Lycopene as effective as statins for artery health: rabbit study

Related topics: Research, Antioxidants, carotenoids, Cardiovascular health

Supplements of lycopene, the carotenoid that give tomatoes their red color, may be as effective as "statin" drugs to reduce the formation of plaques in the arteries that cause atherosclerosis, says a new study with rabbits.

If the results of the study, published in the journal Nutrition, can be repeated in humans, they will add to a significant body of research supporting the potential heart health benefits of lycopene, predominantly associated with benefits for prostate health.

Lycopene is an antioxidant that is present in red and pink-colored fruits and vegetables. As well as being used as a food coloring, it is also used in supplements. The role of lycopene in heart health and in reducing the risk of certain cancers is supported by a body of research.

Min-Yu Hu and co-workers from the Central South University in Changsha, China divided 40 male adult rabbits into five equal groups to consume a standard diet, a high-fat diet, a high-fat diet plus 4 or 12mg per kg of lycopene, or the high-fat diet plus 10mg per kg of a "statin" drug (fluvastatin). The animals consumed the diets for eight weeks.

The researchers used a relatively high dose of lycopene (4 and 12 mg/kg of body weight) because rabbits reportedly do not absorb the nutrient efficiently.  These doses produced blood levels of the carotenoid of 0.19 and 0.24 moles per litre, respectively.

"This corresponds to low plasma levels of lycopene in humans who may achieve five-fold higher levels already with the intake of only 0.3 mg/kg of body weight," explained the authors.

At the end of the study, the animals fed only the high-fat diet had higher levels of total and LDL cholesterol, triglycerides, oxidized low-density lipoprotein, malonaldehyde, and interleukin-1 than animals fed the standard diet.

However, animals fed the high-fat diet and supplemented with lycopene or fluvastatin had improved levels of these biomarkers, and the lycopene of both doses was better than the statin.

"The results of our experiment in the high-fat diet rabbit model showed that lycopene and fluvastatin lowered serum levels of total cholesterol and LDL-cholesterol, improved lipid metabolism, and reduced the amount of triglycerides," wrote the authors.

"Lycopene intervention reduced the increase in oxidized LDL levels in rabbits on the high-fat diet, whereas fluvastatin did not show such an effect. The cause of this difference is at present not known, although the result speaks in favor of lycopene," they added.

"These findings provide a theoretical rationale for the use of lycopene as a preventive in atherosclerosis," they concluded.

Atherosclerosis, or hardening of the arteries, is a major risk factor for cardiovascular disease.  

"Comparison of lycopene and fluvastatin effects on  atherosclerosis induced by a high-fat diet in rabbits"

Authors: M.-Y. Hu, Y.-L. Li, C.-H. Jiang, Z.-Q. Liu, S.-L. Qu, Y.-M. Huang

Trial of Lycopene/Ateronon for Secondary Prevention of Coronary Heart Disease

Optimized Turmeric Extracts have Potent Anti-Amyloidogenic Effects
December 24, 2009

Inhibition of ß-amyloid (Aß) accumulation and Aß fibril (fAß) formation from Aß are attractive therapeutic targets for the treatment of Alzheimer's disease (AD). While previous studies have shown anti-amyloidogenic effects of curcumin in vitro and in vivo, no studies have examined optimized turmeric extracts enriched in curcuminoids or turmerones. Three standardized turmeric extracts, HSS-838, HSS-848, and HSS-888, were prepared with different chemical profiles to investigate their potential therapeutic benefits for AD. These extracts were fingerprinted by DART TOF-MS to reveal the significant chemical complexity. In addition four curcuminoids (curcumin, tetrahydrocurcumin, demethoxycurcumin and bisdemethoxycurcumin) were also examined. We measured the effects of the extracts and curcuminoids, on the aggregation of Aß by using a thioflavin T cell-free assay and the secretion of Aß from human neuronal cells (SweAPP N2A cells) in vitro. All three extracts and the curcuminoids showed dose-dependent inhibition of fAß aggregation from Aß_1-42 in the cell-free assay, with IC₅₀ values of = 5 µg/mL. However, only HSS-888, curcumin and demethoxycurcumin significantly decreased Aß secretion (~20%) in SweAPP N2A cells. Interaction matrices were used to examine possible synergistic interactions between HS-888 and the other extracts and the individual curcuminoids on Aß aggregation. Only simple additive effects were observed for the Aß aggregation inhibition, supporting the notion that the known curcuminoids are not strong inhibitors of this activity. However, HSS-888 showed strong inhibition of Aß aggregation and secretion, thus indicating that there are novel bioactive molecules in this extract that might be important leads for future AD drug discovery efforts.

Can reduced exposure to sunlight contribute to heart disease?

December, 3, 2009

We are fond of grumbling about grey skies, but there may be a good medical reason for doing so. It seems the dreary weather is bad for our hearts - worse, even, than raised cholesterol and an unhealthy diet.

That's the controversial claim being made by Dr David Grimes, a gastroenterologist from Blackburn. He's been gazing at the sky for 20 years for clues about why his patients get more sick than those in the south of the country.

And what he's found turns key assumptions about heart disease on their head. 'It's not diet or cholesterol levels that raise your risk of heart disease,' he claims. 'It's where you live. People in the north are more likely to be ill because they get less sunshine.' 

