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Can chitosan affect the absorption of unsaturated fatty acid?

Can chitosan affect the absorption of unsaturated fatty acid?



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I know chitosan will decrease the absorption of fat. However unsaturated fatty acid, such as DHA, is beneficial. Does chitosan effect the absorption of unsaturated fatty acid?


It seems that there is no difference in the effects of chitosan on the uptake of saturated and unsaturated fatty acids. The study cited below (which has been done on guinea pigs) fed the animals a diet which contained different digestion-resistant fibers (maltodextrin, cellulose and chitosan) and also fats. Then the fatty acid content of the feces was measured afterwards.

Only for the group which got additional feeding of chitosan showed a significant increase in the excreted fatty acids. This was true for the saturated (lauric, myristic and palmitic acid) but also for the unsaturated fatty acids (Oleic, linoleic and α-linolenic acid). This effect is specific for fatty acids, The uptake of neutral sterols and bile acids was not affected in any of the experimental groups.

The second paper suggests that chitosan forms an emulsion with fatty acids under the acidic conditions of the stomach. This emulsion is stable and later excreted with the feces.

References:

  1. Selective in vivo effect of chitosan on fatty acid, neutral sterol and bile acid excretion: a longitudinal study.
  2. Interaction between chitosan and oil under stomach and duodenal
    digestive chemical conditions.

Qualitative and Quantitative Tests for Lipids

A large number of heterogenous compounds are referred to as lipids including fats, steroids, waxes, and related compounds, which are related more by their physical than their chemical prop­erties.

They have the common property of being:

(1) Relatively insoluble in water and

(2) Soluble in polar solvents such as ether and chloroform.

Fatty acids are aliphatic carboxylic acids. If the aliphatic chain contains no double bond then it is called saturated and if it contains one or more double bond it is called unsaturated. Most naturally occurring unsaturated fatty acids have cis-double bonds. Some of the most common fatty acids are palmitic acid and stearic acid. Palmitic has 16 carbon atoms and stearic has 18 carbon atoms.

As it is clear from the formulae, both are saturated fatty acids. Some fatty acids like oleic acid may be unsaturated. Naturally occurring animal fats consist largely of mixed glyceride of oleic, palmitic and stearic acids. They are usually mixture of individual fats. Fats have more saturated fatty acids whereas oils have more of unsaturated ones.

Lipids are simple, complex or derived. Simple lipids are esters of fatty acids with various alcohols, e.g., fats (esters of fatty acids with glycerol) and waxes (esters of fatty acids with higher molecular weight of monohydric alcohols). Complex lipids are esters of fatty acids containing groups in addition to an alcohol and a fatty acid, e.g., phospholipids or glycolipids etc. Derived lipids include fatty acids, glycerol, steroids, other alcohols, fatty aldehydes, and ketone bodies, lipid soluble vitamins, and hormones.

Phospholipids yield in addition to alcohol and fatty acids, phosphate and a nitrogenous base like choline, ethanolamine, etc. Lecithin’s and cephalous are representatives of the phospholipids. Similarly glycolipids contain carbohydrates, and sulpholipids contain sulphate. Lipoproteins are com­binations of lipids with proteins.

Now we will consider some qualitative and quantitative tests for lipids.

Qualitative Tests:

I. Physical Test:

Take a small amount of oil on a piece of paper, a greasy spot penetrating the paper will be formed. This happens because lipid does not wet paper unlike water.

2. Test for free fatty acids:

Take a few drops of phenolphthalein solution in a test tube and add to it one or two drops of very dilute alkali solution, just sufficient to give the solution a pink colour. Now add a few drops of the oil and shake. The colour will disappear as the alkali is neutralized by the free fatty acids present in the oil.

Oil or liquid fat becomes finely divided and is dispersed in water when shaken with water to form emulsification. Emulsification is permanent and complete in the presence of emulsifying agent. The important emulsifying agents are bile salts, proteins, soaps, mono- and diglycerides. Emulsification is important in the processes of fat digestion in the intestine. Emulsifying agents lower surface tension of the liquid.

Take 2 clean and dry test tubes, in one test tube added 2 ml water and in other 2ml dilute bile salt solution. Now to each tube added 2 drops of mustard oil and shaken vigorously for about one minute. Allow the tubes to stands for two minutes and note that the water, oil is broken in small pieces and floats on the surface where as in the bile salt solution, the oil can be seen in minute droplets suspended in the liquid (permanent emulsification).

4. Saponification test:

Esters can be hydrolysed by alkali to yield the parent alcohol and salt. When the fatty acid possesses a long chain the salt formed is a soap which we commonly use. This process is called saponification. Oils and fats usually contain long chain fatty acids and are, therefore, the starting materials for the preparation of soap.

Take 1 ml of the oil in a test tube and add an equal amount of alcoholic KOH solution, mix them thoroughly and keep the mixture during the course of warming and shake up gently with a little distilled water. Appearance of some oil drops will indicate the incomplete saponi­fication. After complete saponification no oil drops will appear.

5. Tests for unsaturation of fatty acids:

Unsaturated fatty acids like oleic acid can react with halogens like bromine and iodine due to presence of double bonds as shown below.

The amount of Br2 or I2 taken up will indicate the amount of unsaturation present in a particu­lar acid. Approximate idea about the unsaturation in a different oils and fats can be obtained by the following test. Set up four clean and dry test tubes each containing 5 ml of CCl4.

To the first, add one drop of shark liver oil, to the second, one drop of coconut oil, to the third, a drop of vegetable ghee and add nothing to the fourth tube. Now test for the unsaturation of the added oil by adding bromine water drop by drop to each tube followed by shaking.

Record the number of drops required to obtain a permanent yellowish red colour in each tube and infer the relative unsaturation in the three samples used. It may be mentioned here, vegetable ghee is prepared by hydrogenating vegetable oil. Hydrogenation means saturation of unsaturated fatty acid by hydrogen.

6. Isolation of free fatty acids from soap:

Take a few ml of 20% H2SO4 in a test tube and gradually add 5 ml of some soap solution. The fatty acids will separate out in a distinct layer due to the hydrolysis of the soap.

Cool the solution which will become hot and skim off the surface layer and wash it several times with water till free from H2SO4. Then dissolve it in some water and add alkaline phenolphthalein solution and shake. The pink colour will be discharged indicating the presence of free fatty acids.

Calcium soap formation:

To a small amount of the soap solution in a test tube add CaCl2 solution. A white precipitate will be formed. The white precipitate is due to insoluble calcium salt of fatty acid. This is referred to as calcium soap.

To a small amount of the soap solution in a test tube add lead acetate solution, a white precipitate will appear. The white ppt is due to insoluble lead salt of fatty acids. This is referred to as lead soap.

Take pure glycerol in a dry test tube add to it a few crystals of potassium hydrogen sulphate. Warm gently to mix and then heat strongly. A very pungent odour of acrolein is produced. Acrolein is formed due to removal of water from glycerol by potassium hydrogen sulphate.

Take in a dry test tube 3 or 4 ml of glycerol solution, to it add a few drops of 5% potassium dichromate solution and 5 ml of conc. HNO3, mix well and note that the brown colour is changed to blue. This test is given by the substances containing primary and secondary alcohol groups. The chromic ions oxidize the glycerol and in this process they are reduced to chromous ions which give the blue colour. This test is also given by reducing sugars, so before confirming glycerol be sure that the reducing sugars are not present.

Quantitative Tests:

1. Determination of Iodine Number:

The iodine number of a fat is the amount in gm. of iodine taken up by 100 gm. of fat. Not only iodine but also equivalent amounts of other halogens will add at double bonds so bromine is often used instead of iodine because it is more reactive. The halogenating reagent used in this method is pyridine sulphate di-bromide. This reagent can be prepared by adding carefully 8.1 ml pyridine in 20 ml glacial acetic acid and making the volume up to 1 litre with glacial acetic acid.

