Showing posts with label allergies. Show all posts
Showing posts with label allergies. Show all posts

Monday, January 25, 2016

Elimination Diet Kickstarts Fatloss in "People Who Cannot Lose Weight" - 16% Body Fat Reduction in 6 Months, But...

Many of these foods contain supposed allergens and have thus to be eliminated from your diet... is it any wonder that this triggers weight loss? Hardly...
While I have to admit that I am a bit skeptical about the reliability of the results of a recent study from the Sifa University, Faculty of Health Sciences in Turkey, I cannot ignore that Meltem Yaman Onmus, Elif Cakirca Avcu, and Ali Saklamaz claim that "people who cannot lose weight by low-calorie diet can lose weight and fat with elimination diet according to the results of FI [food intolerance] test. FIED [FI elimination diet] is also significantly effective in triglyceride levels" (Onmus. 2016). I know that sounds as if it was taken from the latest unreferenced blogpost on a dubious website, but let's not judge prematurely and instead take a closer look at the design and results of the study.
Unlike elimination diets, fasting must be considered a scientifically proven weight loss trick

Breakfast and Circadian Rhythm

Does Meal Timing Matter?

Habits Determine Effects of Fasting

Fasting Works for Obese, Too!?

Does the Break- Fast-Myth Break?

Breakfast? (Un?) Biased Review
82 patients (24 male, 58 female) were included in the study. The mean age was 42.04±11.81 (18-65 years). All of them were "unable to lose weight", i.e. patients who said of themselves that they couldn’t lose weight by diet programs and who had a positive reaction to at least one nutrient in food intolerance test and a BMI value ≥ 25kg/m² - in other words: the subjects were the average Internet bullet-in board dwellers searching for the "magic key" to weight loss.

This is obviously an important fact, because it increases the significance of the study for said group of subjects. Whether the results are significant for anyone else, though, is questionable, since patients who had no food reaction in food intolerance test were excluded from the study. The same goes for subjects who had chronic diseases like diabetes mellitus, coronary heart disease, renal diseases, etc., or individuals who use(d) weight loss drugs and who had allergy to any drug or food and who overuse medications or have pure menstrual migraine or headache that associated a disorder.
No health benefits from "eliminating" foods: Interestingly, the food intolerance elimination diet failed to do what its proponents say it's actually doing: Improve the subjects health. With the exception of a statistically significant decrease in triglyceride levels, there was no improvement in health markers (fasting blood glucose, A1C, total cholesterol, HDL-cholesterol, LDL-cholesterol, AST, and ALT) the scientists didn't observe in the control group, too.
As you can see in Figure 1, this particular group of subjects saw significant benefits from following a diet that did not allow the subjects to consume any of the foods to which they showed an IgG response in the previously conducted food intolerance test for 6 months. Otherwise, the diets of the elimination diet (ED) and control diet were personalized diets with "identical" (according to the size, weight, physical activity, dietary habits and socioeconomic status) energy content.
Figure 1: Pre- and post-intervention weight, body fat, lean body mass, and waist / hip ratio (x10); * indicates significant inter-group difference, this means that everything, but the effect on lean mass was sign. more pronounced in the ED group.
Against that background it is unquestionably striking that the subjects in the elimination diet group lost 16% body fat, while the control group didn't lose either significant amounts of fat or weight. Now, the obvious question is: "Which foods were eliminated?" Unfortunately, this question is neither answered in the study at hand, nor in previous studies showing that elimination diets reduce also reflux disease, chronic fatigue syndrome, and headaches (Selvin. 2007; Akmal. 2009).
Unlike Onmus et al., Akmal et al. publish-ed a list of allergens their IgG test could supposedly identify. A list eliminated foods is yet missing from study, too.
Why's it a problem that we don't know which foods were eliminated? Actually, the answer to this question should be obvious. Let's assume you're "allergic" to sugar, alcohol and high omega-6 vegetable oils like soybean oil. Would you be surprised if you lost significant amounts of body fat if you dropped all sugary and pro-inflammatory high omega-6 foods and stopped drinking alcohol? I, for my part, wouldn't and I guess you wouldn't and you certainly shouldn't be surprised either.

