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The fiber and/or polyphenols present in lingonberries null the

glycemic effect of the sugars present in the berries when

consumed together with added glucose in healthy

human volunteers

Kaisa M. Linderborg a,, Riikka Jrvinena, Henna-Maria Lehtonena,Matti Viitanen b,c, Heikki P.T. Kallioa

a Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finlandb Department of Geriatrics, University of Turku, Turku City Hospital, Turku, Finlandc Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden

A R T I C L E I N F O A B S T R A C T

Article history:

Received 18 October 2011

Revised 7 June 2012

Accepted 8 June 2012

This study was undertakenon thebroad hypothesis that lingonberry (Vaccinium vitis-idaea L.)

has potential to reduce postprandial glycemic and lipemic response. More specifically, 2

postprandial crossover studies with healthy normal-weight male subjects were conducted

to study the influence of commercial lingonberry powder on postprandial glycemia and

lipemia. The test meals contained fat-free yoghurt with either glucose (50 g) or

triacylglycerols (35 g) with or without (control) the lingonberry powder. The lingonberry

powder provided the meals with a known amount of fiber and a known amount and

composition of sugars,and it was a rich sourceof polyphenols. Postprandial glucose, insulin,

and triacylglycerol responses were analyzed. There were no significant differences in the

postprandial glucose concentration between the meals in the glycemia trial despite the fact

that the lingonberry meal contained more glucose and fructose. When the meal did not

contain added sugar but, instead, added triacylglycerol, no glycemia or lipemia-lowering

effect was detected. On the contrary, there were indications of higher glycemic and

insulinemic effect after the lingonberry meal. The results of this study indicate that the

fibers and/or polyphenols present in lingonberries null the glycemic effect of the sugars

present in the berries when consumed together with added glucose. By contrast, the

lingonberry powder did not affect the postprandial lipemic response.

2012 Elsevier Inc. All rights reserved.

Keywords:Lingonberry

Glycemia

Lipemia

Polyphenols

Men

1. Introduction

Lingonberry (Vaccinium vitis-idaea L.) is a wild, semiwoody

chamaephyte that keeps its leaves through the winter and

commonly grows at the northern latitudes. Edible fruits of

lingonberries are the most abundantly picked wild berries in

many Eurasian countries, and they are commercially used in a

wide range of products. Among other northern berries,

lingonberry is associated with a number of bioactive com-

pounds such as phenolics, lignans, vitamins C, inositols,

N U T R I T I O N R E S E A R C H 3 2 ( 2 0 1 2 ) 4 7 1 4 7 8

Corresponding author. Department of Biochemistry and Food Chemistry, University of Turku, FI-20014 Turku, Finland.Tel.: +358 2 333 6874.E-mail address:[email protected](K.M. Linderborg).

0271-5317/$

see front matter 2012 Elsevier Inc. All rights reserved.doi:10.1016/j.nutres.2012.06.004

A v a i l a b l e o n l i n e a t w w w . s c i e n c e d i r e c t . c o m

w w w . n r j o u r n a l . c o m


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triacylglycerols, glycerophospholipids, fatty acids, tocoph-

erols, phytosterols[1], and fibers including pectin, cellulose,

lignin, and cuticular polymers[2].

On the whole, berries contain a wide range of phenolic

compounds in different conjugated forms. From lingon-

berries, a total of 28 different phenolic compounds have

been identified [3]. Based on the weight of the aglycone,

anthocyanidins, mainly cyaniding-containing compounds, as

well as proanthocyanidins represent most phenolic com-

pounds in lingonberries, followed by mainly quercetin-

containing flavonols [1]. Although cyanidin-3-galactoside,

cyaniding-3-glucoside, and cyaniding-3-arabinoside are the

most abundant anthocyanins in lingonberries, the most

abundant flavonols are quercetin glycosides followed by

kaemferol glycosides[3].