Weigh the bottle containing sample of oil plus a medicine dropper and then transfer about 0.1 to 0.3 gm. of oil to a flask. Reweigh the bottle containing oil and dropper to find out the exact quantity of the sample transferred. Add 10 ml of chloroform and then 25 ml of the pyridine sulphate di-bromide reagent.

Shake thoroughly allow standing for 5 minutes and then determining the residual bromine. To do this, add 10 ml of 10% KI and titrate the equivalent amount of iodine liberated by the residual bromine with the help of 0.1 (N) Na2S2O3 (sodium thiosulphate). The titration can be done by adding sodium thiosulphate solution through a burette to the flask.

When the colour of the solution in flask becomes light yellow add 1 ml of starch solution. It will become blue. Slowly add the thiosulphate solution again till it becomes colourless. Note the total volume of thiosulphate used.

The total amount of bromine originally added is found by titrating 25 ml of the pyridine sulphate di-bromide reagent with thiosulphate after adding KI as in the previous case. The amount of bromine taken up by the fat sample can be determined by the difference between the two titers and then the iodine number can be calculated.

Suppose with a sample of 0.2 gm. oil the data obtained are as follows:

0.1 (N) Na2S2O3 used for titration of blank = 47.0 ml

0.1 (N) Na2S2O3 used for titration of sample = 27.0 ml

0.1 (N) Na2S2O3 equivalent to iodine absorbed by the sample = 20.0 ml

As 1 ml 0.1 (N) Na2S2O3 = 1.0 ml of 0.1 (N) Bromine = 1 ml of 0.1 (N) Iodine

Hence, 20 ml of 0.1 (N) Na2S2O3 = 20 ml of 0.1 (N) Iodine = 20合.7/1000 gm Iodine = 0.254 gm Iodine.

Thus 0.2 gm of oil can take up 0.254 gm of iodine.

Therefore, iodine number of oil used = 127.

Qualitative Test of Cholesterol:

Cholesterol is a lipid with a structure quite different from that of phospholipids. It is a steroid, built from four linked hydrocarbon rings. A hydrocarbon tail is linked to the steroid at one end, and a hydroxyl group is attached at the other end. In membranes, the molecule is oriented parallel to the fatty acid chains of the phospholipids, and the hydroxyl group interacts with the nearby phospholi­pid head groups.

Cholesterol is absent from prokaryotes but is found to varying degrees in virtually all animal membranes. It constitutes almost 25% of the membrane lipids in certain nerve cells but is essentially absent from some intracellular membranes.

It is found in bile and a common constituent of gall stones.

The main test for cholesterol is known as Liberman-Burchard test. This is carried in the follow­ing way. In a dry test tube take a small amount of solution of cholesterol in chloroform. Add 1 ml of acetic anhydride and 1 drop of conc. H2SO4. Mix and observe that a purple colour is formed which soon changes to green. It may take 15-30 min for full development and it is advisable to put the tube in dark during this time.

Quantitative Estimation of Cholesterol:

The above mentioned qualitative test has been developed quantitatively for the estimation of cholesterol.

Follow the following protocol for the purpose:

Shake the tubes well and keep them at room temperature for 30 minutes. Blue colour will develop in all the tubes except blank tube. Measure the absorbencies at 625 m|a. against the blank tube and plot these against the amount of cholesterol.

Note: Acetic anhydride-sulphuric acid reagent.

This reagent has to be freshly prepared before use. Acetic anhydride (20 ml) is taken in a glass stoppered flask which is then chilled in ice water. When cold, add 1 ml of conc. H2SO4 to it drop by drop. The contents are mixed and cooled during the addition. After completion of the addition the flask is stoppered and shaken vigorously for a few minutes. The solution has to be kept cold in ice and should be used within an hour.

Enzymatic Methods:

Assays have been developed in which cholesterol oxidase obtained from the bacterium Nocardia erythropolis is used to convert cholesterol into cholest-4-en-3-one with the formation of Hydrogen peroxide. The cholest-4en-3-one formed has been measured by reading at 240 nm after extracting into isopropanol. Alternatively, hydrogen peroxide has been quantified by formation of chelate complex with quadrivalent titanium and xylenol orange.

Other Tests for Cholesterol:

1. Salkowski’s Test (H2SO4 Test):

Dissolve cholesterol in 2 ml of chloroform in dry test tube. Add equal amount of con. H2SO4. Shake gently. The upper layer turns red and the sulphuric acid layer shows a yellow colour with a green fluorescence.

2. Formaldehyde-H2SO4 Test:

Add 2 ml of formaldehyde-sulphuric acid solution (1 part of 40% formaldehyde to 50 parts of the acid) to 2 ml of chloroform solution in a dry test tube. The cherry colour is developed in the chloroform. Pour off the chloroform in another test tube and add 2-3 drops of acid anhydride. The blue colour develops.


Effect of different animal fat and plant oil additives on physicochemical, mechanical, antimicrobial and antioxidant properties of chitosan films

Practical application of chitosan-essential oil blend films is limited due to the uneconomical extraction procedure of essential oils from plants. This study aimed to produce chitosan films blended with low cost and commercially available oils and fats consumed in daily human diet (olive, corn and sunflower oils, butter and animal fats). The study also focused on how physicochemical, biological and mechanical properties of chitosan blend films were influenced by the incorporation of oils and fats with varying unsaturation degrees. Possible interactions of chitosan film matrix with incorporated oils or fats were investigated. Chitosan-olive oil film showed better surface morphology and higher thermal stability than the films with other unsaturated oils. Tensile strength, Young's modulus and elongation at break were improved by 57.2%, 25.1% and 31.7% for chitosan-olive oil film, respectively. Chitosan-olive oil blend film had the highest antibacterial activity (almost equal to that of commercial antibiotic gentamicin). Edible films obtained from by incorporation of natural oils and fats into chitosan can help produce an environmentally friendly packaging material that is low cost and easily manufactured.


Abstract

The unsaturated fatty acid (linoleic acid) sustained-release microspheres were prepared with linoleic acid (LA) using alginate–chitosan microcapsule technology. These LA sustained-release microspheres had a high encapsulation efficiency (up to 62%) tested by high performance liquid chromatography with a photo diode array. The dry microspheres were characterized by a scanning electron microscope, X-ray diffraction measurement, dynamic thermogravimetric analysis and Fourier transform infrared spectral analysis. The results of characterization showed that the microspheres had good thermal stability (decomposition temperature of 236 °C), stable and temperature independent release properties (release time of more than 40 d). Compared to direct dosing of LA, LA sustained-released microspheres could inhibit Microcystis aeruginosa growth to the non-growth state. The results of this study suggested that the LA sustained-release microspheres may be a potential candidate for algal inhibition.