Accordingly, the study at hand does unfortunately not provide enough information to decide whether it provides convincing evidence of using IgG-tests to guide you when you're designing diets for yourself or your clients. Hopefully future research will do just that | Comment!
References:
  • Akmal, Mohammed, Saeed Ahmed Khan, and Abdul Qayyum Khan. "The Effect of the ALCAT Test diet therapy for food sensitivity in patient’s with obesity." Middle East Journal of Family Medicine 7.3 (2009).
  • Onmus, Meltem Yaman, Elif Cakirca Avcu, and Ali Saklamaz. "The Effect of Elimination Diet on Weight and Metabolic Parameters of Overweight or Obese Patients Who Have Food Intolerance." Journal of Food and Nutrition Research 4.1 (2016): 1-5.
  • Selvin, E., Paynter, N. P., Earlinger T. P. "Nutrition and allergy." Arch Intern Med, 167.1 (2007): 31-39.

Friday, July 13, 2012

Are Camels the Better Cows? Cancer, Heart Disease, High LDL and Triglycerides, Diabetes, High Blood Pressure, Allergies, Viral and Bacterial Infections and Trace Mineral Deficiencies, Camel Milk Prevents or Fixes Them All!

Image 1: To the average inhabitant of the Western hemisphere camels are probably not the most beautiful animals under the sun; but hey, cows aren't either, hah?
When muscle-heads think of dairy, they think of whey, they think of casein, they think of cottage cheese... but I bet few of them will think of camels! Even if you just went by the mere amino acid composition (see. figure 2 at the end of the article), of which you, as an educated SuppVersity student should by now be aware that it does not give you the 'whole picture', as far as the biological effects of a given protein and peptide containing foodstuff is concerned, it appears that camel milk would at least make an excellent alternative for cows milk, in case global warming is progressing and Europe and the US turn into desert wastelands... but all jokes aside, muscle is not everything and I bet that after reading this article you will be interested to register for the US' first official camel milking seminar *rofl*

7+1 reasons you may want to slaughter your grass fed cows and replace them with camels

