Plasma lipid lowering effects of wheat germ in ... - Springer Link

1 downloads 0 Views 899KB Size Report
Printed in the Netherlands. Plasma lipid lowering effects of wheat germ in hypercholesterolemic subjects. LOUIS CARA, PATRICK BOREL, MARTINE ARMAND,.
Plant Foods for Human Nutrition 41: 135-150, 1991. 9 1991 Kluwer Academic Publishers. Printed in the Netherlands.

135

Plasma lipid lowering effects of wheat germ in hypercholesterolemic subjects LOUIS CARA, PATRICK

BOREL, MARTINE

ARMAND,

M I C H E L E S E N F T , H U G U E T T E L A F O N T , H E N R I P O R T U G A L ~, A N N E - M A R I E P A U L I I, D A N I E L E B O U L Z E 2, CHRISTIANNE LACOMBE 3 & DENIS LAIRON* UnitO 130-INSERM (Institut National de la Santo et de la Recherche Mddicale), 18 Avenue Mozart, F-13009 Marseille, France (*author for correspondence)," I Laboratoire Central, Hopital Ste- Marguerite, F-131~09 Marseille ; 2Ecole de DidtOtique, F-31400 Toulouse," 3UnitO 317-INSERM, Institut de Physiologie, UniversitO Paul Sabatier, F-31400 Toulouse, France Received 10 June 1990; accepted 19 October 1990

Key words: human, hypercholesterolemia, hypertriglyceridemia, serum lipoproteins, wheat germ Abstract. The present study was performed to investigate the possible effects of wheat germ supplementation on lipid metabolism in humans. Ten free-living adult subjects participated in the study. None was obese or diabetic. They all presented an hypercholesterolemia (from 6.58 to 9.50 raM), associated in 6 over 10 cases to an hypertriglyceridemia (from 1.70 to 5.00 raM). The subjects were studied in three consecutive periods, during which they first were on their usual diet (first week), they then ingested a daily supplement of 30 g wheat germ (4 weeks) and then they returned to their usual basal diet (4 weeks follow-up). Dietary records were obtained for 7 and 3 consecutive days before and during wheat germ supplementation, respectively. Fasting blood samples were taken at the end of each period. After 4 weeks of wheat germ intake, glycemia did not change while total plasma cholesterol significantly decreased (paired Student's t test, p ~< 0.05) from 7.80 to 7.15 mM. LDL and HDL cholesterol values did not show marked changes, but VLDL cholesterol significantly dropped by 40.6%. Thus, the plasma/HDL total cholesterol ratio was significantly lower. Apoprotein B and A1 decreased. In the hypertriglyceridemic subjects, this was accompanied by a significant reduction of plasma triglycerides (1.64 vs. 2.68 mM) and a marked drop of VLDL triglycerides ( - 5 1 % ) . Taken as a whole, the present results obtained in humans are very close to those previously obtained in the rat [40] and point out that wheat germ may play a beneficial role in the dietary management of hyperlipidemia.

Introduction A c c o r d i n g to several e p i d e m i o l o g i c a l studies, the c o n c e p t t h a t a high d i e t a r y fibre i n t a k e m a y p r o t e c t a g a i n s t c o r o n a r y h e a r t disease b e c o m e s m o r e a n d * Part of the data communicated in this paper was presented at the Fibre 90 Conference, 17-20 April 1990, Norwich, UK.

136 more plausible [31, 35, 36, 42, 44]. It is generally assumed that hypercholesterolemia and especially elevated LDL and VLDL-cholesterol [47, 64], hypertriglyceridemia [1, 11, 18] and hypertension [1] are major risk factors for cardiovascular diseases. In fact, many studies have been devoted in the past 15 years to experimentally evaluating whether or not fractions rich in dietary fibres possess some lipid lowering properties in laboratory animals [34] and in humans [4]. To summarize, it seems that purified soluble fibres, i.e. pectins, guar gums or some food fractions rich in soluble fibres such as oat bran or legume seeds may decrease blood cholesterol, and especially LDL-cholesterol, whereas these fibres sources have generally no effect on triglycerides. Conversely, wheat fibre sources such as wheat bran have variable effects on blood cholesterol while exhibiting more marked effects on plasma triglycerides and triglyceride-rich lipoproteins. These different metabolic effects may arise from differences in their mechanisms of action due to the chemical composition, the physico-chemical properties and the possible presence of additional compounds in the fiber-rich fractions tested. In addition to wheat bran, another by-product of wheat milling is wheat germ, which contains about 8-12% total dietary fibres, most of them being hemi-celluloses and cellulose as in wheat bran [16]. We have already shown in the adult rat [40] that the addition of 7% wheat germ to a high fatcholesterol diet significantly decreased the VLDL-cholesterol and the VLDL-triglycerides and increased the HDL-cholesterol after 7 weeks feeding. Wheat germ could alter the processing of dietary fat in the stomach and the small intestine as shown in vivo in the rat, thus decreasing the intestinal uptake of dietary lipids and cholesterol [6]. This may result from an inhibition of lipases [38, 39] and protein components associated to fibres might play a key role in this inhibition [6, 7]. Another possibility is that wheat germ directly acts on the intestinal uptake of cholesterol, as observed in the rat [9]. Given the beneficial effects previously observed in the rat on the liver and blood parameters, the present study was performed in hypercholesterolemic subjects in view to experiment the effects of a 30 g daily intake of wheat germ on various parameters of lipid metabolism.