2. Chitosan

Chitosan is a linear polysaccharide composed of two repeated units, D-glucosamine and N-acetyl-D-glucosamine, linked by β-(1𡤤)-linkages, characterized in terms of intrinsic properties such as molecular weight, viscosity, and degree of deacetylation ( Figure 1 ). Chitosan is a collective name for a group of the partially or fully deacetylated biopolymer chitin, it is a natural compound, non-toxic, biocompatible, biodegradable, bioactive, muco-adhesive, and has been identified as safe for use in food in Japan (1983), Korea (1995), and the United States (Food and Drug Administration 2012). It is a high molecular weight poly-cationic polymer, the second most abundant polysaccharide in nature, and is present in the structural exoskeleton of insects, crustaceans, mollusks, cell walls of fungi, and certain algae, but largely obtained from marine crustaceans [30]. Several gigatons of crustacean shell are produced annually and the extraction of chitin (10 6 � 7 tons), chitosan, and protein from this waste has added value [31,32]. It has antimicrobial properties against bacteria, filamentous fungi, and yeast, and even has virus, anti-inflammatory, antitumor activity, antioxidative activity, anticholesterolemic, hemostatic, and analgesic effects [33,34]. The application of chitosan either alone or blended with other natural polymers can be done in several ways such as silage inoculants, food processing, food preservation, textile, biotechnology, water treatment, pharmaceutical, tissue engineering, and the cosmetics industry [35,36]. Recent research in animal nutrition has focused on its potential to modulate rumen fermentation in beef or dairy cattle [37,38,39,40,41] and nutrient digestibility in cattle. The chitosan extraction process can be carried out in a chemical or biological way. The chemical method at an industrial scale starts with demineralization to eliminate the calcium carbonate and calcium chloride deproteinization decolorization (mainly astaxanthin and β-carotene) and finally alkaline deacetylation using sodium or potassium hydroxide [35,42,43]. The biological way, which is considered environmentally safe, uses lactic acid for demineralization, deproteinization by proteases, decoloration with acetone or organic solvents, and finally deacetylation by bacteria. In recent years, new extraction methodologies have been developed with the use of microwave irradiation [43]. The quality of the final product depends upon the raw material (crustaceans species), process of extraction, and seasonal variations [36,44].


Chitosan: a marine dietary fibre to fight lifestyle-related diseases

In recent years, consumers have become increasingly interested in the use of natural, alternative approaches to enhance their health and quality of life, reports Dr Hélène L. Lauzon, R&D Director, Primex

Lifestyle-related diseases such as obesity, hyperlipidaemia, atherosclerosis, type 2 diabetes and hypertension are widespread in industrialised countries, threatening our cardiovascular health. As a result, our ageing population is facing major medical and socioeconomic problems.

Metabolic syndrome has emerged as a combination of metabolic disorders, including abdominal obesity, hypertriglyceridaemia, a low level of high-density lipoprotein (HDL) cholesterol, hypertension and a high fasting-glucose level, leading to an increase in cardiovascular morbidity and mortality. 1

There are many risk factors associated with cardiovascular disease: some cannot be altered whereas others can be modified by direct action. Several of these modifiable risk factors are diet-related, which means that it’s possible to voluntarily enhance an individual’s health by making basic changes that lead to a healthier lifestyle. A diet that’s high in saturated fat increases the risk of heart disease and stroke. It is estimated to cause about 31% of coronary heart disease and 11% of strokes worldwide.

Abnormal blood lipid levels, including high total cholesterol, high levels of triglycerides, high levels of low-density lipoprotein (LDL) or low levels of HDL-cholesterol, all increase the risk of heart disease and stroke. Hypertension is the single biggest risk factor for stroke, playing a significant role in heart attacks. Obesity is a major risk for cardiovascular disease and predisposes you to diabetes. 2 Ways and means to control these health-related parameters are being sought.

A marine dietary fibre for cardiovascular health

An increased focus has been put on the use of natural, alternative approaches for disease prevention and therapeutic applications to enhance health and quality of life. Marine biomolecules are among those being considered. Chitosan is a natural dietary fibre and a deacetylated form of chitin that can be obtained from the shells of crustaceans as a by-product.

Chitin is one of the world’s most abundant natural polymers. Chitosan is a copolymer of glucosamine and N-acetyl-glucosamine, which is soluble in acidic media following protonation, resulting in its unique cationic and bioactive nature. Chitosan has been demonstrated to possess several biological properties. 3

Dietary fibres are differentiated according to their water solubility, which is related to their structure. Soluble fibres increase viscosity and reduce both plasma cholesterol and the glycaemic response, whereas insoluble fibres are porous, contributing to faecal bulk and decreased intestinal transit time. 4 Chitosan is considered to be an insoluble fibre but, it will dissolve in stomach acid and become soluble and viscous, behaving like a soluble fibre.

On transiting to the intestine, the higher pH will cause it to gel and become less soluble, contributing to faster transit times and reduced putrefactive activity. This is advantageous because rapid intestinal transit is linked to higher energy recoveries by the host owing to increased bacterial metabolite production in the colon. 5 Chitosan chelates fat and reduces cholesterol. 6,7

Xu, et al. suggest that chitosan improves lipid metabolism in rats by modifying total cholesterol and LDL-cholesterol levels by up-regulating hepatic LDL receptor mRNA expression, increasing the excretion of faecal bile acids. 5 In fact, the European Commission Panel on Dietetic Products, Nutrition and Allergies has concluded that a cause and effect relationship has been established between the consumption of chitosan (3g daily) and the maintenance of normal blood LDL-cholesterol concentrations. 8

Fat-binding capacity and fat selectivity of chitosan

In the dietary supplement industry, chitosan is used to prevent dietary fat absorption. Fat complexation or entrapment is a function of chitosan solubility in the acidic stomach environment and insolubility at basic intestinal pH levels.

Solubilised chitosan mixes with fat and subsequently forms a semi-solid emulsion under basic pH conditions that is resistant to digestion and absorption. A useful in vitro test to evaluate the fat-binding capacity of chitosan under acidic condition should be based on well-defined quantity and time-dependent solubility parameters to allow a fair comparison between commercial products. Further, the type of fat/oil tested may influence the outcome owing to the varying fatty acid composition of oils and fat commonly used in the food industry.

Saturated fatty acids (SFAs) are known to have a hypercholesterolaemic effect on lipid metabolism, whereas polyunsaturated fatty acids (PUFAs) are hypocholesterolaemic and of major importance in normal physiological functions. Despite the fact that some PUFAs are not produced in our body and therefore considered to be essential, excessive amounts of omega-6 PUFAs and very high omega-6/omega-3 ratios in our diet may promote the pathogenesis of many diseases associated with pro-inflammatory and prothrombic mediators. 9

Therefore, the reduction of this ratio is important to approach more desirable levels. The common use of fat that is rich in omega-6 PUFAs in our food production renders this task difficult. Linoleic acid (C18:2omega-6) as well as oleic acid (C18:1omega-9) are the major unsaturated fatty acids present in all oils. 10 It is noteworthy that ChitoClear chitosan, produced by PRIMEX, has been shown to selectively bind to cholesterol and fats, specifically SFAs and omega-6 PUFAs. 11

Intestinal bioconversion of cholesterol and bile acids is therefore inhibited by ChitoClear chitosan. Furthermore, the study reported that the ratio of omega-6/omega-3 fatty acids in faeces was significantly increased by ChitoClear chitosan, indicating that ChitoClear chitosan could contribute to balancing the ratio of omega-6/omega-3 essential fatty acids in our body.

Efficacy of Liposan Ultra as a fat binder

As the composition of dietary fat can influence our lipid metabolism, it is important to consider how chitosan will perform in binding different types of fats. This was recently investigated by PRIMEX, an Icelandic marine biotech company manufacturing high quality chitin and chitosan products.

LipoSan Ultra chitosan, a safe and effective dietary supplement for weight management and cholesterol control, is a unique, patented product (US Patent No. 6,130,321) produced by PRIMEX. LipoSan Ultra rapidly dissolves in the stomach, complexes and traps fats and oils, thereby reducing the digestion of dietary fat and limiting the calorie intake. The superior efficacy of LipoSan Ultra compared with other products implies that it can be taken just before or during a meal as a convenient weight management product.

