Archive for the ‘triglycerides’ Category

Doctor:    Therein the patient

  Must minister to himself.

Macbeth: Throw physic [medicine] to the dogs; I’ll none of it.

— William Shakespeare, Macbeth

The quality of nutrition papers even in the major scientific and medical journals is so variable and the lack of restraint in the popular media is so great that it is hard to see how the general public or even scientists can find out anything at all. Editors and reviewers are the traditional gate-keepers in science but in an area where rigid dogma has reached Galilean proportions, it is questionable that any meaningful judgement was made: it is easy to publish papers that conform to the party line (“Because of the deleterious effects of dietary fructose, we hypothesized that…”) and hard to publish others: when JAMA published George Bray’s “calorie-is-a-calorie” paper and I pointed out that the study more accurately supported the importance of carbohydrate as a controlling variable, the editor declined to publish my letter.  In this, the blogs have performed a valuable service in providing an alternative POV but if the unreliability is a problem in the scientific literature, that problem is multiplied in internet sources. In the end, the consumer may feel that they are pretty much out there on their own. I will try to help.  The following was posted on FatHead’s Facebook page:

 How does one know if a study is ‘flawed’? I see a lot of posts on here that say a lot of major studies are flawed. How? Why? What’s the difference if I am gullible and believe all the flawed studies, or if I (am hopefully not a sucker) believe what the Fat Heads are saying and not to believe the flawed studies — eat bacon.

Where are the true studies that are NOT flawed…. and how do I differentiate? : /

 My comment was that it was a great question and that it would be in the next post so I will try to give some of the principles that reviewers should adhere to.  Here’s a couple of guides to get started. More in future posts:

 1“Healthy” (or “healthful”) is not a scientific term. If a study describes a diet as “healthy,” it is almost guaranteed to be a flawed study.  If we knew which diets were “healthy,” we wouldn’t have an obesity epidemic. A good example is the paper by Appel, et al. (2005). “Effects of protein, monounsaturated fat, and carbohydrate intake on blood pressure and serum lipids: results of the OmniHeart randomized trial,” whose conclusion is:

“In the setting of a healthful diet, partial substitution of carbohydrate with either protein or monounsaturated fat can further lower blood pressure, improve lipid levels, and reduce estimated cardiovascular risk.”

 It’s hard to know how healthful the original diet, a “carbohydrate-rich diet used in the DASH trials … currently advocated in several scientific reports” really is if removing carbohydrate improved everything.

Generally, understatement  is good.  One of the more famous is from Watson & Cricks’s 1953 paper in which they proposed the DNA double helix structure. They said “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”  A study with the word “healthy” is an infomercial.

2. Look for the pictures (figures).  Presentation in graphic form usually means the author wants to explain things to you, rather than snow you.  This is part of the Golden Rule of Statistics that I mentioned in my blogpost “The Seventh Egg”  which discusses a very flawed study from Harvard on egg consumption. The rule comes from the book PDQ Statistics:

“The important point…is that the onus is on the author to convey to the reader an accurate impression of what the data look like, using graphs or standard measures, before beginning the statistical shenanigans.  Any paper that doesn’t do this should be viewed from the outset with considerable suspicion.”

The Watson-Crick  paper cited above had the diagram of the double-helix  which essentially became the symbol of modern biology.  It was drawn by Odile, Francis’s wife, who is described as being famous for her nudes, only one of which I could find on the internet.

Krauss, et. al. Separate effects of reduced carbohydrate intake and weight loss on atherogenic dyslipidemia.

The absence of a figure may indicate that the authors are not giving you a chance to actually see the results, that is, the experiment may not be flawed but the interpretation may be misleading, intentionally or otherwise.  An important illustration of the principle is a paper published by Krauss. It is worth looking at this paper in detail because the experimental work is very good and the paper directly — or almost directly — confronts a big question in diet studies: when you reduce calories by cutting out carbohydrate, is the effect due simply  to lowering calories or is there a specific effect of carbohydrate restriction.  The problem is important since many studies compare low-carbohydrate and low-fat diets where calories are reduced on both. Because the low-carbohydrate diet generally has the better weight loss and better improvement in HDL and triglycerides, it is said that it was the weight loss that caused the lipid improvements.