Subjects and methods

Subjects Ten adult volunteers participated in the study after giving informed consent to a protocol approved by the local Human Ethics Committee (Medical Ethics Committee, Regional University Hospital Center, Marseille). With

137 Table 1. Characteristics of the subjects on admission to the study Subject

1 2 3 4 5 6 7 8 9 10

Age (years)

Sex

60 35 60 64 62 52 68 54 53 38

F F F F F F M F F M

BMI a (W/H 2)

Fasting plasma levelsb Cholesterol (mM)

Triglycerides c (mM)

Glucose (mM)

25.8 19.2 27.2 22.9 30.0 22.5 25.0 23.4 23.9 22.0

8.02 7.61 9.46 7.37 9.50 6.58 7.00 7.54 6.70 8.25

1.90* 0.99 2.27* 1.70" 5.00* 0.79 1.32 1.99" 0.71 3.23*

4.91 5.02 5.80 4.83 5 67 4.62 5.53 5.77 4.73 5.66

a The body mass index (BMI) was determined as weight/height 2, according to Keys et al. (1972). b Plasma parameters measured during the first week of basal diet. ~ Triglyceride values bearing an asterisk are those above normal range (1-15 mM).

the help of Drs G. Vigneron and J.L. Ode (84160 Cadenet, France) and D. Chanal (84120 Pertuis, France), volunteers with serum cholesterol concentrations exeeding 6.5mM cholesterol were selected. Fasting serum triglycerides were variable. Among the ten subjects, 4 subjects had normal values and 6 subjects presented mild to severe hypertriglyceridemia (1.7 to 5.0 mM). Glycemia levels were in the normal range. The individual values of fasting serum cholesterol, triglycerides and glucose at the beginning of the study are given in Table 1. Given the lipoprotein patterns obtained by lipoprotein electrophoresis, we considered all subject to have hypercholesterolemia that fit the criteria for either type IIa or type IIb hyperlipoproteinemia [17]. Free-living subjects (8 females and 2 males) were from 35 to 68 years old (Table 1). Based on body mass index values [30], none was obese. None had a diagnosis of chronic alcoholism or ethanol abuse; the daily alcohol consumption was 6.14 (_+2.94) and 5.88 (_+2.89)g before and during wheat germ supplementation, respectively. None was a cigarette smoker. None had received hypolipidemic agents in the 3 months before the study.

Experimental design and diets The protocol consisted of 1 week basal diet, 4 weeks experimental diet (basal diet plus wheat germ) and a 4 weeks follow-up coming back to basal diet. Fasting blood samples for glucose, lipid and lipoprotein analysis were taken at baseline (2 collections at the beginning and the end of the first week

138 Table 2. Nutrient, energy and dietary fibre composition of food consumed on basal diet or on wheat germ supplemented basal diet

Daily intake a

Energy (KJ)c Protein (g) Carbohydrates (g) Complex Refined Fat (g) Cholesterol (mg) e-tocopherol (mg) Dietary fibres (g)

Basal diet

Diet during supplementation

(Control period)

(Basal diet)

(Basal diet + germ)b

7846 67.6 233.3 192.5 40.8 74.9 262.6 20.9 13.6

7947 68.4 225.4 191.6 33.8 80.7 310.2 24.3 12.3

8346 78.4 231.6 195.7 35.9 83.8 310.2 30.7 15.1

_+ 557 + 3.2" _ 6.8 • 6.8 • 6.4 • 3.4 + 23.2 • 5.1 • 1.8~b

• 651 _+ 4.4~ _+ 9.3 _+ 6.4 + 5.8 -I- 3.5 + 38.5 _ 6.0 • 1.3a

• 651 --}-4.5b • 9.9 _ 7.9 • 5.9 ___ 3.8 • 38.5 + 6.0 _ 1.2b

a Values are mean ___ SEM (10 subjects) for daily intake over 7 days on basal diet (left column) and 3 days on wheat germ supplemented diet (middle and right columns). b Calculation including a 30 g daily intake of wheat germ with the composition given in the text (Methods section). Values bearing different superscript letters in the same row are significantly different (p < 0.05).

o f basal diet), after the 4 experimental weeks and after the 4-week follow-up period. The usual diet (basal diet) o f each subject was m o n i t o r e d by a 7-day f o o d recall during the first week o f the experiment. A n o t h e r 3-day f o o d recall was carried out during the last week o f the experimental period in order to check possible changes in dietary habits during w h e a t g e r m intake. The daily intake o f nutrients and fibres was calculated using a c o m p u t e r d a t a base [5, 56] and is given in T a b l e 2. The d a t a indicate that the subjects h a d a rather p r u d e n t diet [23] with m o d e r a t e energy, fat and cholesterol intake and a conventional p o l y u n s a t u r a t e d : s a t u r a t e d fatty acid ratio (1.20 _+ 0.11). The ratio o f animal to vegetable proteins (1.71 _+ 0.21) was rather low. D u r i n g the control week, complex a n d refined c a r b o h y d r a t e s , fat a n d proteins represented 41.0 ( + 1.4)%, 8.7 (_+ 1.3)%, 35.9 (_+ 1.63)% a n d 14.4 (_+ 0.7)% o f the total energy intake, respectively. T h e m e a n alcohol intake was very low (mean: 6.14 g/day). D u r i n g the 4-week experimental period, the subjects were instructed to take a daily dose o f 30 g w h e a t germ, which was fractionated into three rations i n c o r p o r a t e d e x t e m p o r a n e o u s l y to their m a i n meals. The actual daily ingestion o f w h e a t g e r m was estimated and the variability between subjects was in the range o f +_ 10% o f the r e c o m m e n d e d dose. The wheat g e r m used in the present study was " S u p e r g e r m e s " c o m i n g f r o m Diepal, Villefranche sur Sa6ne, France. It was p r e p a r e d f r o m soft