This is demonstrated in Figure 1 in which an in vitro fat-binding test performed at room temperature and based on 0.1g of chitosan product and 10g of oil is used to assess fat-binding, with a maximum binding efficacy of 100g of oil by 1g of chitosan product. As shown, the solubilisation time (1–60 min) can be varied to mimic the time in the stomach acid before oil or fat addition and to demonstrate different fat-binding capacities among different products, reflecting the time needed before intake for maximal activity during a meal.

Figure 1: Fat-binding capacity of different chitosan products as influenced by solubilisation time in acid

Using this test, LipoSan Ultra's fat-binding capacity is found to be 99–100g (oil)/g(product). Any higher (>100g [oil]/g[chitosan]) fat-binding capacity cannot be evidenced unless a slightly lower ( 11 They observed that ChitoClear chitosan, used in the preparation of LipoSan Ultra, selectively reduced fat absorption and had a greater binding affinity to fatty acids with higher polarities.

ChitoClear chitosan significantly increased the excretion of lauric (C12:0) and myristic (C14:0) acids, highly atherogenic saturated fatty acids, compared with other dietary fibres (cellulose and digestion-resistant maltodextrin). It also bound well to palmitic (C16:0), stearic (C18:0) and linoleic (C18:2omega-6) acids based on the fatty acid profile given for the diet and that recovered from faeces.

The fact that a fat or oil that is rich in oleic acid (olive oil) or medium-chain fatty acids (coconut oil) is less well captured by LipoSan Ultra is noteworthy, considering their health promoting effects. A higher intake of oleic acid decreases LDL-cholesterol but does not affect HDL-cholesterol levels. 10

Coconut oil contains caprylic and capric acids, which are referred to as medium-chain fatty acids (MCFAs) and found in medium-chain triglycerides (MCTs) as MCFA esters of glycerol. MCTs are hydrolysed rapidly and the resulting MCFAs are absorbed directly into the liver and used as an energy source. A recent review indicated that experimental studies in animal and human subjects have shown that dietary MCFAs/MCTs suppress fat deposition through enhanced thermogenesis and fat oxidation. Furthermore, several reports suggest that MCFAs/MCTs offer the therapeutic advantage of preserving insulin sensitivity in animal models and patients with type 2 diabetes. 12

Health benefits of ChitoClear Chitosan and Liposan Ultra

Both the in vitro fat-binding test and the Santas, et al. study provide additional information regarding the usefulness of chitosan in preventive and therapeutic treatments. Earlier studies have shown that a daily dose (3g) of LipoSan Ultra led to a significant weight loss (1kg) and reduced body mass index (BMI) in treated subjects (overweight, mildly obese women, 21-55 years old) adhering to a non-restrictive diet for 8 weeks compared with a 1.5kg weight gain and increased BMI in the placebo group. 13

A 6-month study assessed the supplementation of a low calorie diet (1000kcal/day) with ChitoClear chitosan (1.5g, three times a day). 14 Significantly higher body weight loss and a decrease in systolic and diastolic blood pressure were noted in the chitosan group. Therefore, chitosan can be used as a valuable and safe adjunct in the long-term dietary treatment of obesity, enhancing the reduction of blood pressure associated with weight reduction. Further, regular consumption will contribute to the maintenance of normal blood LDL-cholesterol concentrations. 8 Considering these benefits, supplementing our diet with chitosan can contribute to cardiovascular health.

1. S.M. Grundy, et al., 'Diagnosis and Management of Metabolic Syndrome,' Circulation 112, 2735–2752 (2005).

3. I. Aranaz, et al., 'Functional Characterization of Chitin and Chitosan,' Curr. Chem. Biol. 3, 203–230 (2009).

4. K.L. Roehrig, 'The Physiological Effects of Dietary Fiber: A Review,' Food Hydrocolloids 2, 1–18 (1988).

5. G.T. Macfarlane and S. Macfarlane, 'Fermentation in the Human Large Intestine: Its Physiologic Consequences and the Potential Contribution of Prebiotics,' J. Clin. Gastroenterol. 45, S120–S127 (2011).

6. R.M.N.V. Kumar, “A Review of Chitin and Chitosan Applications,” React. Funct. Polym. 46, 1–27 (2000).

7. G. Xu, et al., 'Mechanism Study of Chitosan on Lipid Metabolism in Hyperlipidemic Rats,' Asia Pac. J. Clin. Nutr. 16, 313–317 (2007).

8. EFSA Journal 9(6),2214-2235: doi:10.2903/j.efsa.2011.2214 (2011).

9. F.H. Chilton, et al., 'Mechanisms by Which Botanical Lipids Affect Inflammatory Disorders,' Amer. J. Clin. Nutrition 87, 498S (2008).

10. R.C. Zambiazi, et al., 'Fatty Acid Composition of Vegetable Oils and Fats,' B.CEPPA, Curitiba 25(1), 111–120 (2007): http://www.nononsensecosmethic.org/wp-content/uploads/2015/01/fatty-acid-oil-composition.pdf.

11. J. Santas, et al., 'Selective In Vivo Effect of Chitosan on Fatty Acid, Neutral Sterol and Bile Acid Excretion: A Longitudinal Study,' Food Chem. 134, 940–947 (2012).

12. K. Nagao and T. Yanagita, 'Medium-Chain Fatty Acids: Functional Lipids for the Prevention and Treatment of the Metabolic Syndrome,' Pharmacol. Res. 61, 208–212 (2010).

13. R.N. Schiller, et al., 'A Randomized, Double-Blind, Placebo-Controlled Study Examining the Effects of a Rapidly Soluble Chitosan Dietary Supplement on Weight Loss and Body Composition in Overweight and Mildly Obese Individuals,' J. Amer. Nutrac. Ass. 4(1), 42–49 (2001).

14. B. Zahorska-Markiewicz, et al., 'Effect of Chitosan in Complex Management of Obesity,' Pol. Merkur Lekarski. 13(74), 129-132 (2002).


Mobility of Membrane Lipids and Lipid Asymmetry (With Diagram)

Much more is known about the specific lipid composi­tion of cell membranes, because the lipids are more readily extracted from the membranes using a variety of organic solvents.

Once extracted from isolated membranes, the lipids may be separated and identi­fied using chromatographic or other procedures.

Nearly all the membranes studied so far appear to contain the same types of lipid molecules.

Phospholip­ids such as phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidyl choline (lecithin), and sphingomyelin are the most common constituents, but cholesterol is also present.

Table 15-2 lists the most common lipids found in a variety of cell membranes and also shows their protein-to-lipid weight ratios the latter vary considerably. In addition to its widespread occurrence in plasma membranes, cholesterol is also found in many intracellular membranes. The rigid (i.e., planar) nature of cholesterol imparts an ordering effect to those cellular membranes that contain this lipid.

Mobility of Membrane Lipids:

Lipids exhibit a higher degree of mobility in mem­branes than do proteins, although lateral mobility is very much greater than transverse (“flip-flop”) mobil­ity. A single lipid molecule may move several microns laterally through the membrane in just 1 or 2 seconds! The mobility of lipid and protein molecules in the plasma membrane attests to the membrane’s fluidity. C. F. Fox and H. M. McConnell have shown that the degree of fluidity is dependent, in turn, on the fatty acid contents of side chains of phospholipids in the membrane.

Fatty acid side chains of membrane phospholipids can be either saturated or unsaturated. In saturated side chains, all the carbon-carbon bonds are single, with the remaining carbon bonds carrying hydrogen atoms in unsaturated side chains, one or more pairs of neighboring carbon atoms are linked by double bonds.

In phospholipid layers consist­ing exclusively of saturated fatty acids, the side chains are aligned next to one another in an ordered, crystal­line array the result is a relatively rigid structure (Fig. 15-15). In phospholipid layers consisting of a mixture of saturated and unsaturated fatty acid side chains, the packing of neighboring molecules is less orderly (and therefore more fluid).

