting, numbness, coldness) and signs (e.g., reduced sensation, absent tendon reflexes) for at least 10 months. All patients had arteriosclerosis obliterans (ASO) as determined by Doppler echography, digital subtraction angiography, and absent foot pulses. None of them had severe cardiovascular or renal diseases, or had nephropathy due to alternative causes such as alcohol dependence or nerve entrapment syndrome were excluded. All patients gave their informed consent to the study, which was conducted according to the guidelines expressed in the Declaration of Helsinki.
TABLE 2. CHANGES
IN VIBRATORY PERCEPTION DURING TREATMENT Before -. 0 weeks
VPT Tibia (km) Ulna (km)
THRESHOLD (VPT) OF TIBIA WITH EPA-E During
32.1 2 8.5 13.3 r 1.4 _----... ..- -
..-
AND ULNA
_..
12 weeks
24 weeks
48 weeks
19.1 2 3.6* Y.5 2 0.4* ---
22.1 : 6.P+ Y.6 210.7
16.1 x 4.8** 9.8 - 2.y
EPA-E, eicosapmiaenoic acid. Mea11 L SEM. *p < 0.05, ** p < 0.07 us. before (0 zuerksi.
Study Design and Treatment. EPA-E (EpadelB, obtained from the pharmacy at the University of Tsukuba hospital, batch number 1017) was administered orally at a dose of 1800 mg; 21.8 kcal/day, in three divided doses, immediately after meals, over a period of 48 weeks. The calories of EPA-E (21.8 kcal/day) were added to existing diet of each patients. Blood pressure and pulse rate were measured before, and 12, 24, and 48 weeks after the administration of EPA-E. Prior to the examination, all subjects had to rest in the supine position for 30 min. The critical measurements were the cross-sectional area of the dorsalis pedis artery and the blood flow index, which were assessed noninvasively using a new ultrasonic color Doppler duplex system (Toshiba SSA-270A, Toshiba Co., Yokohama, Japan).” This system consists of a B-mode scanner and an ultrasonic range-gated pulsed Doppler echography. The blood flow index was obtained by multiplying the mean blood flow velocity by the cross-sectional area of the dorsalis pedis artery. The blood velocity and cross-sectional area of the artery were the mean of two or more measurements taken after allowing the patients to rest for 20 min in the supine position at room temperature (25”-27°C). The vibratory perception threshold (VET), as an indicator of sensory disturbance, was measured at both the medial malleolus of the tibia and styloid process of both ulnae using a vibratory sensation meter model TM-31 (Medic
TABLE
3. HEMODYNAMICS
International Co., Tokyo, Japan). Neurological subjective symptoms (coldness, numbness) were classified into four levels in increasing order of severity (0.1.2.3). At 0, 12, 24, and 48 weeks, venous blood was obtained from all patients before breakfast to determine fasting plasma glucose, hemoglobin (Hb) A,,, serum lipids (triglyceride, total cholesterol, high-density lipoprotein (HDL) cholesterol, apolipoproteins), blood urea nitrogen (BUN), creatinine (Cr), and the plasma composition of unsaturated fatty acids (not determined at 48 weeks). Urine samples were also collected to determine albuminuria. Data Analysis and Statistical Methods. All data in this paper are presented as the mean + SEM and were analyzed for statistical significance by the paired f test or Wilcoxon test. A p value of 0.05 or less was considered significant. RESULTS Effect on Clinical Symptoms. EPA-E significantly improved clinical symptoms such as coldness and numbness (p < 0.01, Wilcoxon test) (Figure 1). Measurement of the Vibratory Perception Threshold. VM was significantly improved at both arm and leg during treatment with EPA-E (Table 2). Improvement was detected by 12 weeks of treatment but showed no further progression thereafter.
DURING
TREATMENT -
Before
Blood pressure Systolic (mm Hg) Diastolic (mm Hg) Pulse rate (beats/min) Area (mm*) Blood flow index
-
0 weeks
12 weeks
129 73 73 2.3 17
128 72 71 3.4 25
EPA-E, eicosapenfaenoic acid. Mean +- SEM. “p < 0.05, ““p < 0.01 ZIS.before (0 wwks).
‘+ 3 +- 3 ? 2 -c 0.2 -c 2 -
= -t ? ? -t
3 2 3 0.3** 4**
.