139 wheats by using conventional milling processes. The wheat germ used contained 8.5% moisture and its average composition (per 100g fresh matter) was as follows: 27.5g protein, 27.2g carbohydrate, l l . 0 g fat, 21.4 mg alpha-tocopherol, 327 mg phytosterols, 9.7 g total dietary fiber [50]. Its pancreatic lipase inhibitory capacity was 497 inhibitory units per g, as measured in vitro [6]. In consequence, the daily ingestion of 30g wheat germ provided an additional daily intake of energy (398 Kj, 95.1 Kcal), 8.2 g protein, 8.2 g carbohydrate, 3.3 g fat, 6.4mg alpha-tocopherol, 98 mg phytosterols and 2.9 g dietary fibers. As shown in Table 2, no significant change (p ~< 0.05) was observed in the basal diet composition before or during the wheat germ supplementation period. Moreover (Table 2), when comparing the basal diet plus wheat germ with the basal diet consumed during the week before supplementation, only the protein daily intake was significantly higher (p ~< 0.05).

Plasma analysis Blood samples were taken after an overnight fast. Plasma total and free cholesterol [57], triglycerides [10], phospholipids [59] and glucose [25] were measured by enzymatic procedure. Total plasma Apoprotein A1 [55] and Apoprotein B [54] were assayed by immunonephelometry. The lipoprotein classes (VLDL: d ~< 1.006, LDL: 1.006 ~< d ~< 1.060; HDL: 1.060 ~< d ~< 1.21) were separated from 1.5 ml plasma by ultracentrifugation (38 000 rpm at 15 ~ for 24h in a Beckman SW41 rotor) on a KBr discontinuous density gradient as previously described [37]. Total cholesterol was determined in 0.5ml collected fractions. Total and free cholesterol, triglycerides and phospholipids were quantified in each pure lipoprotein class by using the enzymatic procedures mentioned above. Lipoprotein lipid concentrations were adjusted for ultracentrifugation recovery (85-90%) by assuming that the (VLDL + LDL + HDL) value equals the total plasma value.

Statistical analysis Student's t test for paired values was used to assess the statistical significance of the differences observed between the experimental periods at the probability level of 95%. Results

The wheat germ supplementation, i.e. 30 g per day, was well tolerated by all the subjects, without any intestinal discomfort such as flatulence or any

140 Table 3. Changes in fasting plasma and lipoprotein lipids (mM) before and after wheat germ intake Measure"

Basal period

After 4-week wheat germ

After 4-week follow up

7.80 1.80 4.93 1.07

_+ +_ +_ _+

0.33" 0.37 ~ 0.26 0.15

7.15 t.07 4.87 1,21

_ +_ _+ _+

0.39 b 0.15 b 0.40 0.18

7.35 1.04 5.03 1.28

_+ + _+ +

0.39 ab 0.18 b 0.39 0.18

1,99 1.41 0.42 0.16

_+ _+ _+ _

0.38" 0.39 ~ 0.05" 0.02

1.32 0.80 0.40 0.12

_ 0.168 • 0.18 b 4-0.05" _+ 0.03

1.60 0.93 0.53 0.14

_+ • • __

0.24 a8 0.24 b 0.06 b 0,02

2.27 0.65 0,90 0.72

_+ _+ _+ _+

0.24" 0.21" 1.07 ~ 0.09

1.91 0.40 0.78 0.74

_+ • q_+

1.88 0.39 0.72 0.77

_+ 0.148 ___ 0.108 +_ 0.07 b _+ 0.08

Total cholesterol Plasma VLDL b LDL HDL

Triglycerides Plasma VLDL LDL HDL

Phospholipids Plasma VLDL LDL HDL

0.10 "8 0.09 "8

0.048 0.09

a Values (mean _+ S E M o f ten subjects) bearing different superscript letters in the same row are significantly different ( p < 0.05). b V L D L : Very Low Density Lipoproteins; LDL: Low Density lipoproteins; H D L : High Density Lipoproteins.

other disturbance. No significant change in body weight was observed over the experimental period. Mean plasma glucose concentration was 5.26 (+__4.62)mM, 5.31 ( • 4.64) mM and 5.25 (+ 4.45) mM at baseline, after wheat germ intake and after the follow-up, respectively. Thus, normal glycemia did not change after wheat germ supplementation. Plasma cholesterol

The changes observed after wheat germ supplementation in plasma lipids and lipoproteins are presented in Table 3. Ingesting 30 g per day wheat germ significantly decreased plasma total cholesterol from 7.80 to 7.15 mM. This change was mainly due to the decrease observed for plasma esterified cholesterol after wheat germ intake (5.04 _ 0.3 vs. 5.37 _+ 0.3 mM). After going back to the basal diet for one month, total plasma cholesterol showed a middle value which was not significantly different from the initial value measured before wheat germ intake. After wheat germ supplementation, HDL cholesterol tended to increase slightly (+15.2%) and LDL cholesterol did not noticeably change

141 ( - 1 . 2 % ) . The VLDL total cholesterol significantly dropped by 40.6%, from 1.80 to 1.07mM; this was mainly due to a significant decrease of esterified cholesterol, from 1.11 (+ 02) to 0.59 (+_ 0.1) mM. In consequence, the plasma/HDL total cholesterol ratio significantly decreased from 8.74 (+_ 1.24) to 7.24 (_+ 1.09) after 4 weeks of wheat germ intake. This value remained low even after the 4-week follow-up period.