The double bonds of the unsaturated side chains produce bends in the hydrocarbon chains, and these give rise to structural deformations that prevent formation of the more rigid crystalline structure. The greater the number of dou­ble bonds, the more disordered (and fluid) is the lipid bilayer (Fig. 15-15).

The rigidity of lipid layers is also affected by tem­perature. Almost everyone is familiar with the “melt­ing” of fats and waxes at elevated temperatures. To maintain membrane fluidity, cells living at low tem­peratures have higher proportions of unsaturated fatty acids in their membranes than do cells at higher temperatures. Evidence also exists suggesting that cells can alter the balance of saturated and unsatu­rated fatty acids in their membranes as an adjustment to changing temperature or other factors.

In recent years, the degree of membrane fluidity has been linked to the capability of various metabo­lites and hormones to bind to surface receptors. An in­crease in membrane fluidity may be accompanied by the withdrawal of exposed receptors (i.e., they are drawn deeper into the lipid bilayer), whereas a de­crease in membrane fluidity is accompanied by greater accessibility of the receptor through in­creased exposure above the bilayer.

Lipid Asymmetry:

The various membrane lipids are not equally distrib­uted in both monolayers, although the asymmetry is not nearly as marked as in the case of protein. The dis­tribution of lipids in the erythrocyte membrane is shown in Table 15-3 and reveals that the choline phos­phatides are primarily in the outer monolayer and the amino phosphatides are in the inner monolayer.

Al­though lipid asymmetry is a general property of mem­branes, the type of asymmetry varies considerably from one membrane to another. Asymmetry, once es­tablished, is most likely maintained because of the high activation energy that would be required to move the polar groups through the hydrophobic center of the bilayer.

Just as proteins are differentially distrib­uted in the plasma membrane areas comprising the various functional faces of a tissue cell, so are the lip­ids. This is vividly seen in Figure 15-16, which shows the distributions of phosphatidyl choline, sphingo­myelin, phosphatidyl ethanolamine, and phosphatidyl serine in the three major plasma membrane regions (faces) of the liver parenchymal cell.


RESULTS

Fatty Acid Composition of Wild-Type anddesA + Cells

We investigated changes in the fatty acid composition of glycerolipids after transformation of Synechococcus cells with the desA + gene for Δ12 desaturase (Table I). The most abundant fatty acids in wild-type cells were 16:0 (49% of the total fatty acids) and 16:1(9) (41%). However, we also found low levels of 18:0, 18:1(9), and 18:1(11) in wild-type cells. In desA + cells 16:2(9, 12) appeared (15%) at the expense of 16:1(9), suggesting that some of the 16:1(9) had been desaturated to 16:2(9, 12). IndesA + cells, 18:2(9, 12) accounted for 3% of the total fatty acids. These results indicate that the wild-type cells contained only saturated and mono-unsaturated fatty acids, whereas the desA + cells contained, in addition, di-unsaturated fatty acids such as 16:2(9, 12) and 18:2(9, 12).

Fatty acid composition of wild-type Synechococcus sp. PCC 7942 and of desA + cells after growth at 32°C

Strain . Fatty Acid .
16:0 . 16:1 (9) . 16:2 (9,12) . 18:0 . 18:1 (9) . 18:1 (11) . 18:2 (9,12) . 18:2 (?) .
mol %
Wild type 49 41 0 2 4 4 0 0
desA +48 25 15 2 3 1 3 3
Strain . Fatty Acid .
16:0 . 16:1 (9) . 16:2 (9,12) . 18:0 . 18:1 (9) . 18:1 (11) . 18:2 (9,12) . 18:2 (?) .
mol %
Wild type 49 41 0 2 4 4 0 0
desA +48 25 15 2 3 1 3 3

Positions of double bonds were not determined for 18:2(?). Each value represents the average of results from four independent experiments. Experimental deviations were within 2% for 16:0, 16:1 (9), and 16:2 (9,12) and within 0.5% for the other fatty acids.

Fatty acid composition of wild-type Synechococcus sp. PCC 7942 and of desA + cells after growth at 32°C

Strain . Fatty Acid .
16:0 . 16:1 (9) . 16:2 (9,12) . 18:0 . 18:1 (9) . 18:1 (11) . 18:2 (9,12) . 18:2 (?) .
mol %
Wild type 49 41 0 2 4 4 0 0
desA +48 25 15 2 3 1 3 3
Strain . Fatty Acid .
16:0 . 16:1 (9) . 16:2 (9,12) . 18:0 . 18:1 (9) . 18:1 (11) . 18:2 (9,12) . 18:2 (?) .
mol %
Wild type 49 41 0 2 4 4 0 0
desA +48 25 15 2 3 1 3 3

Positions of double bonds were not determined for 18:2(?). Each value represents the average of results from four independent experiments. Experimental deviations were within 2% for 16:0, 16:1 (9), and 16:2 (9,12) and within 0.5% for the other fatty acids.

Inactivation of Photosystem II under Salt Stress and Osmotic Stress

Changes in the oxygen-evolving activity of PSII in wild-type and desA + cells during incubation with NaCl, LiCl, and sorbitol. Cells were incubated in darkness or in light at 70 μE m −2 s −1 in the presence of 0.5 m NaCl (A), 0.5 m LiCl (B), or 1.0 m sorbitol (C). At designated times, a portion of the cell suspension was withdrawn. The oxygen-evolving activity was measured after addition of 1.0 m m BQ to the suspension. The activities of wild-type and desA + cells that corresponded to 100% were 548 ± 30 and 576 ± 35 μmol O2 mg −1 Chl h −1 , respectively. ▪, Wild-type cells in darkness ■, wild-type cells in light ●,desA + cells in darkness ○,desA + cells in light. Each point and bar represent the average ± se of results from four independent experiments.

Changes in the oxygen-evolving activity of PSII in wild-type and desA + cells during incubation with NaCl, LiCl, and sorbitol. Cells were incubated in darkness or in light at 70 μE m −2 s −1 in the presence of 0.5 m NaCl (A), 0.5 m LiCl (B), or 1.0 m sorbitol (C). At designated times, a portion of the cell suspension was withdrawn. The oxygen-evolving activity was measured after addition of 1.0 m m BQ to the suspension. The activities of wild-type and desA + cells that corresponded to 100% were 548 ± 30 and 576 ± 35 μmol O2 mg −1 Chl h −1 , respectively. ▪, Wild-type cells in darkness ■, wild-type cells in light ●,desA + cells in darkness ○,desA + cells in light. Each point and bar represent the average ± se of results from four independent experiments.

When similar experiments were performed with illumination at 70 μE m −2 s −1 , the oxygen-evolving activity in both wild-type anddesA + cells declined rapidly, as observed in darkness. However, light had a striking effect, namely, restoration of the oxygen-evolving activity after the initial decline. This effect was more pronounced in desA + than in wild-type cells, and in desA + cells the oxygen-evolving activity was fully restored within 2 h. In wild-type cells, by contrast, the extent of the restoration of activity was more limited. These observations indicated that PSII ofdesA + cells was more resistant to salt stress than the PSII of wild-type cells and that the difference was especially pronounced under illumination.

We obtained similar results when cells were incubated in the presence of 0.5 m LiCl in darkness or in light (Fig. 1B). However, the PSII complex in both wild-type anddesA + cells was inactivated to a greater extent by 0.5 m LiCl than by 0.5 m NaCl. The ability of light to restore the oxygen-evolving activity was minimal in wild-type cells. By contrast, in desA + cells, activity returned to approximately 50% of the original level within 3 h and remained at this level for the remainder of the 10-h incubation.