While the idea of drinking the milk of an ugly desert ship may appear hilarious at first, I guess my compilation of purported and scientifically established benefits of camels milk will have you reconsider if the Sheikh Hamdan bin Mohammed bin Rashed Al-Maktoum, Crown Prince of Dubai, may not have made a very good investment, when he spent $16.5 million dirham ($4.5 million USD) on the winner of a 2008 beauty pageant in the United Arab Emirates’ capital city of Abu Dhabi (FYI, the guy in image 1 is not the Sheikh, just tom make sure I don't get sued, here ;-):
    Image 2 (DrCate.com): Homogenization makes milk more convenient, but it disrupts the natural structure of the fat globules and releases the otherwise bound xanthine oxidase of which scientists hypothesized that it could trigger heart disease, a hypothesis, btw., that was not disproven, but simply kept quiet for the past 25+ years (Deeth. 1983)
  • Little to no xanthine oxidase (=reductase) - While the idea that XOR (xanthine oxidoreductase), which is supposedly released during homogenization of bovine milk, could be a potential contributor to overall inflammation and cardiovascular disease has disappeared from the 'scientific radar' within the past couple of years (cf. Deeth. 1983; Berry. 2004) , it may still be of interest (and for certain populations such as people with increased gut permeability even of great importance) that camel milk apparently contains little to no xanthine oxidase - irrespective of whether you drink it raw, pasteurized or homogenized (Baghiani. 2003).
  • Anticancer effects - Camels milk has been shown to trigger apoptosis (controlled cell death) in human breast cancer and liver cancer cells via epigenetic mechanisms (Korashi. Feb 2012; Korashi. May 2012).
  • Antibacterial & antiviral effects -Camel milk prevents gram positive bacteria from growing and reduces the amounts and activity of gram-negative cultures (el Agamy. 1992). With the latter being among the primary drivers of lipopolysaccharide (LPS) and endotoxin induced inflammation (Ulevitch. 1999), camel milk could thus help to reduce local and systemic inflammation. Aside from its activity against rotavirus, the lactoferrin faction from camel milk appears to have protective effects against hepatitis C, as well (Redwan. 2007).
  • Camel milk whole- & beta-caseins act as natural anti-oxidants and ACE-inhibitors - As Salami et al have shown the whole casein and beta-casein (β-CN) faction(s) of camel milk exert Angiotensin Converting Enzyme (ACE)-inhibitory (=blood pressure reducing) and antioxidant activity after they were hydrolyzed in the stomach (Salami. 2011). In a previous study, the same researchers had already determined that the whey fraction of camel milk exhibits significant anti-oxidant and antimicrobial activities, as well, and that those were up to 100% greater (depending on the essay and fraction the scientists used) than those of bovine whey protein (Salami. 2010).
  • Figure 1: A certain part of the population in Rajasthan (India) who consumes camel milk on a daily basis has been found to have a significantly reduced incidence of diabetes (not a single one!), impaired fasting glucose (-6%/-11%) and impaired glucose tolerance (-10%/-10%) than both non-camel milk drinking parts of the Raica community or other non-camel milk drinkers from the same region (based on Agrawal. 2007)
  • Profound and long-lasting anti-diabetic effects - Camel milk has a long history of being used to tread type 1 diabetes in the Middle East (see figure 1; cf. Mohamad. 2009). Studies from animal models (dogs, Sbou. 2010) and humans (Agrawal. 2009; Mohamad. 2009) improved blood glucose, microalbumenia and secondary symptoms such as diabetic neuropathy. Probably also as a consequence of the small, but biologically active natural insulin content of camels milk (Malik .2012), the type 1 diabetics in a 2009 study by Agrawal could even reduce their insulin medication by 32% from 41µ/day to 28µ/day. In the 2-year follow up, the researchers report that "out of 12 subjects receiving camel milk, insulin requirement in 3 subjects reduced to zero" (Agrawal. 2011) - try that with metformin, let alone some of the other 'diabetes prolongation drugs'.
  • Improved lipid metabolism - The 24 type one diabetics who consumed 500ml of plain camel milk per day for 6 months in the aforementioned 2009 study by Agrawal et al. for example exhibited -30% decreases in LDL and -66% decrease in triglycerides.
  • Camel milk is an extraordinary good source of trace minerals - According to Al-Awidi et al. Camel milk contains 7-20x and 1-10x higher levels of manganese and iron than human milk, more zinc and comparable amounts of selenium, copper and other protein bound and thus highly bioavailable trace minerals (Al-Awadi. 2001).
And best of all, based on studies on people with cow's milk allergy, we know that the incidence of allergic reactions to camel milk is not only much lower, but also that 80% of cow's milk allergy sufferers can actually ingest camel milk without any unwanted side-effects (Cardoso. 2010; Ehlayel. 2011).

Figure 2: Even if you go solely by the amino acid composition (here expressed relative to the total amino acids), camel milk protein could be a valid replacement for bovine proteins (data based on Davis. 1994 & Beg. 1987)
This is also worth mentioning, because the host of great effects I listed above do - at least in parts - also occur with the unwarrantedly vilified bovine (=cow's) milk, which also contains ACE precursors (Saito. 2008), exert anti-cancer effects (Gill. 2000), and so much more (see "suggested readings" at the end of this article).

So, in the unfortunate case that you "ain't got no camel handy" at the moment and, due to "the current economy" (I hate when people say that) lack the $1300 to $1700 (Debacle. 2006) to buy your own, obviously not  pageant winning $16.5 million dirham camel, and tolerate bovine milk, just stick to the milk of the farmer you trust. After all, even if Camels were the better cows, you better have a gallon of cows milk in the fridge than a camel in the Arabian desert, right? Ah, wait that was a bird in the hand, right? ... ah, whatever ;-)

Suggested readings:

References
:
  1. Agrawal RP, Budania S, Sharma P, Gupta R, Kochar DK, Panwar RB, Sahani MS. Zero prevalence of diabetes in camel milk consuming Raica community of north-west Rajasthan, India. Diabetes Res Clin Pract. 2007 May;76(2):290-6.
  2. Agrawal RP, Dogra R, Mohta N, Tiwari R, Singhal S, Sultania S. Beneficial effect of camel milk in diabetic nephropathy. Acta Biomed. 2009 Aug;80(2):131-4. 
  3. Agrawal RP, Jain S, Shah S, Chopra A, Agarwal V. Effect of camel milk on glycemic control and insulin requirement in patients with type 1 diabetes: 2-years randomized controlled trial. Eur J Clin Nutr. 2011 Sep;65(9):1048-52. doi: 10.1038/ejcn.2011.98. Epub 2011 Jun 1.
  4. Al-Awadi FM, Srikumar TS. Trace elements and their distribution in protein fractions of camel milk in comparison to other commonly consumed milks. J Dairy Res. 2001 Aug;68(3):463-9.
  5. Baghiani A, Harrison R, Benboubetra M. Purification and partial characterisation of camel milk xanthine oxidoreductase. Arch Physiol Biochem. 2003 Dec;111(5):407-14.
  6. Beg OU, von Bahr-Lindström H, Zaidi ZH, Jörnvall H. Characterization of a heterogeneous camel milk whey non-casein protein. FEBS Lett. 1987 Jun 1;216(2):270-4.
  7. Berry CE, Hare JM. Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications. J Physiol. 2004 Mar 16;555(Pt 3):589-606.
  8. Cardoso RR, Santos RM, Cardoso CR, Carvalho MO. Consumption of camel's milk by patients intolerant to lactose. A preliminary study. Rev Alerg Mex. 2010 Jan-Feb;57(1):26-32.
  9. Davis TA, Nguyen HV, Garcia-Bravo R, Fiorotto ML, Jackson EM, Lewis DS, Lee DR, Reeds PJ. Amino acid composition of human milk is not unique. J Nutr. 1994 Jul;124(7):1126-32.
  10. Deeth HC. Homogenized milk and atherosclerotic disease: a review. J Dairy Sci. 1983 Jul;66(7):1419-35.
  11. Gill HS, Cross ML. Anticancer properties of bovine milk. Br J Nutr. 2000 Nov;84 Suppl 1:S161-6. Review.
  12. Ehlayel MS, Hazeima KA, Al-Mesaifri F, Bener A. Camel milk: an alternative for cow's milk allergy in children. Allergy Asthma Proc. 2011 May-Jun;32(3):255-8.
  13. el Agamy EI, Ruppanner R, Ismail A, Champagne CP, Assaf R. Antibacterial and antiviral activity of camel milk protective proteins. J Dairy Res. 1992 May;59(2):169-75. 
  14. Gill HS, Cross ML. Anticancer properties of bovine milk. Br J Nutr. 2000 Nov;84 Suppl 1:S161-6. Review.
  15. Korashy HM, El Gendy MA, Alhaider AA, El-Kadi AO. Camel milk modulates the expression of aryl hydrocarbon receptor-regulated genes, Cyp1a1, Nqo1, and Gsta1, in murine hepatoma Hepa 1c1c7 cells. J Biomed Biotechnol. 2012;2012:782642. Epub 2012 Feb 27.
  16. Korashy HM, Maayah ZH, Abd-Allah AR, El-Kadi AO, Alhaider AA. Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism. J Biomed Biotechnol. 2012;2012:593195. Epub 2012 May 13.
  17. Debacle J. Long or Short Capital. Camels, The Next Big Thing. August 1, 2006. < http://longorshortcapital.com/camels-the-next-big-thing.htm > received on July 13, 2012
  18. Malik A, Al-Senaidy A, Skrzypczak-Jankun E, Jankun J. A study of the anti-diabetic agents of camel milk. Int J Mol Med. 2012 Sep;30(3):585-92.
  19. Mohamad RH, Zekry ZK, Al-Mehdar HA, Salama O, El-Shaieb SE, El-Basmy AA, Al-said MG, Sharawy SM. Camel milk as an adjuvant therapy for the treatment of type 1 diabetes: verification of a traditional ethnomedical practice. J Med Food. 2009 Apr;12(2):461-5.
  20. Salami M, Moosavi-Movahedi AA, Ehsani MR, Yousefi R, Haertlé T, Chobert JM, Razavi SH, Henrich R, Balalaie S, Ebadi SA, Pourtakdoost S, Niasari-Naslaji A. Improvement of the antimicrobial and antioxidant activities of camel and bovine whey proteins by limited proteolysis. J Agric Food Chem. 2010 Mar 24;58(6):3297-302.
  21. Salami M, Moosavi-Movahedi AA, Moosavi-Movahedi F, Ehsani MR, Yousefi R, Farhadi M, Niasari-Naslaji A, Saboury AA, Chobert JM, Haertlé T. Biological activity of camel milk casein following enzymatic digestion. J Dairy Res. 2011 Nov;78(4):471-8.
  22. Redwan el-RM, Tabll A. Camel lactoferrin markedly inhibits hepatitis C virus genotype 4 infection of human peripheral blood leukocytes. J Immunoassay Immunochem. 2007;28(3):267-77.
  23. Saito T. Antihypertensive peptides derived from bovine casein and whey proteins. Adv Exp Med Biol. 2008;606:295-317. Review.
  24. Sboui A, Khorchani T, Djegham M, Agrebi A, Elhatmi H, Belhadj O. Anti-diabetic effect of camel milk in alloxan-induced diabetic dogs: a dose-response experiment. J Anim Physiol Anim Nutr (Berl). 2010 Aug 1;94(4):540-6.
  25. Ulevitch RJ, Tobias PS. Recognition of gram-negative bacteria and endotoxin by the innate immune system. Curr Opin Immunol. 1999 Feb;11(1):19-22.