Plasma triglycerides As shown in Table 3, mean plasma triglycerides of ten subjects markedly decreased ( - 33.7%) after one month feeding wheat germ. An intermediate value was observed after the follow-up period. The LDL and the HDLtriglyceride concentrations did not change over the experimental period, while the mean VLDL-triglycerides significantly dropped by 43.3%. In fact, as presented in Fig. la, the mean triglyceridemia of the four normotriglyceridemic subjects did not change (0.83 _ 0.20 vs. 0.95 _+ 0.14mM) whereas that of the six hypertriglyceridemic subjects significantly dropped from 2.68 (_+ 0.51) to 1.64 (_+ 0.18)mM. This effect was not significant after the 4-week follow-up period. At the same time, as shown in Fig. lb, the VLDL-triglycerides of the hypertriglyceridemic subjects markedly dropped ( - 5 1 % ) from 2.06 (+0.49) to 1.01 ( • after wheat germ intake while the VLDL-triglycerides of the normotriglyceridemic subjects did not change. This lowering effect remained marked (.-37.4%) even after the follow-up period. Cholesterol and triglyceride decreases (total and VLDL) observed after wheat germ supplementation were accompanied by a significant plasma apolipoprotein B decrease (Table 4). A concomitant plasma apolipoprotein A1 decrease was also observed (Table 4) without great change in HDL lipid levels. Therefore, the Apo-B:Apo-A1 ratio remained almost constant. Data about lipoprotein composition are presented in Table 5. No marked change was observed in VLDL, LDL or HDL lipid composition after wheat germ supplementation of the diet. Only a slight decrease in cholesterol esters of LDL associated with an increase in triglycerides occurred after the follow-up period.

Discussion

This short-term study on the effects of wheat germ supplementation on plasma lipids and lipoproteins was carried out in adult hypercholesterolemic subjects. Six out of the ten subjects had an associated hypertriglyceridemia

142 PLASMA TRIGLYCERIDES (mM) 3.5-

3.5

3.0-

3.0"

2.5-

2.5-'

2.0-

2.0 .~

1.5-

1.5-

1.0-

1.0 i

0.5-

o, D D

0.0-

0.0

basal

w.germ

followup

basal

HTG SUBJECTS

w.germ

followup

NTG SUBJECTS

VLDL TRIGLYCERIDES (raM) 3.0-

3.0 2.5

2.5-

2.0

2.0-

1.5

1.5-

1.0

1.0-

0.5

0.5-

0.0

o

basal

w.germ

followup

HTG SUBJECTS

basal

w.germ

followup

NTG SUBJECTS

Fig. 1. Changes in plasma total triglycerides (upper pannel) and in Very Low Density Lipoprotein (VLDL) triglycerides (lower pannel) in six hypertriglyceridemic (HTG) subjects and in four subjects (NTG) with normal plasma triglyceride values.

Table 4. Changes in plasma Apolipoproteins (g/l) before and after wheat germ intake

Measure"

Basal period

After 4-week wheat germ

After 4-week follow-up

Apo A1 Apo B

1.37 -I- 0.08 a 1.75 + 0.11 a

1.12 + 0.06 b 1.59 -I- 0.13 b

1.47 + 0.12 ~ 1.71 _ 0.12 a

a Values (mean +_ SEM of ten subjects) bearing different superscript letters (a, b) are significantly different (p < 0.05).

143 Table 5. Percentage changes in lipid lipoprotein composition (weight percentage)

Lipoproteins

Triglycerides Phospholipids Cholesterol esters Free cholesterol

VLDL" Basal period b 40.0 _+ 3.35 After 4 wks wheat germ 43.2 _+ 3.61 After 4 wks follow-up 42.9 +_ 3.41

19.1 +_ 2.13 19.7 _ 1.90 19.5 _+ 2.45

30.4 _+ 2.82 26.8 _+ 1.95 26.6 + 2.90

10.5 + 0.94 10.4 _+ 1.25 11.0 _+ 1.22 13.5 __ 0.85 13.8 _+ 0.50 14.9 _+ 1.01

LDL Basal period After 4 wks wheat germ After 4 wks follow-up

9.7 _+ 1.08 a 18.0 _+ 0.90 9.6 _+ 1.18 a 16.4 _+ 0.90 12.5 -t- 1.03 b 15.4 + 1.59

58.1 + 1.73 ab 60.1 _+ 1.42a 57.2 -I- 1.46b

HDL Basal period After 4 wks wheat germ After 4 wks follow-up

11.3 _+ 1.05 8.1 _+ 1.43 9.6 _+ 1.74

39.3 _+ 1.66 41.9 _+ 1.1 41.0 _+ 2.11

42.0 __. 1.94 41.9 + 2.52 41.7 +_ 2.01

7.4 _+ 0.66 7.9 _+ 0.45 7.7 _+ 0.41

a VLDL: Very Low Density Lipoprotein; LDL: Low Density Lipoprotein; HDL: High Density Lipoprotein. b Values (mean _+ SEM of ten subjects) bearing different superscript letters in the same row are not significantly different (p ~< 0.05).