Weather woes: It is claimed dreary weather is bad for our health

Basically they are suffering from 'latitude' sickness. The link is vitamin D. While we get some from our diet, the main source is the sun - sunlight converts a compound in the skin into vitamin D, so the amount you make is directly related to the amount of sunshine you get.

In a new book Dr Grimes argues the higher the level of vitamin D in your blood, the lower your risk of heart disease and a range of other illnesses.

If he's right, what we need is not diet and lifestyle advice, but food fortified with vitamin D. For years the vitamin was thought to be useful only for preventing rickets.

Dr Grimes, who works at the Royal Blackburn Hospital, was one of the first to ask whether it had a wider role.

This was prompted by his observation that many Asian patients became ill after arriving in Britain, often suffering from a severe vitamin D deficiency.

His first clue was a 1988 study of three towns showing that if you lived in Ipswich you were least likely to have heart disease; your risk rose further north in Stoke-on-Trent and even further in Wakefield.

'If you believe poor diet strongly raises the risk of heart disease, then the obvious explanation is that the further north of London you go, the more unhealthily people eat -lots of fat and processed foods and too little fruit and vegetables,' says Grimes. 

But that didn't fit because the study also analysed diet in the three towns and found there was no major difference. In fact, those in Ipswich actually ate more fat, carbohydrates and sugar than people in the more northerly towns.

Most heart experts ignored the study because everyone 'knew' poor diet led to heart disease.

But for Grimes it was a lightbulb moment. He obtained figures for annual hours of sunshine in each of the three towns and discovered that the one with the lowest risk of heart disease - Ipswich - also had the most sunshine, 1,591 hours a year.

And the one with highest risk, Wakefield, had the least: 1,259 hours a year. But that was only three towns.

So Grimes checked sunshine levels in several of his neighbouring towns and found that moving from east to west produced as much variation as going north. While there's less sunlight generally in the north, local factors can also make a big difference.

Furthest west with most sunshine (1,476 hours) was Blackpool, which had the fewest deaths from heart disease. The fewest hours of sunshine (1,196 hours) were to be found 40 miles east in Burnley, which had the most heart disease deaths.

Blackburn is between the two and so were its hours of sunshine (1,281) and the death toll. When it came to local factors, he found that living on higher ground usually also means more intense sunlight energy compared with towns such as Blackburn or Burnley, which sit at the foot of some hills (as these encourage clouds).

Soak up the sun: We need the vitamin d to prevent a wide range of diseases

Anyone with darker skin is particularly at risk because they need longer sun exposure. 'We have a large Asian community in Blackburn, and they have a higher risk of heart disease and diabetes,' says Grimes.

'Of course, these are only correlations,' he acknowledges. 'They don't prove lack of sunshine causes heart disease.'

His theory would have to be tested by comparing what happened to heart rates when one group gets extra vitamin D while the other is given a placebo. In the meantime, he's been testing his patients' vitamin D levels.

'We've been doing many more vitamin D tests in recent years and finding a staggering level of deficiency.'

As a guideline, a reading below ten puts you at risk of rickets and some experts, including Grimes, believe you need more than 30 to be healthy. 'More than 90 per cent of my patients are below 30 and, shockingly, 40 per cent don't reach ten.'

So how does he treat them? 'You can do it with diet,' he says 'One Bangladeshi woman eats oily fish every day and now has a vitamin D blood level of 40. 'We give supplements of 1,000 international units (IU) a day or we can give an injection of 300,000 IU that lasts for a year.

'The patients respond well,' says Grimes 'but what's needed is a proper controlled, long-term trial and who is going to fund that? Not a drug company.'

Grimes had to stop his own research in 1990 when he became medical director of the hospital. But when he started again five years ago and began writing his book, he found he was no longer a lone voice.

Others were pointing to the damage too few hours of sunlight could do. Research has linked lack of vitamin D to a wide range of diseases, from cancer to diabetes and inflammatory conditions.

Only last week, researchers told the annual meeting of the American Heart Association that a study of more than 27,000 people aged 50- plus found that those with very low levels of vitamin D were 77 per cent more likely to die early than those with normal levels and 45 per cent more likely to have heart disease.

Last month researchers reported that giving the vitamin to pregnant women in a controlled trial reduced the risk of premature births and boosted the health of newborn babies.

The women also had a 25 per cent reduction in infections. Nutrition experts in the UK are moving slowly in Grimes's direction.

'His research ties in with other studies suggesting that higher levels of vitamin D are linked with better health outcomes,' says Dr Inez Schoenmaker, an expert in bone health with the Medical Research Council nutrition unit. 'But we need more research.

The Food Standards Agency is running studies in the North of Scotland and Surrey to discover the factors that most affect vitamin D levels in the blood.

'How much people get from sunlight, how much food contributes and what difference does being overweight make? Once there is more of an evidence base, higher daily amounts might be recommended.'

But if Grimes is right, it suggests we may need to rethink our approach to heart disease at least.

'At the moment public policy on many diseases involves blaming the victim,' he says. 'Patients are seen as architects of their own sickness because they didn't eat and live right. But what if they are just casualties of their geography?'