So Krauss compared the effects of carbohydrate restriction and weight loss on the collection of lipid markers known collectively as atherogenic dyslipidemia.  The markers of atherogenic dyslipidemia, which are assumed to predispose to cardiovascular disease, include high triglycerides (triacylglycerol), low HDL and high concentrations of the small dense LDL.

Here is how the experiment was set up: subjects first consumed a baseline diet of  54% of energy as carbohydrate, for 1 week. They were then assigned to one of four groups.  Either they continued the baseline diet, or they kept calories constant but reduced carbohydrate by putting fat in its place.  The three lower carbohydrate diets had 39 % or 26 % carbohydrate or 26 % carbohydrate with higher saturated fat.  After 3 weeks on constant calories but reduced carbohydrate, calories were decreased by 1000 kcal/d for 5 week and, finally, energy was stabilized for 4 weeks and the features of atherogenic dyslidemia were measured at week 13.  The protocol is shown in the figure from Krauss’s paper:

The Abstract of the paper describes the outcomes and the authors’ conclusions.

Results: The 26%-carbohydrate, low-saturated-fat diet reduced [atherogenic dylipidemia]. These changes were significantly different from those with the 54%-carbohydrate diet. After subsequent weight loss, the changes in all these variables were significantly greater…(my italics)

 Conclusions: Moderate carbohydrate restriction and weight loss provide equivalent but non-additive approaches to improving atherogenic dyslipidemia. Moreover, beneficial lipid changes resulting from a reduced carbohydrate intake were not significant after weight loss.

Now there is something odd about this.  It is the last line of the conclusion that is really weird. If you lose weight, the effect of carbohydrate is not significant?  As described below, Jeff Volek and I re-analyzed this paper so I have read that line a dozen times and I have no idea what it means.  In fact, the whole abstract is strange.  It will turn out that the lower (26 %) is certainly “significantly different from.. the 54%-carbohydrate diet” but it is not just different but much better. Why would you not say that?  The Abstract is generally written so that it sounds negative about low carbohydrate effects but it is already known from Krauss’s previous work and others that carbohydrate restriction has a beneficial effect on lipids and the improvements in lipid markers occur on low-carbohydrate diets whether weight is lost or not.  The last sentence doesn’t seem to make any sense at all.    For one thing, the experiment wasn’t done that way.  As set up, weight loss came after carbohydrate restriction.  So, let’s look at the data in the paper.  There are few figures in the paper and Table 2 in the paper presents the results in a totally mind-numbing layout.  Confronted with data like this, I sometimes stop reading.  After all, if the author doesn’t want to conform to the Golden Rule of Statistics, if they don’t want to really explain what they accomplished, how much impact is the paper going to have.  In this case, however, it is clear that the experiment was designed correctly and it just seems impossible from previous work that this wouldn’t support the benefits of carbohydrate restriction and the negative tone of the Abstract needs to be explained.  So we all had to slog through those tables.  Let’s just look at the triglycerides since this is one of the more telling attributes of atherogenic dyslpidemia.  Here’s the section from the Table:

Well this looks odd in that the biggest change is in the lowest carb group with high SF but it’s hard to tell what the data look like.  First it is reported as logarithms. You sometime take logs of your data in order to do a statistical determination but that doesn’t change the data and it is better to report the actual value.  In any case, it’s easy enough to take antilogs and we can plot the data.  This is what it looks like:

It’s not hard to see what the data really show: Reducing carbohydrate has an overwhelming effect on triglycerides even without weight loss.  When weight loss is introduced, the high carbohydrate diets still can’t equal the performance of the carbohydrate reduction phase.  (The dotted line in the figure are data from Volek’s earlier work which Krauss forgot to cite).

The statements in the Conclusion from the Abstract are false and totally misrepresent the data.  It is not true as it says “carbohydrate restriction and weight loss provide equivalent…” effects. The carbohydrate-reduction phase is dramatically better than the calorie restriction phase and it is not true that they are “non-additive”  Is this an oversight?  Poor writing?  Well, nobody knows what Krauss’s motivations were but Volek and I plotted all of the data from Krauss’s paper and we published a paper in Nutrition & Metabolism providing an interpretation of Krauss’s work (with pictures).  Our conclusion:

Summary Although some effort is required to disentangle the data and interpretation, the recent publication from Krauss et al. should be recognized as a breakthrough. Their findings… make it clear that the salutary effects of CR on dyslipidemia do not require weight loss, a benefit that is not a feature of strategies based on fat reduction. As such, Krauss et al.  provides one of the strongest arguments to date for CR as a fundamental approach to diet, especially for treating atherogenic dyslipidemia.