It is likely that polyphenols affect the glucose metabolism

via a multifaceted mechanism. Many polyphenols have

inhibited intestinal-glucosidase activity or glucose transport

in vitro and suppressed the elevation of blood glucose

concentration after oral administration of glucose in animal

models, as reviewed by Hanhineva et al [4]. Phytochemicals

found in grapes or grape plantderived products have shown

inhibitory effects in the chemically induced diabetic models,

possibly via reducing oxidative stress in the pancreas and

aiding in the preservation of the -cell mass[5]. Recently, the

administration of polyphenols was associated with the

prevention of fatty lipid disease in mice possibly at least via

the activation of 5-adenosine monophosphateactivated

protein kinase[6].

The mechanisms through which berries could attenuate

lipemia remain speculative. In the case of grape seed

procyanidins, the suggested mechanism has been the regula-

tion of bile acid pathway[7], whereas black tea polyphenols

have been indicated to suppress the lymphatic transport of

dietary fat [8]. In an in vitro trial, apple, but not wine,

polyphenol extract decreased the enterocyte secretion of

lipoproteins[9]. The cardioprotective potential of cranberries

has been reviewed, and one of the suggested mechanisms is

the decrease of the amount of oxidized low-density lipopro-

tein in human plasma [10]. In addition to polyphenols,

different dietary fibers influence the postprandial glucose

and lipid response at least via the gastric-emptying rates[11].

In humans, mixed berries have been found to suppress

postprandial hyperglycemia[12,13], and we have reported the

beneficial effects of seabuckthorn on postprandial glucose and

insulin response [14]. The inclusion of cinnamon in a rice

pudding meal has lowered the postprandial glucose response

[15]. Regarding postprandial lipemia, we are aware of only 3

studies investigating the effects of berries or polyphenols on

human subjects; first, strawberry polyphenols were reported

to lower postprandial lipemia in overweight hyperlipidemic

men and women[16]; second, red wine polyphenols attenu-

ated postprandial chylomicron and chylomicron remnant

levels in postmenopausal women[17]; and third, our recent

study showed that sea buckthorn extraction residues delayed

postprandial lipemia [18]. In animals, black [8], oolong[19],and

green tea have found to decrease postprandial lipemia[20].

To our knowledge, the present study pioneers an investi-

gationinto the effects of thefibers and/or polyphenols present

in lingonberries in human subjects. More specifically, the aim

of the study was to assess how a commercial lingonberry

powder affects postprandial hyperglycemia, insulinemia, and

lipemia after either a high-glucose or a high-fat meal. We

hypothesized that the addition of fiber and polyphenol-

containing lingonberry powder could attenuate the glycemic

and lipemic responses to the meals. Fat-free yoghurt, glucose,

and canola oil were chosen as components of the study meal

to mimic a common use of lingonberries in Scandinavia.

Healthy young menwere recruitedas volunteers to establisha

starting point in postprandial lingonberry research.

2. Methods and materials

2.1. Subjects

Healthy normal-weight nonsmoking men aged between 18

and 40 years were recruited for the study. The subjects had

normal liver, kidney, and thyroid functions indicated by

plasma alanine aminotransferase level lower than 60 U/L,

creatinine level lower than 115 mol/L, and a thyroid-

stimulating hormone level of 0.3 to 4.2 mU/L.

Tensubjects were recruitedfor the glycemia trial and13 for

the lipemia trial. All subjects finished the study. The baseline

characteristics of the subjects are explained inTable 1.

2.2. Study design

The study subjects acted as their own controls and consumed

the meals at a random order on 2 distinct study days, which

were at least 6 days apart from each other. Regarding the

evening preceding the study visits, the subjects were

instructed to eat a standardized evening snack with a lowflavonoid content, which consisted of wheat bread, cucumber,

water, and a banana. Before the study, thesubjects were given

information about the investigation, and they had an oppor-

tunity to ask questions. They were also informed of their right

to discontinue the study at any time without explanation. All

subjectsprovideda written consent.The study wasperformed

in accordance with the ethical standards laid down in the

Declaration of Helsinki. The protocol was evaluated and

approved by the ethics committee of the Hospital District of

Southwestern Finland.