VITAMIN D And Cholesterol: The Importance Of The Sun, by Dr David Grimes 

Vitamin C Lowers Levels Of Inflammation Biomarker Considered Predictor Of Heart Disease

 A study led by researchers at the University of California, Berkeley, adds to the evidence that vitamin C supplements can lower concentrations of C-reactive protein (CRP), a central biomarker of inflammation that has been shown to be a powerful predictor of heart disease and diabetes. The same study found no benefit from daily doses of vitamin E, another antioxidant.

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That trial does not necessarily close the books on the benefits of vitamin C for cardiovascular health, according to Gladys Block, UC Berkeley professor emeritus of epidemiology and public health nutrition and lead author of the study looking at vitamins C and E and their impact on CRP levels. She pointed out that the Brigham and Women's Hospital study did not screen study participants for elevations in CRP - defined by the American Heart Association as 1 milligram per liter or greater - which is an important distinction in determining who might benefit from taking vitamin C.

The study led by Block, currently online and scheduled to appear in the Jan. 1 issue of the journal Free Radical Biology and Medicine, shows that for healthy, non-smoking adults with an elevated level of CRP, a daily dose of vitamin C lowered levels of the inflammation biomarker after two months compared with those who took a placebo. However, participants who did not start out with elevated CRP levels saw no benefit from vitamin C supplementation.

"This is an important distinction; treatment with vitamin C is ineffective in persons whose levels of CRP are less than 1 milligram per liter, but very effective for those with higher levels," said Block. "Grouping people with elevated CRP levels with those who have lower levels can mask the effects of vitamin C. Common sense suggests, and our study confirms, that biomarkers are only likely to be reduced if they are not already low."

The researchers said that for people with elevated CRP levels, the amount of CRP reduction achieved by taking vitamin C supplements in this study is comparable to that in many other studies of cholesterol-lowering drugs called statins. They noted that several larger statin trials lowered CRP levels by about 0.2 milligrams per liter; in this latest study, vitamin C lowered CRP by 0.25 milligrams per liter.

"This finding of an effect of vitamin C is important because it shows in a carefully conducted randomized, controlled trial that for people with moderately elevated levels of inflammation, vitamin C may be able to reduce CRP as much as statins have done in other studies," said Block.

Evidence of the link between elevated CRP levels and a greater risk of heart disease has grown in recent years, but it had been unclear whether the beneficial effects of lowering CRP were independent of the effects of lowering cholesterol.

Newly released results from a multinational clinical trial help answer that question. Led by researchers at Harvard Medical School, the study, known as the Jupiter trial, found that statins reduced cardiovascular mortality and morbidity among people whose cholesterol levels were normal, but whose levels of CRP were greater than 2 milligrams per liter. The Jupiter trial found that among people who had such high levels of CRP at baseline, levels of CRP were 37 percent lower with statins compared with a placebo.

"One of the strengths of the Jupiter trial is that only persons with CRP levels greater than 2 milligrams per liter were enrolled," Block added. "Researchers found very important effects of lowering CRP in people who had high levels to begin with."

In the UC Berkeley study on vitamin C, participants who started out with CRP levels greater than 2 milligrams per liter had 34 percent lower levels of CRP with vitamin C compared with a placebo.

The UC Berkeley study also found a strong link between obesity and elevated levels of CRP. The researchers found that while 25 percent of normal-weight people had elevated levels of CRP, those levels were found in 50 percent of overweight and 75 percent of obese participants.

"The low-grade inflammation that characterizes obesity is believed to contribute to a number of disorders, including atherosclerosis and insulin resistance," said Nina Holland, adjunct professor at UC Berkeley's Division of Environmental Health Sciences and co-investigator on the study. Holland's biorepository at UC Berkeley processed and stored the thousands of blood samples involved in this study.

Notably, the American Heart Association and the U.S. Centers for Disease Control and Prevention recommends that clinicians measure CRP levels in patients who have a moderately elevated risk of cardiovascular problems, as determined by other established risk factors such as high cholesterol levels and smoking.

"Major studies have found that the level of CRP in the body predicts future risk of cardiovascular disease, including myocardial infarction, stroke and peripheral artery disease, as well as diabetes," said Block. "Some believe CRP to be as important a predictor of future heart problems as high levels of LDL and low levels of HDL cholesterol."

The UC Berkeley-led study looked at the separate effects of two antioxidants: vitamin C and vitamin E. The researchers randomly divided 396 healthy, non-smoking adults from the San Francisco Bay Area into groups taking daily doses of either 1,000 milligrams of vitamin C, 800 international units of vitamin E or a placebo. The recommended dietary allowance (RDA) for vitamin C is 90 milligrams per day for men and 75 milligrams per day for women. The researchers noted that the suggested upper limit for vitamin C is 2,000 milligrams per day, or twice the level used in the study.

They compared participants' baseline CRP levels with their levels two months later, at the end of the study. Fewer than half of the participants in the study started with elevated levels of CRP.

Participants who had baseline CRP levels less than 1 milligram per liter saw no significant effect on CRP levels after taking vitamin C supplements. However, those who started off with CRP levels of 1 milligram per liter or higher saw a 16.7 percent drop in levels after two months of treatment with vitamin C.