An important question in this experiment, however, is whether even in the calorie reduction phase, calories are actually the important variable.  This is a general problem in calorie restriction studies if for no other reason than that there is no identified calorie receptor.  When we published this data, Mike Eades pointed out that in the phase in which Krauss reduced calories, it was done by reducing macronutrients across the board so carbohydrate was also reduced and that might be the actual controlling variable so we plotted the TAG against carbohydrate in each phase (low, medium and high carb (LC, MC, HC) without or with weight loss (+WL) and the results are shown below

This is remarkably good agreement for a nutrition study. When you consider carbohydrates as the independent variable, you can see what’s going on.  Or can you?  After all, by changing the variables you have only made an association between carbohydrate and outcome  of an experiment. So does this imply a causal relation between carbohydrate and triglycerides or not?  It is widely said that observational studies do not imply causality, that observational studies can only provide hypothesis for future testing. It certainly seems like causality is implied here.  It will turn out that a more accurate description is that observational studies do not necessarily imply causality and I will discuss that in the next posts.  The bottom line will be that there is flaw in grand principles like “Random controlled trials are the gold standard.” “Observational studies are only good for generating hypotheses,”  “Metabolic Ward Studies are the gold standard.” Science doesn’t run on such arbitrary rules.

(Answers to last week’s organic puzzler at the end of this post).

One of the more remarkable results from Jeff Volek’s laboratory in the past few years was the demonstration that when the blood of volunteers was assayed for saturated fatty acids, those who had been on a low carbohydrate diet had lower levels than those on an isocaloric low-fat diet. This, despite the fact that the low-carbohydrate diet had three times the amount of saturated fat as the low-fat diet. How is this possible? What happened to the saturated fat in the low-carbohydrate diet? Well, that’s what metabolism does. The saturated fat in the low-carbohydrate arm was oxidized while (the real impact of the study) the low-fat arm is making new saturated fatty acid. Volek’s former student Cassandra Forsythe extended the idea by showing how, even under eucaloric conditions (no weight loss) dietary fat has relatively small impact on plasma fat.

The essential point of what I now call the Volek-Westman principle — we should be speaking of basic principles because the idea is more important than specific diets where it is impossible to get any agreement on definitions — the principle is that carbohydrate, directly or indirectly through insulin and other hormones, controls what happens to ingested (or stored) fatty acids. The motto of the Nutrition & Metabolism Society is: “A high fat diet in the presence of carbohydrate is different than a high fat diet in the presence of low carbohydrate.” Widely attributed to me, it is almost certainly something I once said although it has been said by others and the studies from Volek’s lab give you the most telling evidence.

The question is critical. Whereas the scientific evidence now establishes that dietary saturated fat has no effect on cardiovascular disease, obesity or anything else, plasma saturated fatty acids can be a cellular signal and if you study the effect of dietary saturated fatty acids under conditions where carbohydrate is high and/or in rodents where plasma fat better correlates with dietary fat, then you will confuse plasma fat with dietary fat. An important study identified potential cellular elements in control of gene transcription that bear on lipid metabolism.

So, it is important to know about plasma saturated fatty acids. First, recall that strictly speaking there are only saturated fatty acids (SFA) — this is explained in detail in an earlier post.  What is called saturated fats simply mean those fats that have a high percentage of SFAs — things that we identify as “saturated fats,” like butter, are usually only 50 % saturated fatty acids (coconut oil is probably the only fat that is almost entirely saturated fatty acids but because they are medium chain length, they are usually considered a special case).

In Volek’s study, 40 overweight subjects were randomly assigned either to a carbohydrate-restricted diet (abbreviated CRD; %CHO:fat:protein = 12:59:28) or to a low fat diet, (LFD; %CHO:fat:protein = 56:24:20). The group was unusual in that they were all overweight would be characterized as having metabolic syndrome, in particular they all had, atherogenic dyslipidemia, which is the term given to a poor lipid profile (high triacylglycerol (TAG), low HDL-C, high small-dense LDL (so-called pattern B)). Metabolic syndrome (MetS) is the predisposition to CVD and diabetes and is characterized by the constellation of overweight, atherogenic dyslipidemia and, by now, a dozen other markers.