but none had hyperglycemia or was overweight. They all relied on a rather balanced diet (35.9% of energy provided by fat, 262-310 mg/d cholesterol) and dietary records indicate that the composition of the basal diet did not change during the 4-week period of wheat germ intake. Adding each day 30 g wheat germ to their basal diet for 4 weeks induced several significant modifications in their plasma lipid pattern. Total plasma cholesterol significantly decreased by 8.3% while among lipoproteins, VLDL-total and esterified cholesterol dropped by 40.6% and 46.8% respectively. In the six hypertriglyceridemic subjects, this was accompanied by a marked significant drop of total plasma triglyceride concentration. These data are strikingly comparable to those previously obtained in the adult rat [40]. Indeed, adding 7% wheat germ to rats on a high fat-cholesterol diet for 7 weeks resulted in significant decreases in VLDL-cholesterol (-37.9%), VLDL-triglycerides (--37.7%) and in the plasma/HDL total cholesterol ratio (-32.8%) and a significant increase in HDL-cholesterol ( + 35.2%). In fact, the wheat germ content in the diets was very close in both studies, Since an approximate value of 7.5% is obtained in the present study in humans (30 g/d wheat germ added to about 400 g/d dry matter supplied by the basal diet). Thus, adding wheat germ to the diet affects the lipid and cholesterol metabolism in a very comparable way in rats and in humans with high plasma cholesterol and triglyceride values. Numerous studies have been already performed to investigate the effects of fibre-rich cereal fractions on lipid metabolism. Wheat bran, containing about

144 38-45% dietary fibres, with very variable daily intakes (8-60g/d), showed variable effects on plasma cholesterol values either in normal people [2, 22, 24, 29, 46, 49, 53, 62] or in hypercholesterolemic people [24, 43, 53, 60]. Part of these discrepancies could result from very different experimental lay-outs and diets [63]. More generally, wheat bran intake decreased plasma triglycerides [2, 24, 43, 53, 62] and this was accompanied by a decrease in the VLDLtriglycerides [22, 43, 62]. Other studies carried out with very high oat bran amounts (98-100g/d) showed marked reductions in plasma and LDLcholesterol in hypercholesterolemic subjects with [3] or without [3, 32] lowering effects on plasma triglycerides. Mechanisms involved in the alteration of lipid metabolism by dietary fibre-rich fractions are still debated. According to animal and human studies, it is becoming more and more evident that soluble or insoluble fibres may have different mechanisms of action and that components other than fibres, present in natural fibre-rich food fractions, may exert important metabolic effects. Concerning wheat germ, it is striking to observe a parallel lowering effect on both elevated plasma cholesterol and triglycerides in the rat and in humans; this can account for the drop in the Very Low Density Lipoprotein lipids and in Apoprotein B. These metabolic changes could probably result from a decreased intestinal uptake of dietary fat and cholesterol resulting in a lower intestinal synthesis of chylomicrons, a reduced influx of chylomicrons remnants to the liver and then a lower synthesis and secretion of VLDL by the liver. For instance, a very recent study [15] has shown that a reduction of plasma triglycerides in type IV (and very likely in type IIb) hypertriglyceridemic subjects was accompanied by a drop in VLDL triglycerides and, more specifically, in Apo E-poor VLDL particles whose 15roduction rate was expected to decrease. In fact, we have already demonstrated in the rat [6, 9] that addition of 10% wheat germ into fatty test-meals lowers fat lipolysis and thus the intestinal uptake and the output in the blood stream of dietary lipids and cholesterol. In another study [40], we showed that feeding rats for 7 weeks a diet containing 7% wheat germ significantly decreased the triglyceride and cholesterol accumulation in the liver, in agreement with other data [52]. Several hypothesis about the mechanism of action of wheat germ may be discussed. In the present study, the addition of 30 g/d wheat germ to the basal diet resulted only in limited changes in the mean food intake. For instance, fat, cholesterol and carbohydrate intakes did not change and the addition of 2.9 g dietary fibres provided daily by wheat germ did not significantly increase fibre consumption (15.1 vs. 13.6 g/d). Thus, it does not seem that the low amount of wheat germ fibres, mainly composed of cellulose and hemi-celluloses [16] plays a key role in lipid metabolism. In addition, cellulose and hemi-celluloses

145 did not show any significant effects on dietary fat assimilation or plasma lipids [8, 19, 38, 41, 61,631. During wheat germ supplementation, the protein intake significantly increased because the high protein content of wheat germ (Table 2). Various vegetable proteins are well known to have plasma cholesterol lowering properties [33] as compared to animal proteins. Such effects were generally observed with diets containing only one source of protein (10-20% in the diets). From these data, it is difficult to know whether adding 8.2 g/d wheat germ protein to a basal diet providing 68.4 g/d protein (15.4% increase) could account for the modifications observed in lipid metabolism. However, we have shown that wheat germ inhibits the activity of pancreatic lipase in vitro [39] and that extractable proteins are involved in this enzyme inhibition process [7, 38, 39]. When 10% wheat germ was added to test-meals in the adult rat, the gastric and intestinal fat lipolysis was impaired and the lipid and cholesterol intestinal uptake was thus reduced [6]. When wheat germ was previously depleted of lipolysis inhibitory proteins (LIP), then addition of 10% LIP-depleted wheat germ to test-meals no longer exhibited any effects on dietary fat assimilation. Thus, proteins provided by wheat germ could likely be involved, at least partly, in the metabolic changes observed. Some proteins in soybean may also have the ability to inhibit gastric and pancreatic lipase activity in vitro [20, 21] and some hydrophobic peptides resulting from the enzymatic clivage of soybean proteins have been specifically involved in the hypocholesterolemic effects of soybean protein diets [65]. In fact, hydrophobic proteins can impair fat lipolysis [7, 20, 21]. Other studies on vegetable proteins [14, 45] have shown that the observed decrease in plasma cholesterol was associated to changes in the VLDL and LDL turn-over with possible relation with an increase in the activity of the ApoB receptor, thus giving an additional explanation for the observed results. Other recent data point out that vegetable proteins extracted from wheat or soybean might lower plasma and liver triglyceride levels by decreasing triglyceride synthesis rate in the liver [27, 28]. Thus, the role of wheat germ proteins may be important. The effect of alpha-tocopherol on plasma lipids and lipoprotein has been reported [13, 58]. The additional 6.2 mg/d intake of Vitamin E coming from wheat germ did not lead to a significantly higher daily intake (30.7 vs. 20.9mg) and was negligeable in comparison to the experimental daily intakes (500-600 mg) able to lower plasma lipids [13, 58]. The total amount of phytosterols (98 mg) provided by wheat germ could also contribute to the lowering of plasma cholesterol. Although the exact amount of phytosterols provided by the basal diets of the ten subjects was not determined, we can consider that usual french diets provide about