During incubation of cells with 1.0 m sorbitol for 1.5 h, the oxygen-evolving activity declined both in darkness and in light to approximately 30% and 70% of the original level in wild-type anddesA + cells, respectively (Fig. 1C). No restoration of activity during the subsequent 8.5-h incubation was observed in darkness in either type of cell. However, in light at 70 μE m −2 s −1 , the oxygen-evolving activity returned to the original high level within 3 h in desA + cells. In wild-type cells, only 50% of the original activity was regained. These observations suggest that desA + cells were also more tolerant than wild-type cells to osmotic stress.

Inactivation of Photosystem I under Salt Stress and Osmotic Stress

Changes in PSI activity in wild-type anddesA + cells during incubation with NaCl, LiCl, and sorbitol. Cells were incubated in darkness or in light at 70 μE m −2 s −1 in the presence of 0.5 m NaCl (A), 0.5 m LiCl (B), or 1.0 m sorbitol (C). At designated times, a portion of the cell suspension was withdrawn. The PSI activity was measured by monitoring the uptake of oxygen after addition of 15 μ m DCMU, 0.1 m m DCIP, 5 m m sodium ascorbate, and 0.1 m m MV to the suspension. The activities of wild-type and desA + cells that corresponded to 100% were 314 ± 27 and 332 ± 30 μmol O2 mg −1 Chl h −1 , respectively. ▪, Wild-type cells in darkness ■, wild-type cells in light ●,desA + cells in darkness ○,desA + cells in light. Each point and bar represent the average ± se of results from five independent experiments.

Changes in PSI activity in wild-type anddesA + cells during incubation with NaCl, LiCl, and sorbitol. Cells were incubated in darkness or in light at 70 μE m −2 s −1 in the presence of 0.5 m NaCl (A), 0.5 m LiCl (B), or 1.0 m sorbitol (C). At designated times, a portion of the cell suspension was withdrawn. The PSI activity was measured by monitoring the uptake of oxygen after addition of 15 μ m DCMU, 0.1 m m DCIP, 5 m m sodium ascorbate, and 0.1 m m MV to the suspension. The activities of wild-type and desA + cells that corresponded to 100% were 314 ± 27 and 332 ± 30 μmol O2 mg −1 Chl h −1 , respectively. ▪, Wild-type cells in darkness ■, wild-type cells in light ●,desA + cells in darkness ○,desA + cells in light. Each point and bar represent the average ± se of results from five independent experiments.

Incubation with 0.5 m LiCl markedly inhibited the activity of PSI in both types of cell (Fig. 2B) and the activity in wild-type and desA + cells declined within 2 h to 30% and 45% of the original level, respectively. Light at 70 μE m −2 s −1 restored some activity after the rapid decline but the effect of 0.5 m LiCl was more damaging than that of NaCl. However, it was clear that the PSI complex indesA + cells, in darkness and in light, was much more tolerant to LiCl than that in wild-type cells.

During incubation with 1.0 m sorbitol, PSI activity declined within 2 h to 70% and 85% of the original level in wild-type and desA + cells, respectively (Fig. 2C). During subsequent incubation in light for 4 h, the PSI activity of desA + cells returned to the original level and remained at that level for the remainder of the 10-h incubation. In darkness, the PSI activity ofdesA + cells was always higher than that of wild-type cells.

Effects of Lincomycin on the Salt-Induced Inactivation of PSII and PSI

Effects of lincomycin (Lin) on the NaCl-induced inactivation of PSII and PSI in wild-type anddesA + cells. Cells were incubated with 0.5 m NaCl in light at 70 μE m −2 s −1 in the presence of lincomycin at 200 μg mL −1 (dashed lines) or in its absence (solid lines). At designated times, a portion of the cell suspension was withdrawn. A, The oxygen-evolving activity of PSII was measured after addition of 1.0 m m BQ to the suspension. The oxygen-evolving activities of wild-type anddesA + cells that corresponded to 100% were 568 ± 32 and 576 ± 39 μmol O2mg −1 Chl h −1 , respectively. B, PSI activity was measured by monitoring the uptake of oxygen after addition of 15 μ m DCMU, 0.1 m m DCIP, 5 m m sodium ascorbate, and 0.1 m m MV to the suspension. The oxygen-uptake activities of wild-type anddesA + cells that corresponded to 100% were 286 ± 24 and 288 ± 27 μmol O2mg −1 Chl h −1 , respectively. Wild-type (■) and desA + (○) cells in the absence of lincomycin. Wild-type (▿) anddesA + (▵) cells in the presence of lincomycin. Each point and bar represent the average ± se of results from five independent experiments.

Effects of lincomycin (Lin) on the NaCl-induced inactivation of PSII and PSI in wild-type anddesA + cells. Cells were incubated with 0.5 m NaCl in light at 70 μE m −2 s −1 in the presence of lincomycin at 200 μg mL −1 (dashed lines) or in its absence (solid lines). At designated times, a portion of the cell suspension was withdrawn. A, The oxygen-evolving activity of PSII was measured after addition of 1.0 m m BQ to the suspension. The oxygen-evolving activities of wild-type anddesA + cells that corresponded to 100% were 568 ± 32 and 576 ± 39 μmol O2mg −1 Chl h −1 , respectively. B, PSI activity was measured by monitoring the uptake of oxygen after addition of 15 μ m DCMU, 0.1 m m DCIP, 5 m m sodium ascorbate, and 0.1 m m MV to the suspension. The oxygen-uptake activities of wild-type anddesA + cells that corresponded to 100% were 286 ± 24 and 288 ± 27 μmol O2mg −1 Chl h −1 , respectively. Wild-type (■) and desA + (○) cells in the absence of lincomycin. Wild-type (▿) anddesA + (▵) cells in the presence of lincomycin. Each point and bar represent the average ± se of results from five independent experiments.

Light-Dependent Recovery of PSII and PSI from NaCl-Induced Inactivation

Effects of light and the removal of NaCl on the recovery of PSII and PSI activities in wild-type anddesA + cells after NaCl-induced inactivation. Wild-type and desA + cells were incubated for 7 and 16 h, respectively, in darkness in the presence of 0.5 m NaCl. Then cells were further incubated in light at 70 μE m −2 s −1 in the presence of lincomycin at 200 μg mL −1 or in its absence. After incubation for 30 h in light, the cells were collected by centrifugation, resuspended in fresh BG-11 medium with no added NaCl, and incubated in light for a further 10 h. At designated times, a portion of the cell suspension was withdrawn. A, The oxygen-evolving activity of PS II was measured after addition of 1.0 m m BQ to the suspension. The oxygen-evolving activities of wild-type anddesA + cells that corresponded to 100% were 527 ± 36 and 543 ± 33 μmol O2mg −1 Chl h −1 , respectively. B, PSI activity was measured by monitoring the uptake of oxygen after the addition of 15 μ m DCMU, 0.1 m m DCIP, 5 m m sodium ascorbate, and 0.1 m m MV to the suspension. The oxygen-uptake activities of wild-type anddesA + cells that corresponded to 100% were 315 ± 26 and 309 ± 21 μmol O2mg −1 Chl h −1 , respectively. ▪ and ■, Wild-type cells ● and ○,desA + cells in the absence of lincomycin. Wild-type (▿) and desA + (▵) cells in the presence of lincomycin. Each point and bar represent the average ± se of results from four independent experiments.