Tuesday, July 10, 2012

Childhood Asthma: Kids Born to Mothers Who Consume Semi-Skimmed Milk 8% More, Offspring of Whole Milk Drinking Mothers 15% Less Likely to Develop Asthma

Image 1: Better make sure your "mother's milk" (the milk you drink while you or your significant other are pregnant) is full fat!
I don't know if you have listened to my "brother" Carl Lanore's Super Human Radio interview with Boyd Eaton, yesterday, but if you did, you will probably remember that the two also broached the issue of dairy consumption on a paleo diet. And while many of you may have been surprised to hear that Eaton does consume milk products on a regular basis, it is probably even more intriguing that the show came full-circle only a couple of minutes later, when Carl who had just addressed the issue of iron-overload and how that relates to the relative abundance of meat in an ancestral diet, read my facebook message about the beneficial effects of milk proteins, Zunquin et al. observed in a 2006 trial rodent trial intended to simulate the potential side effects of high dose iron supplementation to replete iron stores in iron-depleted athletes (Zunquin. 2006).

Milk allergy or allergy due to the lack of full-fat milk - is that the question?

Yet while the Zunquin study is by no means the only scientific evidence that nature's original meal replacement, irrespective of whether or not it may be "intended for human consumption" can exert potent health effects (cf. Barfray. 2003), the pros and cons of a high(er) intake of dairy is still a matter of constant debate. Other than within the paleosphere, the public and scientific debate does yet usually revolve around the notion of low- vs. high fat dairy, with the common recommendation to "choose low fat dairy in order to avoid the unhealthy saturated fats that come with the full-fat variety".
Figure 1: Associations between mothers' consumption of milk and dairy during pregnancy and incidence of early childhood asthma (18 months) in children from the Danish national Birth Cohort; data expressed as differences to no-consumption (data based on Masolva. 2012)
Aside from the downside of non-existent yet potentially healthy levels of certain fatty acids, such as conjugated linolic acid (yeah, the one that can kill empty fat cells, cf. "Nasty Insights into the Yo-Yo-Effect: Lower Body Fat Sticks and From Fit2Fat There's no Easy Way Back!"), the reduced absorption and presence of fat-soluble nutrients and the necessity to add sugar or artificial flavors, as well as chemicals to modify the taste and mouth-feel of those watery low-fat dairy products, a recent study from Department of Nutrition at the Harvard School of Public Health in Boston does now provide compelling evidence that either one or all of the former aspects, or a hitherto not elucidated difference between skim and full-fat dairy products increases children who are born to mothers who consume >5 portions of semi-skimmed milk per week during their pregnancy have an 8% increased risk of developing childhood asthma and a 40% increase in risk of developing allergic rhinitis later in life (Maslova. 2012).

Table 1: If you are interested in other sources of CLA (and omega-3), in general, and the difference between grass- and grainfed beef, in particular, check out this table from a Daley et al. (Daily. 2012)
What's even more intriguing, though, is that the consumption of equal amounts of full fat milk was associated with a statistically significant 25%(!) reduction in asthma-risk (and recurrent wheeze symptoms) in the ~60,000 children / mother pairs from the dataset of the Danish National Birth Cohort the US scientists analyzed. Similarly, there was a
"dose–response was present for semiskimmed milk intake, while any intake of full-fat yoghurt appeared to be protective of child asthma" (Maslova. 2012)
that did, just as the aforementioned results, remain significant, even when the data was adjusted for the intake of other allegedly beneficial foods (fruit and vegetables) and nutrients (vitamin E, vitamin D, Se, Zn from diet and supplements).