The researchers found no significant results for those taking vitamin E. They are uncertain as to why vitamin E did not show an effect even though it is also an antioxidant. Block noted that these vitamins have other functions independent of their antioxidant properties. Or, perhaps the difference relates to the fact that vitamin E is fat soluble and thus found in cell membranes while vitamin C is water soluble and found in intercellular fluid, the researchers said.

Although this study ended at two months, Block noted that there is no evidence to date of adverse effects for longer-term use of vitamin C at high levels. At the same time, researchers acknowledged the need to study whether vitamin C's beneficial impact on CRP levels continue past two months.

"This is clearly a line of research worth pursuing," said Block. "It has recently been suggested by some researchers that people with elevated CRP should be put on statins as a preventive measure. For people who have elevated CRP but not elevated LDL cholesterol, our data suggest that vitamin C should be investigated as an alternative to statins, or as something to be used to delay the time when statin use becomes necessary."

The National Institutes of Health helped support this research.

Pain Controlling and Cytokine-regulating Effects of Lyprinol, a Lipid Extract of Perna Canaliculus, in a Rat Adjuvant-induced Arthritis Model

¹State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Shenzhen and Department of Applied Biology and Chemical Technology and ²Department of Health Technology and Informatics, Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China

	Abstract

Top Abstract Introduction Methods Results Discussion Conclusion Acknowledgements References

 
Using an adjuvant-induced arthritis rat model, we investigated the effects of a lipid extract of Perna canaliculus (Lyprinol®) on pain. Radiological examinations, as well as levels of pro- and anti-inflammatory (AI) cytokines, were measured aiming to provide independent objective data to the pain controlling investigation. We confirmed the ability of Lyprinol® to control pain at the initial phase of its administration; with similar efficacy to that observed with Naproxen. The pain scores slowly increased again in the group of rats treated with Lyprinol® after day 9–14. The Naproxen-treated rats remained pain-free while treated. Both Naproxen and Lyprinol® decreased the levels of the pro-inflammatory cytokines TNF- and IFN-, and increased that of IL-10. Extra-virgin olive oil was ineffective on cytokine secretion. Rats treated with Lyprinol® were apparently cured after 1 year. This study confirms the AI efficacy of this lipid extract of P. canaliculus, its initial analgesic effect, its perfect tolerance and its long-term healing properties.

Keywords: adjuvant-induced arthritis – inflammatory cytokines – Lyprinol® – NZGLM, pain score

	Introduction

Top Abstract Introduction Methods Results Discussion Conclusion Acknowledgements References

A lipid-rich and stabilized extract of New Zealand green-lipped mussel Perna canaliculus powder (NZGLM, Lyprinol®), has been shown to have significant anti-inflammatory (AI) activity when given to animals and humans (1–11). This article contributes to the emerging literature on bioprospecting (12). This approach seeks to emphasize for complementary and alternative medicine (CAM) the utility of products derived from animals in treating certain diseases (13–17). Lyprinol® is a mixture of five main lipid classes including sterol esters, triglycerides, free fatty acids, sterols and polar lipids (18,19). There are 90 component fatty acids in Lyprinol® (18,19). Omega-3 polyunsaturated fatty acids are one of the most abundant PUFAs. Eicosapentaenoic acid (20 carbons and 5 double bonds, shorthand = 20 : 5 omega-3) (EPA) and docosahexaenoic acid (22 : 6 omega-3) (DHA) are the major omega-3 PUFAs present in Lyprinol.

Anti-inflammatory Effects

Although the exact active ingredients that brought about this AI effect are unknown, Whitehouse et al. (3) reported that Wistar and Dark Agouti rats treated p.o. with this lipid extract did not develop adjuvant-induced polyarthritis or collagen (II)-induced auto-allergic arthritis. This was achieved with doses inferior to the ones of NSAIDs, and 200 times lower than other seed or fish oils (3). Further, omega-3 PUFA subfractions of this lipid extract inhibited LTB₄ biosynthesis by neutrophils in vitro, and PGE₂ production by activated macrophages (20). Subfractions of this extract containing natural antioxidants (e.g. carotenoids) also exhibited AI activity (5). In contrast to NSAIDs, Lyprinol® is non-gastro-toxic in disease-stressed rats at 300 mg kg^–1 p.o. (3), and does not affect platelet aggregation in both humans and rats (3).

Clinical studies, either controlled or randomized, have demonstrated that Lyprinol® has highly significant AI activity in patients with osteoarthritis (OA) (4,7,8,21), asthma (22) and other inflammatory conditions (10). Therefore, it seems that Lyprinol® is a reproducible, stable source of bioactive lipids with much greater potency than plant/marine oils currently used as nutritional supplements to ameliorate signs of inflammation (3,10,23). More importantly, in humans and animal subjects taking Lyprinol®, there are no reported side-effects, even at doses up to 2500 mg per day in patients.

Given the AI activities of Lyprinol®, we were interested to know if these AI effects could be translated into pain-controlling effects. We conducted a series of experiments to understand the AI mechanism of action of this lipid extract. This article reports our findings on the pain scores and the accepted causes of pain in rats subjected to severe arthritis induced by adjuvant. In comparison to extra-virgin olive oil (vehicle/negative control) and to the NSAID Naproxen (positive control), we confirmed the pain-controlling ability of Lyprinol® at the initial phase of its administration. This ability is similar to the one observed with Naproxen. Cytokine levels in/from splenocytes of these animals, as well as radiological examination of the affected joints, were also measured to bring objective confirmation to the pain controlling investigation.