The paper is one of the more striking for the differences in weight loss between two diet regimens. Although participants were not specifically counseled to reduce calories, there was a reduction in total caloric intake in both two groups. The response in weight loss, however, due to the difference in macronutrient composition, was dramatically different in the two groups. The CRD group (labelled as very low carbohydrate ketogenic diet (VLCKD) in the figure) lost twice as much weight on average as the low-fat controls despite the similar caloric intake. Although there was substantial individual variation, 9 of 20 subjects in the CRD (VLCKD) group lost 10% of their starting weight. more than that lost by any of the subjects in the LFD group. In fact, nobody following the LFD lost as much weight as the average for the low-carbohydrate group and, unlike George Bray’s demonstration of caloric inefficiency, whole body fat mass was where the major differences between the CRD (VLCKD) and LF appeared (5.7 kg vs 3.7 kg). Of significance is the observation that fat mass in the abdominal region decreased more in subjects on the CRD than in subjects following the LFD (-828 g vs -506 g). This is one of the more dramatic effects of carbohydrate restriction on weight loss but many have preceded it and these have been frequently criticized for increasing the amount of saturated fat (whether or not any particular study actually increased saturated fat). Although the original “concern” was that this would lead to increased plasma cholesterol, eventually saturated fat became a generalized villain and, insofar as any science was involved, the effects of plasma saturated fat were assumed to be due to dietary saturated fat. The outcome of Volek’s study was surprising. Surprising because the effect was so clear cut (no statistics needed) and because an underlying mechanism could explain the results.

Saturated Fat

The dietary intake of saturated fat for the people on the VLCKD (36 g/day) was threefold higher than that of the people on the LFD (12 g/day). When the relative proportions of circulating SFAs in the triglyceride and cholesterol ester fractions were determined, they were actually lower in the low carb group. Seventeen of 20 subjects on the CRD (VLCKD) showed a decrease in total saturates (the others had low values at baseline) in comparison to half of the subjects consuming the LFD had a decrease in saturates. When the absolute fasting TAG levels are taken into account (low carbohydrate diets reliably reduce TAB=G), the absolute concentration of total saturates in plasma TAG was reduced by 57% in the low carbohydrate arm compared to 24% reduction in the low fat arm who had, in fact, reduced their saturated fat intake. One of the saturated fatty acids of greatest interest was palmitic acid or, in chemical short-hand, 16:0 (16 means that there are 16 carbons and 0 means there are no double bonds, that is, no unsaturation).

So how could this happen? The low fat group reduced their SFA intake by one-third, yet had more SFA in their blood than the low-carbohydrate group who had actually increased intake. Well, we need to look at the next thing in metabolism.

In the post on An Introduction to Metabolism, we made the generalization that there were roughly two kinds of fuel, glucose and acetyl-CoA (the two carbon derivative of acetic acid). The big principle in metabolism was that you could make acetyl-CoA from glucose, but (with some exceptions) you couldn’t make glucose from acetyl-CoA, or more generally, you can make fat from glucose but you can’t make glucose from fat. How do you make fat from glucose? Part of the picture is making new fatty acids, the process known as De Novo Lipogenesis (DNL) or more accurately de novo fatty acid synthesis. The mechanism then involves successively patching together two carbon acetyl-CoA units until you reach the chain length of 16 carbons, palmitic acid. The first step is formation of a three carbon compound, malonyl-CoA, a process which is under the control of insulin. Malonyl-CoA starts the process of DNL but simultaneously prevents oxidation of any fatty acid since, if you are making it, you don’t want to burn it. This can be further processed, among other things, can be elongated to stearic acid (18:0). So this is a reasonable explanation for the increased saturated fatty acid in the low-fat group: the higher carbohydrate diet has higher insulin levels on average, encouraging diversion of calories into fatty acid synthesis and repressing oxidation. How could this be tested?