2.3. Study meal composition

The meals contained yoghurt (lactose-free and fat-free

nonflavored natural yoghurt, 200 g; Valio Ltd, Helsinki,

Table 1Baseline characteristics of the male subjects

Glycemia Lipemia

Age (y) 24.7 4.6 25.6 5.0

BMI (kg/m2) 23.7 3.1 23.7 2.2

Fasting glucose (mmol/L) 5.25 0.28 5.08 0.36

Fasting triacylglycerols (mmol/L) 1.14 0.26 0.83 0.29

Values are means SD (n = 10 in the glycemia trial and n = 13 in the

lipemia trial). BMI, body mass index.

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Finland), 2.5 dL of water, and commercial dried lingonberry

powder (Mahevi Ltd, Polvijrvi, Finland). The amount of the

lingonberry powder was 40 g in the glycemia trial and 60 g in

the lipemia trial. When converted to fresh lingonberries,

according to the typical water content of the berries, the dose

roughly corresponds to about 270 and 400 g of fresh

lingonberries. The powder was easily incorporated into the

yoghurt. In addition to the lingonberry powder, the meal in

the glycemia trial contained 50 g glucose, and the meal in the

lipemia trial contained 35 g canola oil (Raisio plc, Raisio,

Finland). The control meals contained the above ingredients

excluding the lingonberry powder. The composition of the

meals is presented inTable 2. The subjects were offered an

additional 0.5 L of water to be consumed during the 6-hour

postprandial period. The subjects were advised and moni-

tored to consume the water in a similar manner during both

study visits.

2.4. Lingonberry powder

Commercial lingonberry powder made of Finnish lingon-

berries was obtained from Mahevi Ltd. Whole lingonberries

were used in the drying and milling, but the slight juice loss

during the process was not compensated for. Therefore, the

lingonberry powder was richer in seeds and skin of the berries

compared with whole lingonberries.

The total amount of flavonol glycosides, the main flavo-

nols, sugars, and acids, as well as the amount of dietary fiber

in the dried lingonberry powder, was analyzed as explained in

the following sections.

2.5. Analysis and composition of flavonol glycosides

Flavonol glycosides were analyzed with a method previously

reported by the authors[14]. In short, the homogenized berry

product was extracted once with 0.1% trifluoroacetic acid

water (Fluka, Deisenhofen, Germany) and twice with 0.1%

trifluoroacetic acidmethanol. The flavonol glycosides were

eluted from solid phase extraction tubes (C18 500mg, Supelco,

Bellefonte, PA, USA) with solid-phase extraction tubes with

trifluoroacetic acidmethanolwater (40% of 0.1% trifluoroa-

cetic acidwater, 60% methanol). The samples were analyzed

with ultra-high-performance liquid chromatographytandem

mass spectrometry in the positive-ion mode. Syringetin-3-

glucoside (Extrasynthese, Genay, France) was used as an

internal standard and isorhamnetin-3-O-glucoside and iso-

rhamnetin-3-O-rutinoside (Extrasynthese) as external stan-

dards. The phenolic compounds of lingonberry have also

been characterized in detail elsewhere [3], and their avail-

ability in humans has been assessed [21]. The lingonberry

powder contained 0.22 g of flavonol glycosides per 100 g of

powder. The main flavonol glycosides were kemferol-3-

glucoside, quersetin-3-rhamnoside, quersetin-3-galactoside,

and quersetin-3-glucoside.

2.6. Analysis of sugars and acids

The sugars and acids were analyzed as trifluoroacetic acid

derivatives of dried juice samples by gas chromatography

equipped with a Supelco Simplicity-1fused silica column and

a flame ionization detector [22]. Reference compounds

D-fructose,D-quinic acid, and L-ascorbic acid were purchased

from Sigma Chemical Co (St Louis, MO, USA). D-Glucose and

D-sorbitol (internal standard for sugars) were purchased from

Fluka (Buchs, Switzerland). Malic acid and D-tartaric acid

(internal standard for acids) were purchased from Merck

(Darmstedt, Germany), and sucrose and citric acid were from

JT Baker (Deventer, the Netherlands).

2.7. Analysis of dietary fiber

The amount of dietary fiber was determined by an enzymatic-

gravimetric method[2325]that measures both soluble and

insoluble dietary fibers. In short, the food samples were

defatted,heated to gelatinize thestarch, andthen subjected to

enzymatic digestion by protease, amylase, and glucoamylase

to remove the digestible components of the food. The amount

of protein and ash was determined, and the sum was

subtracted from the total residue. The remaining matter was

taken as dietary fiber.