	Methods

Top Abstract Introduction Methods Results Discussion Conclusion Acknowledgements References

Unless stated otherwise, all chemicals were purchased from Sigma (St Louis, MO, USA). All chemicals were at least of AR grade. Organic solvents used were at least of HPLC grade.

Induction of Inflammation in Sprague-Dawley Rats

Four groups of six 6-week-old male Sprague-Dawley (SD) rats (normal group, adjuvant-induced arthritis (AIA) group, AIA + Naproxen group and AIA + Lyprinol group) and two groups of three 6-week old SD rats (AIA group and AIA + long-term Lyprinol feeding group, respectively) were purchased from the Central Animal Facility (CAF) of Hong Kong Polytechnic University (HKPU). All rats were kept and cared under conditions that fully met requirements of Procedures for the Care of Laboratory Animals or Animals (Control of Experiments) Regulations Chapter 340 of the Hong Kong SAR government. Ethics approval (ASESC No.04/9) was from The Animal Subjects Ethics subcommittee of the HKPU. Arthritis was induced in anesthetized rats by administration of adjuvant according to a method previously described (24,25). Briefly, on Day 0, each rat was injected into the paw of the right hind limb with 100 µl of Freund's complete adjuvant containing 10 mg ml^–1 of Mycobacterium butyricum (Difco, Livonia, MI, USA). Another six rats without arthritis induction were observed as a normal group.

Products/Drug Tested Fed to the Treatment and Control Groups of Rats

Rats in the Lyprinol® (Pharmalink International Ltd, Burleigh Heads, QLD, Australia) group were fed by oral gavage at a dosage of 25 mg Lyprinol kg^–1 body weight. Typically, the required amount of Lyprinol was made up with olive oil (Virgin®, Bertolli, Italy) to 300 µl and then force-fed to the rats with a stainless steel stomach tube. Three hundred micro liters of olive oil, and 20 mg kg^–1 body weight of Naproxen were fed as vehicle and positive control. Normal chow was provided to all rats. Except for the long-term Lyprinol-feeding group, feeding of Lyprinol, olive oil and Naproxen were discontinued after the experimental period of 28 days. For the long-term Lyprinol-feeding group, the required amount of diluted Lyprinol (to a dosage of 25 mg kg^–1 body weight) was added onto 25 g of dry rat chow. These chows were fed to rats daily. Once these chows were completely consumed, normal chows were given.

Pain Score Measurement

The measurement of pain score was performed according to Hayashida et al. (26). The number of pain-related responses, represented by vocalizations, was recorded during 10 flexions of the tarsotibial joints of the adjuvant-injected paw. Results were expressed as the mean number of vocalizations.

Splenocyte Preparation

At Day 14 and 28 after arthritis induction, splenocytes of each rat of group P. canaliculus, Naproxen, olive oil and control were prepared as described previously (27). Briefly, spleens were removed aseptically and minced into tiny pieces. Single cell suspensions were prepared by gentle grinding of spleen pieces in RPMI 1640 medium (Life Technologies, US). Splenocytes (mostly B- and T-cells) from the crude spleen cell suspension were recovered by using Ficoll-Hypaque® Plus lymphocyte isolation kit (Pharmacia Biotech, USA) as described in the manufacturer's manual. Recovered splenocytes (1.5 ml) were transferred into a sterile centrifuge tube and four volumes of pre-chilled 0.83% ammonium chloride (NH₄Cl) was added and incubated for 10 min for lysis of residual erythrocytes (28). Splenocytes were recovered by centrifugation at 3000 rpm for 5 min, washed with 8 ml pre-warmed (37°C) sterile PBS buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄·7H₂O, 1.4 mM KH₂PO₄, pH 7.4) and finally resuspended in appropriate volume of complete RPMI 1640 medium (10% fetal bovine serum, 100 units ml^–1 of penicillin and 100 µg ml^–1 of streptomycin, supplemented with 0.3 mg/ml L-glutamine and 25 mM HEPES).

Cell Count and Viability Staining

Resuspended splenocytes were stained with 0.4% Trypan blue exclusion dye (0.4 g Trypan blue in 100 ml PBS buffer) at ratio 1: 1. Number of splenocytes was counted and calculated with the aid of a hemocytometer. More than 99% of splenocytes were viable. Except stated otherwise, splenocytes were diluted to a working population of 5 x 10⁶ cells ml^–1 in all experiments.

ELISA Assay for Cytokines

A total of 5 x 10⁶ splenocytes ml^–1 were seeded in 24-well plate; a suboptimal concentration (1.25 µg ml^–1) of lipopolysaccharide (LPS) (Sigma, St Louis, MO, USA) was used to prime the splenocyte culture. Splenocytes were incubated in 37°C incubator with 80% humidity and 5% CO₂ atmosphere condition. Supernatant was collected at 10 and 24 h after LPS activation and stored at –80°C until later measurement. Interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) were measured by enzyme-liked immunosorbent assay (ELISA) (BioSource, CA, USA) as described in the user's manual.

Statistical Analysis

Experimental data were compared to the ones of the control group and analyzed by Student's t-test with one way ANOVA.