It turns out that, in addition to elongation, the palmitic acid can be desaturated to make the unsaturated fatty acid, palmitoleic acid (16:1-n7, 16 carbons, one unsaturation at carbon 7) and the same enzyme that catalyzes this reaction will convert stearic acid (18:0) to the unsaturated fatty acid oleic acid (18:1n-7). The enzyme is named for the second reaction stearoyl desaturase-1 (SCD-1; medical students always hate seeing a “-1” since they know 2 and 3 may will have to be learned although, in this case, they are less important). SCD-1 is a membrane-bound enzyme and it seems that it is not swimming around the cell looking for fatty acids but is, rather, closely tied to DNL, that is, it preferentially de-saturates newly formed palmitic acid to palmitoleic acid.

There is very little palmitoleic acid in the diet so its presence in the blood is an indication of SCD-1 activity. The data show a 31% decrease in palmitoleic acid (16:1n-7) in the blood of subjects on the low-carb arm with little overall change in the average response in the low fat group. Saturated fat, in your blood or on your plate?

Forsythe’s paper extended the work by putting men on two different weight-maintaining low-carbohydrate diets for 6 weeks. One of the diets was designed to be high in SFA (high in dairy fat and eggs), and the other, was designed to be higher in unsaturated fat from both polyunsaturated (PUFA) and monounsaturated (MUFA) fatty acids (high in fish, nuts, omega-3 enriched eggs, and olive oil). The relative percentages of SFA:MUFA: PUFA were, for the SFA-carbohydrate-restricted diet, 31: 21:5, and for the UFA diet, 17:25:15. The results showed that the major changes in plasma SFA and MUFA were in the plasma TAG fraction although probably much less than might be expected given the nearly two-fold difference in dietary saturated fat and, as the authors point out: “the most striking finding was the lack of association between dietary SFA intake and plasma SFA concentrations.”

So although it is widely said that the type of fat is more important than the amount, the type is not particularly important. But, what about the amount? A widely cited paper by Raatz, et al. suggested, as indicated by the title, that ‘‘Total fat intake modifies plasma fatty acid composition in humans”, but the data in the paper shows that differences between high fat and low fat were in fact minimal (figure below).

The bottom line is that distribution of types of fatty acid in plasma is more dependent on the level of carbohydrate then the level or type of fat. Volek and Forsythe give you a good reason to focus on the carbohydrate content of your diet. What about the type of carbohydrate? In other words, is glycemic index important? Is fructose as bad as they say? We will look at that in a future post in which I will conclude that no change in the type of carbohydrate will ever have the same kind of effect as replacing carbohydrate across the board with fat. I’ll prove it.


Answers to the organic quiz.

The following question was posted on Facebook:

I had thought that free fatty acids were triglycerides. But I am reading a study that measured both. Can someone enlighten me on free fatty acids? … please.

 I think I can help.  The good news is that, contrary to the college myth, organic chemistry is easy — it is freshman chemistry that is hard because it has more physics and mathematics.  Now, jumping into lipid metabolism is a little bit of starting in the middle of things but the reason organic chemistry is easy is that it has only a few assumptions and basic principles and the basic theory, at least, is logical and you can get pretty far deducing things from simple principles, so with a few basic ideas we may have a shot. I have two YouTube videos that are short, relatively easy and might be a background.  The take home message from the videos, the one big idea in organic, is that organic compounds have two parts: A hydrocarbon backbone and a non-hydrocarbon part that contains the chemically reactive part of the molecule, the functional groups. The assumption is that all compounds with the same functional group have similar chemistry.  So, for example, all carboxylic acids have the carboxyl (-COOH) functional group. In many ways, even a simple acid like acetic acid has chemical properties that are similar to a complicated acid, like the fatty acids.  You may need the YouTube to appreciate this: chemistry is about structure, that is, it is visual.

Bottom line on fatty acids and Triglycerides

All dietary and body fats and oils are triglycerides (TG) or, more correctly, triacylglycerols (TAG).  The term “acyl” (pr. A-sill) is the adjective form of acid (i.e. There are three acids).

Fats have a roughly E-shaped structure. The arms of the E are the fatty acids and there are three of them. The fatty acids provide the real fuel in fats.  The three fatty acids are attached to the compound glycerol which is the vertical stroke of the E.  The chemical bond that attaches the fatty acid  to the glycerol is called an ester bond.  You only need to know the term ester because when the fatty acids are found alone, especially in blood, they are referred to either as free fatty acids (FFA) or, because they are no longer attached to the glycerol by the ester bonds, as non-esterified fatty acids (NEFA): FFA and NEFA are the same thing.