2.8. Clinical analysis

Blood was drawn from the forearm at the fasting state and at

30, 60, 90, 120, 180, 270, and 360 minutes postprandially.

Glucose (VF-053SFX; Oriola, Helsinki, Finland) and serum

tubes with coagulant activator (VF-054SPW) were used in the

glycemia trial and lithium heparin tubes (VF-054SPV; Oriola)

in the lipemia trial. All samples were stored at 80C before

analysis. Plasma triacylglycerol and glucose as well as serum

insulin were analyzed with standard biochemical analyses.

Serum glucose and triacylglycerol were determined by a

photometric method and insulin by an electrochemilumines-

cence immunoassay. All analytes were measured from a

single tube with Roche Modular PPEE analyzer, with commer-

cial reagents provided by Roche Diagnostics GmbH (Mann-

heim, Germany).

Table 2Composition of meals given to men for theglycemic and lipemia trials

Ingredient Glycemia meal Lipemia meal

Lacto se-free, fat-free yoghurt 200 g 200 g

Glucose 5 g 5 g

Galactose 5 g 5 g

Fat 0.8 g 0.8 g Glucose 50 g

Canola oil 35 g

Water 2.5 g 2.5 g

Lingonberry powder 40 g 60 g

Citric acid 2.0 g 3.0 g

Quinic acid 2.0 g 3.0 g

Malic acid 0.1 g 0.2 g

Fructose 7.3 g 11.0 g

Glucose 7.3 g 11.0 g

Dietary fiber 14.7 g 23.9 g

Flavonol glycosides 89 mg 133 mg

Details on the analysis of components and the lingonberry powder

are described in the Methods and materials. The control meal

included all ingredients excluding the lingonberry powder.

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Fig The postprandial response of plasma glucose, insulin, and triacylglycerols to the study meal that contained glucose (A)

and to the study meal that contained triacylglycerols (B) with () and without () lingonberry powder (40 g in the glycemia trial

and 60 g in the lipemia trial). All meals included 200 g of lactose-free and fat-free yoghurt. Values arethe mean (SD) of 10 male

subjects in the glycemia trial and the mean ( SD) of 13 subjects in the lipemia trial. An asterisk (*) represents a significantbetween meal differences (P< .05).

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Fig (continued).

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2.9. Statistical analyses

Normal distribution of the data was tested using the Shapiro-

Wilk test. Paired-samples t test or Wilcoxon matched-pairs

signed rank test, depending on the normality of the data, was

used to compare the measured responses to control. Areas

under the response curves were calculated for insulin values

of the glycemia trial from baseline to 180 and 360 minutes, as

well as triacylglycerol values in the lipemia trial from baseline

to 120, 180, and 360 minutes. Values were expressed as means

SD. Statistical significance was indicated by P < .05.

Statistical analyses were performed with SPSS 18.0 software

(SPSS Inc, Chicago, IL, USA).

3. Results

3.1. Glycemia

The primary result of this study indicates that there were no

significant differences to the responses of the 2 meals in the

glycemia trial, although the sugar content of the lingonberry

meal (64.7 g) was considerably higher than that of the control

meal (50 g). No significant differences were detected in the

plasma glucose concentrations, and the glucose curves were

almost identical. Lingonberry powder somewhat shifted the

shape of the insulin curve, but the effect was not significant

(P = .205 at peak concentration). There were no significant

differences in the incremental areas under the insulin curves.

No significant differences were detected in the plasma

triacylglycerol concentrations, whereas triacylglycerols were

insignificantly higher at every postprandial time point after

the lingonberry meal compared with the control meal.

3.2. Lipemia

The plasma glucose response was significantly higher after

the lingonberry meal compared with the control meal at 30

minutes (P= .003), 60 minutes (P= .022), and 90 minutes (P=

.011) postprandially (Fig).

The insulin values were elevated at every postprandial

time point after the lingonberry meal compared with the

control meal, and the time points of 90 minutes (P= .006), 120

minutes (P = .046), 180 minutes (0.002) and 270 minutes (P

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