So are those the CLAs and other ruminant trans-fats?

While Malsova et al. fail to give a clearcut answer on what the underlying reasons for these observations are, their detailed analysis of the data does yield some evidence that CLAs and other ruminant trans-fatty acids cannot explain the differences, as they showed similar correlations with both full-fat and semi-skimmed milk intake and were "not associated with either the early or later childhood outcomes" (Maslova. 2012).

Image 2: Not, not all full-fat milk drinkers worked on a farm ;-)
An analysis of other potentially confounding lifestyle factors yielded similarly inconclusive results so that this leaves us without a causal explanation for either the protective effect of whole milk and full-fat yogurt intake on early childhood asthma or the increased risk to develop wheeze at 18 months in children born to high consumers of semi-skimmed milk. In the absence of any information of what happened in the course of the following 7-years it appears futile come up with any hypothesis that would explain the direct associations of maternal low-fat yoghurt consumption with child asthma and allergic rhinitis and the "suggestive" relationship of the latter with total milk intake, anyways. And as the scientists say:
The diversity and complexity of our results make it difficult to interpret and propose a single agent mechanism, yet the consistent associations with low-fat yoghurt for later childhood outcomes suggest that compounds specific to this food, such as artificial sweeteners, may play a role.
Interestingly, they specifically refer to the use of aspartame as the "primary" artificial sweetener in the Danish food supply since the 1980s, but cite a - at least in my humble opinion - highly biased source (Gideon. 2010) on that issue without even providing a single scientific study that would implicate aspartame as a factor involved in the etiology of asthma (+my brief research shows, there is none).
Image 3: Too much statistical shenanigan involved in the ER data for my liking.
A note on additional data evaluated in the study: The scientists make use of the actual hospital admissions due to asthmatic reactions as another source for their study data. Unfortunately, those only contribute to the already confusing results, because they suggest that despite the fact that the incidence of early childhood asthma is lower (other data), the chance to end up in hospital, as assessed by data from the Danish National Patient Registry appears to be higher in children born to mothers with a high full fat milk intake (+30%), and minimally reduced for children whose mothers consumed the same amount of skimmed dairy - it is however questionable how reliable this 3rd hand data which had to be linked back to the central person registry first and ran through multiple processing steps actually is (same for data from the Register of Medicinal Product Statistics).
As far as the underlying mechanisms are concerned this leaves us with but one relatively certain conclusion: The scientists' initial hypothesis that ruminant trans-fatty acids could play a causal role in the development of allergic disease development cannot be upheld in view of the inverse associations Maslova et al. found between the CLA content and the risk to develop asthma and/or allergic rhinitis across products with different fat content.

Suggested reading:

References:
  1. Bartfay WJ, Davis MT, Medves JM, Lugowski S. Milk whey protein decreases oxygen free radical production in a murine model of chronic iron-overload cardiomyopathy. Can J Cardiol. 2003 Sep;19(10):1163-8. 
  2. Daley CA, Abbott A, Doyle PS, Nader GA, Larson S. A review of fatty acid profiles and antioxidant content in grass-fed and grain-fed beef. Nutr J. 2010 Mar 10;9:10.
  3. Gideon B (2010) Aspartam: Nu også i ikke ‘Light’ produkter. < http://infowars.dk/content/aspartam-nu-ogs%C3% A5-i-ikke-light-produkter > last accessed on July 10, 2012
  4. Maslova E, Halldorsson TI, Strøm M, Olsen SF. Low-fat yoghurt intake in pregnancy associated with increased child asthma and allergic rhinitis risk: a prospective cohort study. Journal of Nutritional Science (2012), vol. 1, e5.
  5. Zunquin G, Rouleau V, Bouhallab S, Bureau F, Theunynck D, Rousselot P, Arhan P, Bougle D. Iron and exercise induced alterations in antioxidant status. Protection by dietary milk proteins. Free Radic Res. 2006 May;40(5):535-42.