WITH EPA-E . During .--____ 24 weeks 128 72 69 3.3 34
z ? Ifi 2 k
3 2 2 0.3* 3**
-
. 48 weeks 126 68 74 3.9 46
rt z It t 2
2 2 2 0.4* 6”*
/ Did? Camp 7996; 10:280-287
EICOSAPENTAENOIC
TABLE 4. LABORATORY
DATA
BEFORE AND DURING
TREATMENT
WITH
Before No. of Patients Body weight Plasma glucose Hemoglobin A,,. Fructosamine Triglyceride 2 150 mg/dL < 150 mg/dL Total cholesterol 2 220 mg/dL < 220 mg/dL HDL cholesterol Apolipoprotein AI AI1 B CII CIII
(kg) (mg/dL) (‘L) (wol/L) (mg/W (WdL) (mg/dL) (mg/dL) I”$Y
0 weeks 58.0 138 7.3 326 182.8 263.0 84.6 196.6 235.0 183.8 50.6 141.2 31.3 103.7 5.8 12.9
21
21 21 21 21 11 10 21 6 15 21 21 21 21 21 21
(:g/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL)
0.74 5.9 -+ + 0.5 0.05
21
:/A:mg’dL)
2 2.1 F 10 F 0.3 + 13 -+ 33.8 2 48.9 + 13.4 2 6.7 +- 6.9 2 5.6 2 3.6 t 6.0 +- 1.6 -c 6.7 i 1.2 t 1.6
ACID
283
IN NIDDM
EPA-E
During 12 weeks 58.0 147 7.3 328 134.1 174.9 70.0 185.1 210.0 178.4 50.4 138.7 29.3 89.1 4.1 12.4 5.4 0.66
24 weeks
2 2.2 2 13 i- 0.3 f- 11 k 22.2* -t 30.2* -+ 9.5 t 5.9* t 15.3* 2 5.3 f 4.2 -t 5.7 -c 1.4* t- 4.6** i 0.5* i- 1.0 i 0.4 -c 0.04*
58.3 143 7.7 333 174.0 251.5 88.5 197.5 235.6 180.2 46.3 130.6 30.5 97.8 5.8 17.9 7.7 0.76 --~-~~~
48 weeks
5 2.1 k 16 z 0.5 2 15 2 33.6 i 45.1 +- 18.7 5 10.1 2 16.5 2 8.8 t- 5.4 2 7.0* 2 2.2 2 5.9 t 1.5 2 4.8 + 1.3 z 1.3
58.1 166 7.6 329 161.0 214.0 81.5 194.8 215.6 185.3 47.5 131.8 33.3 98.0 5.4 15.1 6.7 0.76 .._..._ ~_
rt t -’ ‘t i t ii 2 1 -’ 5 t it z z ?
2.2 18 0.4 16 35.3** 51.2** 18.2 8.9 17.3 9.6 6.9 7.9* 1.5 6.1 1.0 2.4 0.8 0.04
EPA-E, cicosapcrltncnoic ncid; HDL, hi,@-densit!/ lipoprotci~~. Mcarl t SEM. * P i 0.05, **p < 0.07 vs. bCf0l.Pf0 weeks).
TABLE
5. CHANGES
IN PLASMA DURING
COMPOSITION OF UNSATURATED TREATMENT WITH EPA-E Before
Unsaturated
0 weeks
Fatty Acids
Linoleic acid C18:2 w6 n-Linolenic acid C18:3 03 Dihomo-y-linolenic acid C20:3 w6 Arachidonic acid C20:4 w6 (AA) Eicosapentaenoic acid C20:5 03 (EPA) Docosapentaenoic acid C22:5 03 Docosahexaenoic acid C22:6 w3 Total of 18 acids C16:O w C24:l EPA/AA AEI’A ~_~Meari t SEM.