146 100-200 mg/d phytosterols (J. Ferezou, personal communication). Thus, the part due to wheat germ was important and we can assume that the total amount of ingested phytosterols was close to dietary cholesterol intake (260-310mg/d) during the wheat germ supplementation period. Phytosterols are well known to inhibit the intestinal absorption of cholesterol in a ratio of 1/1 or 2/1 (w/w) phytosterols/cholesterol [26, 48]. In fact, wheat germ did not bind appreciable amounts of cholesterol, fatty acids or monoglycerides, but it significantly decreased the intestinal uptake process of dietary cholesterol in the rat, when there was 10% wheat germ in the test-meal [9]. Moreover, various cereal fractions were evidenced to be rich in non-sterol metabolites of the mevalonate pathway, having the capacity to inhibit H M G - C o A reductase, the key enzyme of cholesterol synthesis [51]. In addition, the hepatic synthesis of cholesterol might be reduced by the short-chain fatty acids which are the major fermentation products of fiber in the colon [12]. The subjects participating in the study had hypercholesterolemia, associated or not with hypertriglyceridemia. Both situations are highly related to elevated risks for coronary heart disease. As pointed out by Anderson and Tietyen-Clark [4], the low-fat, low-cholesterol diets, referred to as "prudent diet", lower serum cholesterol levels only by 4-7%, whereas highcarbohydrate, high-fibre diets can decrease cholesterol levels by 20%. This is illustrated in the present study where the subjects already relied on a low-fat, low-cholesterol, low-fibre diet; adding 30 g/d wheat germ allowed a subsequent decrease of total cholesterol (8.3%) and total triglycerides (33.7%). This reduction was mainly due to a decrease of the VLDL particles. Since the accumulation of apo-B containing lipoproteins is well known to increase the risk for atherosclerothic lesions, wheat germ supplementation could represent a preventing factor. Although the mechanism of action of wheat germ on lipid metabolism remains to be fully elucidated, this short-term study shows that a daily intake of moderate amounts of wheat germ may benefically reduce hyperlipidemia in humans. However, the present results need to be validated by a long-term study.

Acknowledgements We are indebted to the subjects having participated in this study, especially for their full attendance to the experimental planning. We are grateful to Drs. Bergier and Chardon for their medical help. We are thankful to Diepal (BSN group) for kindly providing wheat germ for the study. This work was

147

supported by a grant (No 88-G-0894, Aliment 2000 program) from the French Ministry of Research and Technology and by a grant (AIP on Metabolic effects of dietary fibers) from the National Institute for Agronomic Research (INRA). References 1. Aberg H, Lithell H, Selinus I (1985) Serum triglycerides are a risk factor for myocardial infarction but not for angina pectoris. Atherosclerosis 54:89-97 2. Albrink M J, Newman T, Davidson PC (1979) Effect of high and low fiber diets on plasma lipids and insulin. Am J Clin Nutr 32:1486-1491 3. Anderson JW, Story L, Sieling B (1984) Hypocholesterolemic effects of oat-bran or bean intake for hypercholesterolemic men. Am J Clin Nutr 40:1146 1155 4. Anderson JW, Tietyen-Clark J (1986) Dietary fiber: hyperlipidemia, hypertension, and coronary heart disease. Am J Gastroenterol 81:907-919 5. Astra-Calv6 (1986) Information lipodi~t+tique, Paris 6. Borel P, Lairon D, Senft M, Chautan M, Lafont H (1989a) Effect of wheat bran and wheat germ on the digestion and the intestinal absorption of dietary lipids in the rat. Am J Clin Nutr 49:1192-1202 7. Borel P, Lairon D, Termine E, Martigne M, Lafont H (1989b) Isolation and properties of lipolysis inhibitory proteins from wheat germ and wheat bran. Plant Foods Hum Nutr 39:339-348 8. Borel P, Lairon D, Senft M, Lafont H (1989c) Lack of effect of purified cellulose and hemi-cellulose on the digestion and the intestinal absorption of dietary lipids in the rat. Ann Nutr Metab 33:237-245 9. Borel P, Martigne M, Senft M, Garzino P, Lafont H, Lairon D (1990) Effect of wheat bran on the intestinal uptake of oleic acid, monoolein, and cholesterol in the rat. J Nutr Biochem 1:28-33 10. Buccolo G, David H (1973) Quantitative determination of serum triglycerides by the use of enzymes. Clin Chem 19:476-482 11. Carlson LA, Aberg H, Gustafson VK (1985) Serum triglycerides - an independent risk factor for myocardial infarction but not for angina pectoris. N Engl J Med 312:1127 12. Chen WJL, Anderson JW (1984) Propionate may mediate the hypocholesterolemic effects of plant fibers in cholesterol-fed rats. Proc Soc Exp Biol Med 175:215-218 13. Cloarec M J, Perdriset GM, Lamberdiere FA, Colas-Belcour JF, Sauzi6res JP, Neufeld HN, Goldbourt U (1987) ~-tocopherol: effect on plasma lipoproteins in hypercholesterolemic patients. Isr J Med Sci 23:869-872 14. Cohn JS, Kimpton WG, Nestel PJ (1984) The effect of dietary casein and soy protein on cholesterol and very low density lipoprotein metabolism in the rat. Atherosclerosis 52: 219-231 15. Evans AJ, Huff MW, Wolfe BM (1989) Accumulation of an apo E-poor subfraetion of very low density lipopotein in hypertriglyceridemic men. J Lip Res 30:1691-1701 16. Fisher N (1985) Cereals, milling and fibre. In: Trowell H, Burkitt D, Heaton K (eds), Dietary Fibre, Fibre Depleted Food and Disease. London: Academic Press, pp. 377-390 17. Fredrickson DS, Levy RI, Lees RS (1967) Fat transport in lipoproteins - an integrated approach to mechanisms and disorders. N Engl J Med 276:32-94 18. Freedman DS, Gruchow HW, Anderson AJ (1988) Relation of triglyceride levels to coronary artery disease: The Milwaukee cardiovascular data registry. Am J Epidemiol 127:1118-1130