	Results

Top Abstract Introduction Methods Results Discussion Conclusion Acknowledgements References

Figure 1 shows photographs of the right hind paw of rat at different times after AIA induction. Figure 1A was taken at 14 days after AIA induction. Swelling of the whole right hind paw, especially around the tarsotibial joints, is significant as shown in the photo. X-ray photography of the same paw at Day 21 after AIA reveals severe periarticular soft tissue swelling, joint subluxation and periostal new bone formation (Fig. 1B). The degree of arthritis between control group and Lyprinol® group shows no difference during the whole 28-day course of the experiment. However, 1 year after the induction of AIA, deformation of joints and lesions are still observable in the olive oil-control group (Fig. 1C). Conversely, deformation of joints and lesions are not visible in the Lyprinol®-long-term treatment group (Fig. 1D).

View larger version (94K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 1. Photographic and radiographic analysis of adjuvant-induced arthritis (AIA) rats. (A) Photograph of the right hind paw taken 14 days after AIA induction. Swelling and inflammation of the paws were observed, especially around the ankle region. (B) Radiographic analysis of the same right hind paw at Day 21 after AIA induction. Note the swelling of soft tissues as well as bone deformation around the ankle region. (C) Photograph of the right hind paw in one of the representative rats in the olive oil group at 1 year after AIA induction. Toes and footpad are deformed, with multiple lesions. (D) However, rats fed with Lyprinol®-for 1 year recovered much better. Photograph of the right hind paw in one of the representative rats in the Lyprinol® treatment group showed only mild joint deformation and no lesion.

Pain score measurement is a widely used and reliable method to reflect the effectiveness of different treatments on AIA (26). It is one of the commonly used methodologies to measure level of pain (29,30,31). As shown in Fig. 2, the NSAID Naproxen effectively maintained the pain score of AIA rats at a relatively low level during the whole course of the experiment. Compared to the ones of the control and olive oil-treated groups, Lyprinol® effectively lowered the pain score of AIA rats from Day 4 to Day 26 after induction of arthritis. The effect of Lyprinol® was most pronounced between Day 4 and Day 12, with an effect comparable to that of Naproxen. The effect of Lyprinol® began to wane after Day 14, but still maintained the pain score at a significantly lowered level when compared to the control and olive oil-treated groups. Our results clearly demonstrated that Lyprinol® is very useful in controlling pain of severe arthritis, especially during the early and intermediate phase of AIA.

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 2. Mean pain score measured by 10 flexions of the tarsotibial joints of adjuvant-injected paw (n = 6), as described in Methods. Note that Lyprinol® effectively controlled pain between Day 4 and Day 12 when compared to control and olive oil groups.

At Day 14 after induction of arthritis, splenocytes from Lyprinol®-fed rats were harvested and pro-inflammation cytokines levels were determined. As shown in Fig. 3, TNF- level of Lyprinol® group was greatly decreased from 3.07 ± 0.168 to 1.71 ± 0.405 ng ml^–1. Besides, production of another pro-inflammatory cytokine, IFN-, was significantly decreased. As shown in Fig. 4, the IFN- level of the Lyprinol® group, when compared to control, was dramatically decreased from level 10.7 ± 0.8 to 3.0 ± 0.9 ng ml^–1.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 3. Amount of tumor necrosis factor-alpha (TNF-) produced by LPS-stimulated splenocytes 14 days after arthritis induction. Lyprinol® controlled the amount of TNF- production to a level similar to the one found in rats with no arthritis, and was as effective as the NSAID Naproxen. Values shown here are mean ± SEM (n = 6). *P < 0.05.

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 4. Amount of interferon-gamma (IFN-) produced by LPS-stimulated splenocytes 14 days after arthritis induction. Lyprinol® effectively reduced the amount of IFN- produced, when compared to control and olive oil groups. Strikingly, Lyprinol® was even more potent than the NSAID Naproxen in controlling the IFN- level. Values shown are mean ± SEM (n = 6). *P < 0.05; **P < 0.001.

Figure 5 shows that the AI cytokine IL-10 increased at Day 28 after AIA. Although there is no statistically significant difference between the levels observed in the control group and the Lyprinol® group, the increase of IL-10 in the Lyprinol® group almost reached levels observed in the positive control group (Naproxen).

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 5. Amount of interleukin-10 (IL-10) produced by LPS-stimulated splenocytes after 28 days of arthritis induction. Lyprinol® can increase the amount of IL-10 production, an anti-inflammatory cytokine, although the difference was not statistically significant. Values shown are mean ± SEM (n = 6).

	Discussion

Top Abstract Introduction Methods Results Discussion Conclusion Acknowledgements References

Since the major complaint of patients suffering from acute exacerbations of chronic inflammatory disease is pain, we evaluated the analgesic effect of the lipid extract in the AIA rat model. We also measured levels of pro-inflammatory and anti-inflammatory cytokines. We compared results observed of Lyprinol, with a sham group (no drugs), an olive oil group (negative control), a non-steroid AI drug (NSAID) and a Naproxen (positive control) group. We also observed a group of rats as reference that were not treated with adjuvant.

Recent studies on pain, both in humans (33–36) and animals (34,37,38) have demonstrated the active role of IL-6 in inducing pain, as well as IL-1ß, IL-8, TNF- (TNF-R1 receptor) and IFN-. Conversely, IL-10 is associated with control of pain.