(mol%)
(t&md ‘~Fi%; (mol%) WmL) (mol%) ‘yl%; (iq/mb (mol%) (y!:;;
0
‘“(“m’:5;i0 b.g/mL) (mol%) k/n-U -
27.80 797.2 0.94 33.0 0.90 27.6 4.30 124.0 3.02 84.7 0.88 29.0 3.94 128.4 100.00 2977.2 0.69 0.00 0.00
2 I.03 i 80.0 5 0.08 t 9.0 + 0.06 -c 2.3 -+ 0.27 -+ 5.5 i 0.47 +- 10.2 ? 0.05 + 2.5 t 0.26 i 11.2 1- 381.0 i- 0.08
FATTY ACIDS During
12 weeks 25.79 702.0 0.90 25.9 0.73 21.4 4.28 121.5 5.49 150.6 1.32 41.0 3.79 119.4 100.00 2754.8 1.30 2.46 65.8
24 weeks
f 0.75”* -c 49.5 t 0.07 +- 3.5 -+ 0.05*** +- 1.6”” + 0.25 -+ 5.9 t 0.50*** I 9.3*** i 0.07*** ? 2.7** -c 0.25 + 10.1 + + i t
197.2 0.10*** 0.34 9.1 -~~..
-.-
25.04 761.8 1.03 37.3 0.72 24.1 3.87 122.4 5.73 175.5 1.21 43.3 3.84 144.1 100.00 3166.1 1.47 2.52 86.4
t t ? t i 2 ? L ? t + 2 i t-
1.05* 86.2 0.08 8.9 0.05** 2.9% 0.24* 10.7 0.62*** 19.5*** O.OS** 5.4** 0.23 23.6
2 z 2 r
439.4 0.12*** 0.54 18.2
Changes in Fasting Plasma Glucose and HbA,,. Fast-
ing plasma glucose and the level of glycosylated globin A,, showed no significant ment with EPA-E (Table 4).
Y=-1.53X+1.20 r=-0.643 (p
0
n= 18 -301 -4
’ I 1 I 1 -2 0 2 4 6 EICOSAPENTAENOIC
I
I
1
8
10
12
ACID
mol%
09 Q 0.2 Q
u? .4 Y 0
t
o-
\
Y=-o.o5x-o.o5 r=-0.588 (p.eO.05)
l 00
n= 18
\ 0
l 0
0
5 -0.2 CL $ -0.4 -
0
g ,.,I -4
\
-2 0 2 4 6 EICOSAPENTAENOIC
r=0.557 (pcO.01)
l
I
I
I
8 10 12 ACID mol%
I
1
-2 0 2 4 6 EICOSAPENTAENOIC
I
I
8 10 12 ACID mol’%
FIGURE 2 Correlation hetzueen A eicosapentaenoic acid alzd A linoleic ucid (A), Adihomo-y-linolenic acid (B), and A docosapentaenoic mid CC).
Hemodynamic Effects of EPA-E. Hemodynamic changes are sumrnarizecl in Table 3. No significant changes were observed in blood pressure and pulse rate. However, both the cross-sectional area of the dorsalis pedis artery and blood flow index significantly increased. Changes in Body Weight. There was no significant changes in body weight of the patients (Table 4).
herno-
changes during treat-
Changes in Serum Lipids and the Plasma Composition of Unsaturated Fatty Acids. Compliance with EPA-E was confirmed with questionnaire which suggested full consumption of the prescribed regimen. Serum triglycerides (‘I’G) decreased significantly after treatment with EPA-E for 12 and 48 weeks. Total cholesterol (T-Chol) also showed a significant decrease after 12 weeks of treatment (p < 0.05). There were significant changes in TG among patients with a TG baseline value of greater than or equal to 150 mg/dL, and T-chol among those with a T-chol base line of greater than or equal to 220 mg/dL. HDI, cholesterol did not change significantly. While apolipoprotein (APO) CIII and Apo E, did not change significantly during EPA-E treatment; Apo AI, Apo AII, Apo B, and Apo CIl decreased significantly. The Apo B/APO Al ratio, which is an indicator of atherosclerosis also decreased significantly (p < 0.05) from 0.74 .+ 0.0s to 0.66 % 0.04 (12 weeks), but it returned to 0.76 +- 0.04 (48 weeks) (Table 4). Table 5 summarizes the changes in the plasma composition of unsaturated fatty acids during treatment with EPA-E. The molar ratio (mol%) of eicosapentaenoic and arachidonic acid (EPA/AA) increased significantly from 0.69 2 0.08 to 1.30 -C 0.10 (12 weeks) and 1.47 ? 0.12 (24 weeks) during EPA-E treatment (11< 0.001). While plasma Cl&3 w3 (a-linolenic acid), C22:6 w3 (docosahexaenoic acid) did not change significantly, Cl&2 w6 (linoleic acid), C20:3 w6 (dihomoy-linolenic acid), and C20:4 w6 (arachidonic acid) decreased significantly. On the other hand, C20:5 w3 (eicosapentaenoic acid) increased significantly from 3.02 z 0.47 to 5.49 -z 0.50 (12 weeks) and 5.73 ? 0.62 mol’% (24 weeks) (Table 5). There were significant correlations between A eicosapentaenoic acid and A linoleic acid (r = -O&43), A dihomo-y-linoleic acid (r = -0.588), and A docosapentaenoic acid (r = 0.557) (Figure 2A, B, and C). Renal Parameters. While serum BUN, creatinine, p2 microglobulin, and NAG (N-acetyl-P-D-glucosaminidase) did not change significantly during treatment with EPA-E, urinary albumin excretion rate, which is an indicator of early diabetic nephropathy decreased significantly (p < 0.01) (Table 6). DISCUSSION Bang and Dyerbery’? have suggested that ingestion of o-3 fatty acids may be responsible for the low incidence of cardiovascular disease, especially atherosclerosis, in a well-studied community of Greenland Eskimos. In fact, w-3 and w-6 fatty acids have been reported