148 19. Gallaher D, Schneeman BO (1985) Effect of dietary cellulose on site of lipid absorption. Am J Physiol 249:184-191 20. Gargouri Y, Julien R, Pieroni G, Verger R, Sarda L (1984) Studies on the inhibition of pancreatic and microbial lipases by soybean proteins. J Lipid Res 25:1214-1221 21. Gargouri Y, Pieroni G, Lowe PA, Sarda L, Verger R (1986) Human gastric lipase: the effect of amphiphiles. Eur J Biochem 156:305-310 22. Gariot P, Digy JP. Genton P, Lambert D, Bau RMH, Debry G (1986) Long-term effect of bran ingestion on lipid metabolism in healthy man. Ann Nutr Metab 30:369-373 23. Grundy SM, Bilheimer D, Blackburn H (1982) Rationale of the diet-heart statement of the American Heart Association. Report of the Nutrition Committee. Circulation 65: 839A-854A 24. Heaton KW, Pomare EW (1974) Effect of bran on blood lipids and calcium. Lancet 1: 49-50 25. Horvath C, Pedersen H (1977) Immobilized enzymes in continuous-flow analysis. In: Advances in Automated Analysis. Tehnicon International Congress, 1976, Vol. 1, Tarrytown, NY, Mediad Inc, pp. 86-95 26. Ikeda I, Tanaka K, Sugano M, Vahouny GV, Gallo LL (1988) Inhibition of cholesterol absorption in rats by plant sterols. J Lipid Res 29:1573-1582 27. Iritani N, Nagashima K, Fukuda H, Katsurada A, Tanaka T (1986) Effects of dietary proteins on lipogenic enzymes in rat liver. J Nutr 116:190-197 28. Iritani N, Suga A, Fukuda H, Katsurada A, Tanaka T (1988) Effects of dietary casein and soybean protein on triglyceride turnover in rat liver. J Nutr Sci Vitaminol 34:309-315 29. Jenkins DJA, Hill MS, Cummings JH (1975) Effect of wheat fiber on blood lipids, fecal steroid excretion and serum iron. Am J Clin Nutr 28:1408-1411 30. Keys A, Fidanza F, Karvonen MJ, Kimura N, Taylor HL (1972) J Chronic Dis 25: 329-343 31. Khaw KT, Barrett-Connor E (1987) Dietary fiber and reduced ischemic heart disease mortality rates in men and women: a 12-year prospective study. Amer J Epidemiol 126: 1093-1102 32. Kirby RW, Anderson JW, Sieling B, Rees ED, Chen WJL, Miller RE, Kay RM (1981) Oat-bran selectively lowers serum low density lipoprotein cholesterol concentrations of hypercholesterolemic men. Am J Clin Nutr 34:824-829 33. Kritchevsky D (1979) Vegetable protein and atherosclerosis. J Amer Oil Chem Soc 56: 135-146 34. Kritchevsky D, Story JA (1986) Influence of dietary fiber on cholesterol metabolism in experimental animals. In: Spiller GA (ed.), Handbook of Dietary Fiber in Human Nutrition. CRC Press, pp. 129-142 35. Kromhout D, Bosschieter EB, de Lezenne Coulandre C (1982) Dietary fiber and 10-year mortality for coronary heart disease, cancer and all causes. Lancet 2:518-522 36. Kushi LH, Lew RA, Stare FJ, Ellison CR, E1 Lozy M, Bourke G, Daly L, Graham I, Hickey N, Mulcahy R, Kevaney J (1985) Diet and 20-year mortality from coronary heart disease. The Irelande-Boston diet-heart study. N Eng J Med 312:811-818 37. Lacombe C, Corraze G, Nibbelink M, Boulze D, Douste-Blazy P, Camare R (1986) Effects of a low-energy diet associated with egg supplementation on plasma cholesterol and lipoprotein levels in normal subjects: results of a cross-over study. Br J Nutr 56: 561-575 38. Lairon D, Lafont H, Vigne JL, Nalbone G, Leonardi J, Hauton JC (1985a) Effects of dietary fibers and cholestyramine on activity of pancreatic lipase in vitro. Am J Clin Nutr 42:629-638 39. Lairon D0 Borel P, Termine E, Grataroli R, Chabert C, Hauton JC (1985) Evidence for

149

40.

41.

42.

43. 44.

45.

46. 47.

48. 49.

50.