Lyprinol® exhibits a significant effect controlling pain in several recent clinical studies. The group at the University of Hong Kong (8) conducted the first randomized controlled trial to evaluate the effects of Lyprinol®, on signs and symptoms and quality of life (QOL) of patients with knee OA. Their results showed that Lyprinol® over 6 months in patients with knee OA is perfectly tolerated and associated with a decrease in pain perception after 2 months when compared to placebo. The visual analog scale (VAS) for pain score was significantly reduced from 63.0 at baseline to 55.5 at Week 4 (P = 0.046), 51.2 at Week 8 (P = 0.003) and 49.7 at Week 12 (P = 0.001). There was a greater significant reduction in VAS pain score following adjustment for change in amounts of paracetamol (acetaminophen) used in patients who received the lipid extract when compared with controls at Week 8 (P = 0.035), Week 12 (P = 0.032) and Week 24 (P = 0.045).

Another multi-center clinical study in Korea on patients with OA of the hip and knee found that the average VAS score, Lequesne functional index, global assessment by patients and global assessment by physician were all significantly improved (7). After a 4- and 8-week treatment period, 53–80% of patients experienced significant pain relief, and improvement of joint function. There was no reported adverse effect during this clinical trial (7).

A third study was conducted in Germany on 50 adults with inflammatory rheumatoid arthritis (21). The number of painful joints was reduced from 4.18 ± 2.80 at baseline by 0.30 ± 0.81 points (7.2%) after 6 weeks and 0.60 ± 1.18 points (14.4%) after 12 weeks (P = 0.012 and P = 0.001). For a sub-parameter ‘number of painful small joints’, a highly significant reduction of 9.3% after 6 weeks and 18.6% after 12 weeks was observed (P = 0.022 and P = 0.002). Analgesic medications were reduced or stopped in 21 subjects who required these at the beginning of the trial.

Our study of the mean pain score measurement by ten flexions of the tarsotibial joints of adjuvant-injected paw (n = 6) did demonstrate a significant reduction of pain in AIA rats treated with Lyprinol®, or Naproxen. However, this reduction differed according to the product used to treat the rats: pain scores in Naproxen and Lyprinol® groups initially increased; but they decreased significantly and gradually in the Lyprinol® group to reach a nadir (score = 2) at Day 8, while rats in the Naproxen group experienced a more erratic pain reduction with a significant nadir (score = 0) at Day 9. However, the pain score started differing significantly after Day 9: the Lyprinol® group experienced a progressive increase in pain scores, reaching its initial level at Day 15; while the Naproxen group remained essentially pain-free during the rest (total days = 26) of the experiment. This curve, transposed to the human time scale, is quasi-identical to the pain scale observed by Lau et al. (8) where the maximum reduction in pain was observed after approximately 8–12 weeks of treatment in patients with OA. It may well be due to an ‘escape’ mechanism from the production of pro-inflammatory cytokines that tend to increase despite treatment with Lyprinol®; it may also be due to other inflammatory mechanisms of AIA (and/or OA).

The cause of pain severity was confirmed by aspects of the inflamed paw when compared with the non-injected one, and comparative radiological images. X-rays confirmed the presence of major osteo-cartilaginous destruction as well as major swelling of the affected paw.

Pro-inflammatory cytokine production (TNF-, IFN-) was significantly controlled by Lyprinol® (Day 14), as well if not better than Naproxen. IL-10, an ‘anti-inflammatory’ cytokine was increased in the Lyprinol® and in the Naproxen group, but did not reach statistical significance. Extra-virgin olive oil was ineffective in our study. More importantly, after 1 year, rats that were treated with Lyprinol® had a normal AIA paw, while untreated rats had severe inflammatory, ulcerative lesions.

	Conclusion

Top Abstract Introduction Methods Results Discussion Conclusion Acknowledgements References

 
The lipid extract of P. canaliculus is known as a powerful AI product in animal models and human diseases (asthma, arthritis). We have confirmed in the adjuvant-induced arthritis rat model, that this product (Lyprinol®) is an analgesic, as active as Naproxen during the early phase of treatment. AIA is a loco-regional highly inflammatory experimental condition, with systemic repercussions. Lyprinol® also controls as well as Naproxen the levels of pro-inflammatory cytokines (TNF-, IFN-) in AIA rats; it increases the production of the AI cytokine IL-10 in AIA rats. Lyprinol® was perfectly tolerated, and AIA rats treated with Lyprinol® were lesion-free after 1 year, while untreated controls had major inflammatory, ulcerative lesions. The lipid extract of P. canaliculus seems to be uniquely safe and effective in the treatment of pain, both spontaneous (due to inflammation) and triggered by torsion of the affected joint, in AIA in rats.