Metabolism: the fatty acid-TAG cycle.

The digestion of fat in the intestine involves the progressive removal of the fatty acids from the first and last position of the glycerol.  The process is called lipolysis and the enzyme that catalyzes the reaction is called a lipase. What remains is called 2-monoacylglycerol, or 2-MAG  (fatty acid still attached at the center carbon of glycerol) and  2-MAG and the free fatty acids from digestion are absorbed into the intestinal cells.  Within these cells they are re-formed into TAG which is exported together with cholesterol and other components in particles called chylomicrons.  Chylomicrons, in turn, represent one type of complex structure known as lipoproteins. The lipoproteins transport lipids and some of these are familiar, e.g., LDL (low density lipoprotein), HDL. Triglycerides in the blood are carried in these particles. So this is probably the triglycerides you read about.

These are the transporters of lipids.  TAG, in particular is brought into cells by another lipase (lipoprotein lipase or LPL) on the cell surface that removes the fatty acids.  In other words, to be absorbed the TAG is broken down into fatty acids again.  Once absorbed, the fatty acids can be oxidized for fuel or, once again can be re-synthesized, step-wise: → MAG → diacylglycerol (DAG)  → TAG.  Here’s the summary figure:

Bottom line:

Fat (TAG) is continually broken down and re-synthesized.  The breakdown process is called lipolysis and the lipolysis-synthesis cycle goes on in different places in the body but notably in fat cells.  An interesting thing about fat cells is the way they carry out the cycle. Lipolysis is a simple process but synthesis is complicated.  Speaking in energy terms, it is easy to break down nutrients. It requires energy to put them back together.  To make TAG, either the glycerol or the fatty acid has to be “activated”: so the actual reactive form is a molecule called fatty acyl-coenzyme A or fatty acyl-CoA (pr. Co-A).

Biochemical reactions almost never run by themselves even if energetically favorable but are rather controlled by catalysts, that is, enzymes.  The enzyme that catalyzes the first step in the reaction, a transferase, will not work with glycerol itself.  The enzyme requires a particular form of glycerol, glycerol-phosphate.  The special characteristic of the fat cell is that the required glycerol-phosphate cannot be made directly from glycerol as it can, for example, in the liver which also has an active fatty acid-TAG cycle.  In order to make glycerol phosphate, fat cells require glucose. In the absence of glucose, as in starvation or a low carbohydrate diet, fat synthesis is repressed.  At the same time the enzyme that catalyzes breakdown, hormone-sensitive lipase, is enhanced because it is turned on by glucagon and turned offby insulin (these are the hormones in the term “hormone-sensitive lipase”).  This was the original rationalization for the apparent advantage in a low-carbohydrate diet: without carbohydrate the adipocyte would not be able to supply glycerol-phosphate and the fatty acid-TAG cycle would go largely in one direction: breakdown to produce fatty acids and this is undoubtedly one of the major effects.

It turns out, however, that the fat cells protect stores of energy in fat by other methods. We now understand that cells run a process called glyceroneogenesis which is a truncated form of gluconeogenesis, the process whereby glucose is synthesized from other nutrients, mostly protein, that is, the process supplies an intermediate in the synthesis of glucose and this can be converted to glycerol-phosphate. Generally, especially if the diet is hypocaloric, the net effect is to break down fat and supply fatty acids as a fuel for other cells.  Fatty acids circulate in the blood bound to a protein called albumin. Under conditions where there is higher carbohydrate, however, and the fatty acids are not being used for fuel, they can stimulate insulin resistance. So, fatty acids in the blood are a good thing if you are breaking down fat to supply energy.  They are not so good if you are over-consuming energy or carbohydrates because, in the presence of insulin, they can lead to insulin resistance.

Summary: triglycerides are made of three fatty acids.  There is a continual fatty acid-TAG cycle that goes on all the time in different cells.  Triglycerides in the blood are carried in lipoprotein particles, chylomicrons, LDL, HDL.  Fatty acids in the blood are carried by the protein albumin.