51. 52. 53. 54. 55. 56. 57. 58. 59. 60.

a proteinic inhibitor of pancreatic lipase in cereals, wheat bran and wheat germ. Nutr Rep lnt 32:1107-1113 Lairon D, Lacombe C, Borel P, Corraze G, Nibbelink M, Chautan M, Chanussot F, Lafont H (1987) Beneficial effect of wheat germ on circulating lipoproteins and tissue lipids in rats fed a high fat, cholesterol-containing diet. J Nutr 117:838-845 Lafont H, Lairon D, Vigne JL, Chanussot F, Chabert C, Portugal H, Pauli AM, Crotte C, Hauton JC (1985) Effect of wheat bran, pectin and cellulose on the secretion of bile lipids in rats. J Nutr 115:849-855 Lichtenstein M J, Burr ML, Fehily AM, Yarnell JWG (1986) Heart rate, employment status and prevalent ischaemic heart disease confound relation between cereal fibre intake and blood pressure. J Epidemiol Comm Health 40:330-333 Lingarde F, Larsson L (1984) Effects of a concentrated bran fibre preparation on HDL cholesterol in hypercholesterolemic men. Hum Nutr Clin Nutr 38C: 39-45 Liu K, Stamler J, Trevisan M (1982) Dietary lipids, sugar, fiber and mortality from coronary heart disease. Bivariate analysis of international data. Artherosclerosis 2: 221-227 Lovati MR, Manzoni C, Canavesi A, Sirtori M, Vaccarino V, Marchi M, Gaddi G, Sirtori CR (1987) Soybean protein diet increases low density lipoprotein receptor activity in mononuclear cells from hypercholesterolemic patients. J Clin Invest 80:1498-1502 MacDougall RM, Shim YL, Walker L, Thurston OG (1978) Effect of wheat bran on serum lipoproteins and biliary lipids. Can J Surg 21:433-435 Mahtey RW (t982) Atherogenic hyperlipoproteinemia. The cellular and molecular biology of plasma lipoproteins altered by dietary fat and cholesterol. Med Clinics North America 66:375-401 Mattson H, Grundy SM, Crouse R (1982) Optimizing the effect of plant sterols on cholesterol absorption in man. Am J Clin Nutr 35:697-700 Munoz JM, Sandstead HH, Jacob RA, Logan GM, Reck SJ, Klevay LM, Dintzis FR, Inglett GE, Shuey WC (1979) Effects of some cereal brans and textured vegetable protein on plasma lipids. Am J Clin Nutr 32:580-592 Prosky L, Asp NG, Furda I, Devries JW, Schweizer TF, Harland BF (1984) Vitamins and other nutrients. Determination of total dietary fiber in foods, food products, and total diets: interlaboratory study. J Assoc Off Anal Chem 67:1044-1052 Qureschi AA, Burger WC, Peterson DM, Elso~ C (1985) Suppression ofcholesterogenesis by plant constituents: review of Wisconsin contributions to NC-167. Lipids 20:817-824 Ranhotra GS (1973) Effect of cellulose and wheat mill fractions on plasma and liver cholesterol levels in cholesterol-fed rats. Cereal Chem 50:358-363 Salvioli G, Lugli R, Pradelli JM (1985) Cholesterol absorption and sterol balance in normal subjects receiving dietary fiber or ursodeoxycholic acid. Dig Dis Sci 30:301-307 Sievet-Desrumeaux C, Dedomder-Decoopman E, Fruchart JC, Dewailli P, Sezille G (1979) Clin Chim Acta 95:405408 Sievet-Desrumeaux C, Dedomder-Decoopman E, Fruchart JC, Dewailli P, Sezille G, Jaillard J (1980) Clin Chim Acta 107:145-148 Souci SW, Fachmann W, Kraut H (1981) Composition des aliments. Verlag ed, Stuttgart Stahler F, Gruber W, Stinshoff K, Roschlau P (1977) Eine praxisgerechte enzymatische cholesterin-Bestimmung. Med Lag 30:29-37 Sundaram GS, London R, Manimekalai S, Nair PP, Goldstein P (1981) ~-tocopherol and serum lipoproteins. Lipids 16:223-227 Takayama M, Itoh S, Nagasaki T, Tanimizu I (1977) A new enzymatic method for choline containing phospholipids. Clin Chim Acta 79:93-98 Tarpila S, Miettinen TA, Metsaranta L (1978) Effect of bran on serum cholesterol, fecal

150

61. 62.

63. 64.

65.

mass, fat, bile acids and neutral sterols, and biliary lipids in patients with diverticular disease of the colon. Gut: 137-145 Tsai AC, Elias J, Kelley JJ, Lin RSC, Robson R K (1976) Influence of certain dietary fibers on serum and tissue cholesterol levels in rats. J Nutr 106:118-123 Van Berge-Henegouven GP, Huybregts AW, Van de Werf S, Demaker P, Shade RW (1979) Effect of a standardized wheat bran preparation on serum lipids in young healthy males. Am J Clin Nutr 32:794-798 Vigne JL, Lairon D, Borel P (1987) Effect of pectin, wheat bran and cellulose on serum lipids and lipoproteins in rat fed on a low-fat or high-fat diet. Br J Nutr 58:405-413 Wilson PW, Garrison RJ, Castelli WP, Feinleib M, McNamara PM, Kannel WB (1980) Prevalence of coronary heart disease in the Framingham offspring study: role of lipoprotein cholesterol. Am J Cardiol 46:649-654 Yashiro A, Oda S, Sugano M (1985) Hypocholesterolemic effect of soybean protein in rats and mice after peptic digestion. J Nutr 115:1325-1336