	Footnotes

	Acknowledgements

Top Abstract Introduction Methods Results Discussion Conclusion Acknowledgements References

	References

Top Abstract Introduction Methods Results Discussion Conclusion Acknowledgements References

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33. Beilin B, Bessler H, Mayburd E, Smirnov G, Dekel A, Yardeni I, et al. Effects of preemptive analgesia on pain and cytokine production in the postoperative period. Anesthesiology ( 2003;) 98:: 151–5.[CrossRef][Web of Science][Medline]
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    Dietary Omega-3 Fatty Acids, Cyclooxygenase-2 Genetic Variation, and Aggressive Prostate Cancer Risk

Authors' Affiliations: Departments of ¹ Urology and ² Epidemiology and Biostatistics and ³ Institute for Human Genetics, University of California at San Francisco, San Francisco, California and ⁴ Department of Preventive Medicine, University of Southern California, Los Angeles, California

Abstract

Purpose: Dietary intake of long-chain -3 (LC n-3) polyunsaturated fatty acids may reduce inflammation and in turn decrease risk of prostate cancer development and progression. This potential effect may be modified by genetic variation in cyclooxygenase-2 (COX-2), a key enzyme in fatty acid metabolism and inflammation.

Experimental Design: We used a case-control study of 466 men diagnosed with aggressive prostate cancer and 478 age- and ethnicity-matched controls. Diet was assessed with a semiquantitative food frequency questionnaire, and nine COX-2 tag single nucleotide polymorphisms (SNP) were genotyped. We used logistic regression models to estimate odds ratios (OR) for association and interaction.

Results: Increasing intake of LC n-3 was strongly associated with a decreased risk of aggressive prostate cancer (P_trend 0.0001). The OR (95% confidence interval) for prostate cancer comparing the highest with the lowest quartile of n-3 intake was of 0.37 (0.25-0.54). The LC n-3 association was modified by SNP rs4648310 (+8897 A/G), flanking the 3' region of COX-2 (P_interaction = 0.02). In particular, the inverse association was even stronger among men with this variant SNP. This reflected the observation that men with low LC n-3 intake and the variant rs4648310 SNP had an increased risk of disease (OR, 5.49; 95% confidence interval, 1.80-16.7), which was reversed by increasing intake of LC n-3.

Conclusions: Dietary LC n-3 polyunsaturated fatty acids appear protective for aggressive prostate cancer, and this effect is modified by the COX-2 SNP rs4648310. Our findings support the hypothesis that LC n-3 may impact prostate inflammation and carcinogenesis through the COX-2 enzymatic pathway. 
 

PROTECT YOURSELF AND YOUR FAMILY

Consumer Guide to Mercury in Fish

The list below shows the amount of various types of fish that a woman who is pregnant or planning to become pregnant can safely eat, according to the Environmental Protection Agency. People with small children who want to use the list as a guide should reduce portion sizes. Adult men, and women who are not planning to become pregnant, are less at risk from mercury exposure but may wish to refer to the list for low-mercury choices.

Protecting yourself -- and the fish: Certain fish, even some that are low in mercury, make poor choices for other reasons, most often because they have been fished so extensively that their numbers are perilously low. These fish are marked with an asterisk (read more below).

This list applies to fish caught and sold commercially. For information about fish you catch yourself, check for  advisories in your state.

LEAST MERCURY
Enjoy these fish:
Anchovies
Butterfish
Catfish
Clam
Crab (Domestic)
Crawfish/Crayfish
Croaker (Atlantic)
Flounder*
Haddock (Atlantic)*
Hake
Herring
Mackerel (N. Atlantic, Chub)
Mullet
Oyster
Perch (Ocean)
Plaice
Pollock
Salmon (Canned)**
Salmon (Fresh)**
Sardine
Scallop*
Shad (American)
Shrimp*
Sole (Pacific)
Squid (Calamari)
Tilapia
Trout (Freshwater)
Whitefish
Whiting

MODERATE MERCURY
Eat six servings or less per month:
Bass (Striped, Black)
Carp
Cod (Alaskan)*
Croaker (White Pacific)
Halibut (Atlantic)*
Halibut (Pacific)
Jacksmelt
(Silverside)
Lobster
Mahi Mahi
Monkfish*
Perch (Freshwater)
Sablefish
Skate*
Snapper*
Tuna (Canned
chunk light)
Tuna (Skipjack)*
Weakfish (Sea Trout)

HIGH MERCURY
Eat three servings or less per month:
Bluefish
Grouper*
Mackerel (Spanish, Gulf)
Sea Bass (Chilean)*
Tuna (Canned Albacore)
Tuna (Yellowfin)*

HIGHEST MERCURY
Avoid eating:
Mackerel (King)
Marlin*
Orange Roughy*
Shark*
Swordfish*
Tilefish*
Tuna
(Bigeye, Ahi)*

* Fish in Trouble! These fish are perilously low in numbers or are caught using environmentally destructive methods. To learn more, see the Monterey Bay Aquarium and the Blue Ocean Institute, both of which provide guides to fish to enjoy or avoid on the basis of environmental factors.

** Farmed Salmon may contain PCB's, chemicals with serious long-term health effects.

Sources for NRDC's guide: The data for this guide to mercury in fish comes from two federal agencies: the Food and Drug Administration, which tests fish for mercury, and the Environmental Protection Agency, which determines mercury levels that it considers safe for women of childbearing age.

About the mercury-level categories: The categories on the list (least mercury to highest mercury) are determined according to the following mercury levels in the flesh of tested fish.

• Least mercury: Less than 0.09 parts per million
• Moderate mercury: From 0.09 to 0.29 parts per million
• High mercury: From 0.3 to 0.49 parts per million
• Highest mercury: More than .5 parts per million