Effects of light and the removal of NaCl on the recovery of PSII and PSI activities in wild-type anddesA + cells after NaCl-induced inactivation. Wild-type and desA + cells were incubated for 7 and 16 h, respectively, in darkness in the presence of 0.5 m NaCl. Then cells were further incubated in light at 70 μE m −2 s −1 in the presence of lincomycin at 200 μg mL −1 or in its absence. After incubation for 30 h in light, the cells were collected by centrifugation, resuspended in fresh BG-11 medium with no added NaCl, and incubated in light for a further 10 h. At designated times, a portion of the cell suspension was withdrawn. A, The oxygen-evolving activity of PS II was measured after addition of 1.0 m m BQ to the suspension. The oxygen-evolving activities of wild-type anddesA + cells that corresponded to 100% were 527 ± 36 and 543 ± 33 μmol O2mg −1 Chl h −1 , respectively. B, PSI activity was measured by monitoring the uptake of oxygen after the addition of 15 μ m DCMU, 0.1 m m DCIP, 5 m m sodium ascorbate, and 0.1 m m MV to the suspension. The oxygen-uptake activities of wild-type anddesA + cells that corresponded to 100% were 315 ± 26 and 309 ± 21 μmol O2mg −1 Chl h −1 , respectively. ▪ and ■, Wild-type cells ● and ○,desA + cells in the absence of lincomycin. Wild-type (▿) and desA + (▵) cells in the presence of lincomycin. Each point and bar represent the average ± se of results from four independent experiments.

When NaCl was removed at 30 h by pelleting and resuspension of cells, the PSII activity in desA + cells recovered to a small but significant extent with restoration of approximately 25% of the original activity in 2 h and then started to decrease again. No similar recovery was observed in wild-type cells. The presence of lincomycin (200 μg mL −1 ) completely eliminated the recovery of the oxygen-evolving activity in both types of cell (Fig. 4A). These results clearly suggested that protein synthesis was required for the recovery of PSII activity in light and after removal of NaCl.

Figure 4B shows the recovery of PSI activity after NaCl-induced inactivation in wild-type and desA + cells. Wild-type and desA + cells were incubated with 0.5 m NaCl for 7 and 16 h, respectively, in darkness, which caused approximately 75% and 20% inactivation of PSI, respectively. Then they were exposed to light at 70 μE m −2 s −1 . Under these conditions, the PSI activity in desA + cells returned almost to the original level within 3 h. In wild-type cells, the extent of recovery was less than 5%, but then the PSI activity started to decrease again and disappeared completely within 20 h. By contrast, in desA + cells 60% of the original activity was detectable at 30 h.

When NaCl was removed at 30 h, approximately 80% of the original activity was detected in desA + cells within 2 h but no recovery was observed in wild-type cells (Fig. 4B). Lincomycin prevented the recovery of PSI activity in light and upon removal of NaCl in desA + cells (Fig. 4B), results that suggested that protein synthesis was required for the recovery of PSI activity under these conditions.

NaCl-Induced Inactivation of the Oxygen-Evolving Machinery in Vitro

Changes in the PSII activity of thylakoid membranes isolated from wild-type and desA + cells. Thylakoid membranes (10 μg Chl mL −1 ) were incubated in darkness in the presence of 0.5 m NaCl or in its absence. At designated times, a portion of the suspension was withdrawn and the light-induced reduction of DCIP was measured after addition of 0.1 m m DCIP or 0.1 m m DCIP and 0.5 m m DPC to the suspension. A, The transport of electrons from water to DCIP. The activities that corresponded to 100% were 173 ± 15 and 180 ± 17 μmol DCIP reduced mg −1 Chl h −1 in thylakoid membranes from wild-type and desA + cells, respectively. B, The transport of electrons from DPC to DCIP. The activities that corresponded to 100% were 344 ± 20 and 332 ± 25 μmol DCIP reduced mg −1 Chl h −1 in thylakoid membranes from wild-type anddesA + cells, respectively. ■, Thylakoid membranes from wild-type cells ○, thylakoid membranes fromdesA + cells both were incubated with 0.5 m NaCl. ▪, Thylakoid membranes from wild-type cells ●, thylakoid membranes from desA + cells both were incubated in the absence of added NaCl. Each point and bar represent the average ± se of results from four independent experiments.

Changes in the PSII activity of thylakoid membranes isolated from wild-type and desA + cells. Thylakoid membranes (10 μg Chl mL −1 ) were incubated in darkness in the presence of 0.5 m NaCl or in its absence. At designated times, a portion of the suspension was withdrawn and the light-induced reduction of DCIP was measured after addition of 0.1 m m DCIP or 0.1 m m DCIP and 0.5 m m DPC to the suspension. A, The transport of electrons from water to DCIP. The activities that corresponded to 100% were 173 ± 15 and 180 ± 17 μmol DCIP reduced mg −1 Chl h −1 in thylakoid membranes from wild-type and desA + cells, respectively. B, The transport of electrons from DPC to DCIP. The activities that corresponded to 100% were 344 ± 20 and 332 ± 25 μmol DCIP reduced mg −1 Chl h −1 in thylakoid membranes from wild-type anddesA + cells, respectively. ■, Thylakoid membranes from wild-type cells ○, thylakoid membranes fromdesA + cells both were incubated with 0.5 m NaCl. ▪, Thylakoid membranes from wild-type cells ●, thylakoid membranes from desA + cells both were incubated in the absence of added NaCl. Each point and bar represent the average ± se of results from four independent experiments.

NaCl-Induced Inhibition of the Reduction of P700 +

Changes in the rate of reduction of P700 + in wild-type anddesA + cells during incubation in light at 70 μE m −2 s −1 in the presence of 0.5 m NaCl. At designated times, a portion of the cell suspension was withdrawn, and the rate of reduction of P700 + was measured after addition of 15 μ m DCMU, 0.1 m m DCIP, 5 m m sodium ascorbate, and 0.1 m m MV to the suspension. The oxidation-reduction kinetics of P700 were examined at 820 nm with 10 flashes of 5-ms duration at a saturating light intensity of 4.5 mE m −2 s −1 from a xenon discharge lamp (XMT 103 Walz, Germany) in a multiple-turnover mode with 20-s intervals. The results were averaged. ■, Wild-type cells ○, desA + cells. Each point and bar represent the average ± se of results from five independent experiments.

Changes in the rate of reduction of P700 + in wild-type anddesA + cells during incubation in light at 70 μE m −2 s −1 in the presence of 0.5 m NaCl. At designated times, a portion of the cell suspension was withdrawn, and the rate of reduction of P700 + was measured after addition of 15 μ m DCMU, 0.1 m m DCIP, 5 m m sodium ascorbate, and 0.1 m m MV to the suspension. The oxidation-reduction kinetics of P700 were examined at 820 nm with 10 flashes of 5-ms duration at a saturating light intensity of 4.5 mE m −2 s −1 from a xenon discharge lamp (XMT 103 Walz, Germany) in a multiple-turnover mode with 20-s intervals. The results were averaged. ■, Wild-type cells ○, desA + cells. Each point and bar represent the average ± se of results from five independent experiments.

We observed a similar delay in the reduction of P700 + when both types of cell were treated with 1 m m KCN or HgCl2 (data not shown), both of which inhibit the transport of electrons from plastocyanin to P700 + ( Izawa, 1980 Trebst, 1980). These findings suggested that incubation with NaCl might primarily have inactivated the transport of electrons from plastocyanin to P700 + . This reaction indesA + cells was more resistant to the damaging effects of NaCl than that in wild-type cells.