/ Diab Cowlp 2996; 70:280-287
TABLE
6. CHANGES
EICOSAPENTAENOIC
IN RENAL
PARAMETERS
DURING
TREATMENT
Before Renal function
markers
Urinary components Albumin bg/gW Bz-Microglobulin (mg/gCr) NAG (U/gW Serum components BUN (mg/dL) Cr (mg/dL)
WITH
ACID
IN NIDDM
285
EPA-E
During
0 weeks
12 weeks
24 weeks
48 weeks
24.4 +- 3.3 0.26 -c 0.08 10.7 k 1.4
11.7 ? 1.0** 0.22 -+ 0.06 10.6 I 1.7
12.7 i 1.3** 0.23 5 0.04 9.7 -t 1.2
13.9 i 1.8* 0.21 -t 0.03
15.3 t 0.7 0.71 ? 0.03
14.7 ? 0.6 0.72 t 0.03
16.3 t 0.6 0.73 -+ 0.03
16.2 I 0.8 0.70 f 0.03
EPA-E, eicosapenfaenoic acid; NAG, N-acefyl-@-tqhcosanzinidase;
10.0 t 0.9
BUN, blood mea nifrogen; Cr, creafinine.
Mean + SEM. *p < 0.05, **p < 0.01 vs. before 10 weeks)
to have a wide variety of pharmacologic actions, including inhibition of platelet aggregation, reduction of thromboxane production, reduction of adhering capacity of whole blood, and increase of red cell deformability, thereby preventing microcirculation abnormalities.7JJ3 Various products containing fish-oil concentrates are now commercially available. However, the EPA used in previous studies were not highly purified (purity: less than 30%), and EPA was administered during a short-term period (less than 12 weeks).‘” Thus, these studies could not have elucidated the effects of EPA. In this study, we used a new EPA (EPA-E) for 48 weeks, which was highly purified (purity greater than 91%) by ethyl esterification. The results of this study indicated that treatment with EPA-E resulted in a clear improvement of diabetic neuropathy. This favorable effect of EPA-E is considered a consequence of the circulatory insufficiency of the lower extremities. Impaired blood rheological properties have been reported to be closely related to microcirculatory disturbances observed in patients with diabetes mellitus.‘2~‘h In this study, an increase of the crosssectional area of the dorsalis pedis artery was observed after EPA-E treatment. Thus, it is indicated that EPA-E caused the dilatation of peripheral arteries, which in turn resulted in a reduction of vascular resistance and an increase in blood flow. The biochemical mechanisms underlying these processes can be explained as follows: After EPA-E is incorporated into cell membrane in antagonism to arachidonic acid (AA), EPA is released from the membrane by the action of phospholipase Al. Then, EPA is oxygenated by cyclooxygenase and lipoxygenase in the same manner as AA. It also gives rise to prostanoids having one more double bond in the acyl side chain [i.e., prostaglandin I1 (PGI,), thromboxane A? (TXA1), PGDR, PGE?, and leukotrienes (LT) such as LT& Cs, D5, and Es], which decreases the relative amounts of eicosanoids that would otherwise be produced from AA. In addition, PG13produced from
EPA is reported to have a more potent relaxant action on vascular smooth muscles than PGIZ17(Figure 3). Fish oils have a dose-dependent blood-pressure lowering effect in healthy individuals and in patients with essenIn this study, diastolic blood prestial hypertension. ‘8~19 sure was slightly decreased after 48 weeks of treatment. EPA-E induced inhibition of platelet function resulted in an improved blood rheology and vasodilatation with subsequent improvement of small neural blood vessels. This improvement might in turn contribute to a noticeable decrease of the vibratory perception threshold, a parameter that reflects the status of deep sensory function. In addition, we have already demonstrated that EPA-E prevents the inhibition of myo-inositol uptake
Gluco\r
.. I ,Na
._ ---&
r--EPA I=Fibrmectm
t
+LNa+ ,y
AA, PYC‘t
Na*~K*ATPa\e
1
1
I
t
FIGURE 3 Hypothetical mechanisms ofEPA on endothelial cells and peripheral neurons (Ml, myo-inositol; PIP, phosphatidylinositol-4-phosphate; PIPL, phosphatidylinositol-4,5-bisphosphate; IPi, inositol-1,4,5triphosphate; DG, diacylglycerol; PKC, protein kinase C; PLA?, phospholipase A,: ------*, inhibition).
286
OKIJI>A
F’I‘ AI
induced by a high glucose concentration in cultured human endothelial cells (ECS) (20). Moreover, since EPA-E potentiated Na’-K’ ATPase activity of ECS, the mechanism of increment of myo-inositol uptake indicates that EPA-E might affect the influx of myo-inositol through the action of Na’-K’ ATPase. Thus, it is suggested that EPA-E might ameliorate diabetic neuropathy by increasing the content of myo-inositol. On the other hand, long chain polyunsaturated fatty acids also form key structural components of the neural membrane21 23and influence membrane functions (including fluidity,‘.% the behavior of several membrane-associated enzymes and receptors) and the properties of myelin.2“,2i In addition, we have demonstrated that EPA inhibits fibronectin production induced by high glucose2h(Figure 3). The molar ratio (mol %) of eicosapentaenoic acid and arachidonic acid (EPA/AA) increased significantly in patients with NIDDM treated with EPA-E. In addition, the level of docosapentaenoic acid increased in response to EPA-E treatment (Table 5). Thus, compliance was considered to be good. As shown in Figure 2, there were significant inverse correlations between A eicosapentaenoic acid and A linoleic and (Y = -0.643), and A dihomo-y-linolenic acid (Y = -0.588). Linoleic acid is metabolized along a variety of pathways, one of which is its conversion to y-linolenic acid by A-6-desaturase. y-Linoleic acid is rapidly elongated to dihomo-y-linoleic acid which is subsequently desaturated by A-5-desaturase to arachidonic acid. It has been demonstrated that EPA strongly interferes with the metabolism of linoleic acid and dihomo-y-linolenic acid. The A-5-desaturase step converting dihomoy-linoleic acid to arachidonic acid is a rate-limiting step. Previous reports have shown that w-3 fatty acids inhibit the metabolism of o-6 acids, especially at the desaturation step.?7,28 A significant lowering of serum triglycerides was also noted during EPA-E administration. Similar results have been reported by several investigators.28”’ Although the exact mechanisms of the hypolipidemic effects of EPA have not been determined yet, a reduced rate of hepatic synthesis of very-low-density lipoproteins (VLDL) and the inhibition of the synthesis of VLDL-TG and VLDL apoprotein B have been thought to be the most likely causes8 EPA-E may prevent vascular disorders by reducing platelet aggregation and counteract thrombosis by virtue of its vasodilating action and its effect on lipids. Treatment with EPA-E was also followed by a reduction of urinary excretion of albumin, an index of early diabetic nephropathy, which suggests that EPA-E might also be effective in diabetic nephropathy, another manifestation of diabetic microangiopathy. In summary, the results of this study suggest that EPA-E might save as a useful treatment for diabetic
neuropathy and altered serum lipids composition well as other diabetic complications.
as
ACKNOWLEDGMENT The authors wish to thank Miss Y’oko Yokokura for technical assistance. This study was supported in part by a grant from the JapaneseMinistry of Education, Science and Culture, and another from the University
of Tsukuba Project Research.
REFERENCES 1. Gabbay KH: The sorbitol pathway and the complications of diabetes. N Et@ 7 Med 288:831-836, 1973. 2.
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