NaCl-Induced Inactivation of the Na + /H + Antiport System

In a previous study ( Allakhverdiev et al., 1999), we demonstrated that the activity of Na + /H + antiporters is important in the protection of cyanobacterial cells against salt stress. Therefore, we examined the activity of the Na + /H + antiport system in wild-type and desA + cells. When a small amount of a suspension of cells in 0.5 m NaCl (20 μL) was added to 2 mL of Na + -free medium that contained acridine orange, the fluorescence of acridine orange was quenched. This phenomenon was due to the efflux of Na + ions from cells and the influx of H + ions into cells via the activity of the Na + /H + antiport system ( Blumwald et al., 1984 Garbarino and DuPont, 1989). Addition of 100 m m NaCl to the suspension suppressed the quenching of fluorescence, perhaps because of an increase in intracellular alkalization due to the efflux of H + ions coupled with the influx of Na + ions as a result of the activity of Na + /H + antiport system ( Blumwald et al., 1984 Garbarino and DuPont, 1989 for review, see Padan and Schuldiner, 1994, and references therein). Further addition of Triton X-100 to a final concentration of 0.04% (v/v) rapidly increased the fluorescence to a final steady-state level.

Changes in the activity of the Na + /H + antiport system in wild-type and desA + cells during incubation in the presence of 0.5 m NaCl. Cells were incubated in the presence of 0.5 m NaCl in darkness or in light at 70 μE m −2 s −1 . At designated times, 20 μL of the suspension of cells was withdrawn and diluted 100-fold with Na + -free medium that contained 5 μ m acridine orange. Then the fluorescence of acridine orange was monitored as described in “Materials and Methods.” The activity of the Na + /H + antiport system was calculated from the initial rate of recovery of fluorescence quenching upon addition of NaCl, divided by the difference between the fluorescence before the addition of NaCl and the steady-state level of fluorescence 1 min after the addition of Triton X-100 at a final concentration of 0.04% (v/v). ▪, Wild-type cells in darkness ■, wild-type cells in light ●, desA + cells in darkness ○, desA + cells in light. Each point and bar represent the average ± se of results from five independent experiments.

Changes in the activity of the Na + /H + antiport system in wild-type and desA + cells during incubation in the presence of 0.5 m NaCl. Cells were incubated in the presence of 0.5 m NaCl in darkness or in light at 70 μE m −2 s −1 . At designated times, 20 μL of the suspension of cells was withdrawn and diluted 100-fold with Na + -free medium that contained 5 μ m acridine orange. Then the fluorescence of acridine orange was monitored as described in “Materials and Methods.” The activity of the Na + /H + antiport system was calculated from the initial rate of recovery of fluorescence quenching upon addition of NaCl, divided by the difference between the fluorescence before the addition of NaCl and the steady-state level of fluorescence 1 min after the addition of Triton X-100 at a final concentration of 0.04% (v/v). ▪, Wild-type cells in darkness ■, wild-type cells in light ●, desA + cells in darkness ○, desA + cells in light. Each point and bar represent the average ± se of results from five independent experiments.


Dr. Johanna Budwig’s Major Discovery

In 1952, Dr. Budwig wrote in a paper entitled "On Fat Biology V. Paper Chromatography of Blood Lipoids, the Tumour Problem and Fat Research":

"It is basically proven that highly unsaturated fatty acids are the heretofore undiscovered decisive factor in respiratory enzyme function", i.e. constitute the second part of the "equation" that nobelist Otto Warburg [1] had been unable to find.

What sounds insignificant to the layman’s ears, is arguably one of the greatest breakthroughs in medical science: from that moment onward we have known that the highly unsaturated fatty acid is the decisive factor achieving the desired effect of cellular respiratory stimulation.

Working in conjunction with sulfurated amino acids (protein) [2] , the highly unsaturated fatty acid plays a part, even the critical part, in the "bridging" taking place between fats and protein, in the absorption AND utilization of oxygen, in all growth processes, in the formation of blood and in many other processes.

Working from this theory, Dr. Budwig was able to help a great many cancer patients with the scientific oil-protein diet of flaxoil plus cottage cheese she designed (the "Budwig diet"), which allows cancer cells to start "breathing" again. A few physicians followed in her footsteps, such as Dr. Dan C. Roehm from Florida and Dr. Robert E. Willner (Miami). [3]

Based upon her research findings, Dr. Budwig spoke out not only against processed foods and supplements ("no pills") but also against chemotherapy, radiation and drugs, and in a less categorical manner, surgery (see interview). And, rare as that may be, she also was aware of the critical importance of sunlight as well as the spiritual, mental and emotional factors in healing cancer and other illness.

That said, it is important to keep in mind that the fuller details of her published thought on healing cancer are not currently known/accessible in the English- and much of the German-speaking world, thus making all efforts presently undertaken at implementing and spreading the word about Dr. Budwig’s discoveries in English a grassroots movement liable to be enlarged as more details become known to the general public via correctly translated editions of more of her books.

1 Dr. Otto Warburg, twice Nobel Laureate, awarded the Nobel Prize for Physiology or Medicine in 1931 for his research on cellular respiration, explains: "The growth of cancer cells is initiated by a relative lack of oxygen. Cancer cannot live in an oxygen-rich environment. Cancer has only one prime cause. It is the replacement of normal oxygen respiration of the body’s cells by an anaerobic (i.e., oxygen-deficient) cell respiration."

Going into greater detail in The Prime Cause and Prevention of Cancer, he writes: ". the cause of cancer is no longer a mystery, we know it occurs whenever any cell is denied 60% of its oxygen requirements. Cancer, above all other diseases, has countless secondary causes. But, even for cancer, there is only one prime cause. Summarized in a few words, the prime cause of cancer is the replacement of the respiration of oxygen in normal body cells by a fermentation of sugar.

All normal body cells meet their energy needs by respiration of oxygen, whereas cancer cells meet their energy needs in great part by fermentation. All normal body cells are thus obligate aerobes, whereas all cancer cells are partial anaerobes." Compare Otto Warburg On The Prime Cause & Prevention of Cancer: Respiration of Oxygen in Normal Body Cells vs. Fermentation of Sugar in Cancer Cells.

2 Methionine, cystine, and cysteine and their derivatives owe their designation of "sulphurated amino acids" to the fact that they contain sulfur in addition to carbon, hydrogen, nitrogen and oxygen. Incidentally, they are also well-known as an effective cleaning "squad" for all toxic substances that we ingest because they attach themselves to harmful substances and pollutants and carry them out of the body. One example: methionine and cysteine aid in lead elimination (Karen Vago).

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Effect of Salicylic Acid Incorporated Chitosan Coating on Shelf Life Extension of Fresh In-Hull Pistachio Fruit

The effect of edible coating on the postharvest quality of fresh pistachio covered by a yellow-red colored soft shell (hull) was evaluated. Fresh pistachio fruit were immersed in different surface treatment solutions of 2% (w/v) chitosan (CT) in 0.5% acetic acid solution, 2 mmol L −1 salicylic acid (SA), and the combination of CT and SA (CT-SA), then packed in perforated polyethylene terephthalate (PET), and stored in a refrigerator for 28 days at 4 °C. Distilled water and 0.5% acetic acid solution containing 0.1% Tween were considered as control treatments. The weight loss and also the peroxide and free fatty acid values of treated fresh pistachio fruit were lower than the controls at the end of the storage period. The activity of superoxide dismutase, catalase, and peroxidase enzymes in the treated pistachio fruit was significantly higher compared to controls (p < 0.05). The SA-treated pistachio fruit shows the lowest activity of polyphenol oxidase. Also, the pistachio fruit treated with CT and SA were lighter (L*), redder (a*), and more yellow (b*) in color as compared with controls, so that the highest sensory scores of color, texture, and overall acceptance were attributed to these treatments. Interestingly, SA treatment resulted in a remarkable superiority of the fruit color score among the samples. Furthermore, the CT and SA treatments alone or in combination significantly reduced the growth of bacteria and fungi. Overall, it can be concluded that SA and CT-SA treatments can assure the safety and quality of fresh pistachio fruit in refrigerated storage.

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