[Thank you for all the responses to my request for information on increasing bone mineral density! I received a lot of responses and am still working my way through them.]

Because I’m planning to write a more reader-friendly version of VeganHealth.org’s Bones, Vitamin D, and Calcium, I decided to check in on the research on protein and bone health. In so doing, I found a 2012 review from a group of researchers in France who declared having no conflicts of interest (1).

There has been an enormous amount of research on protein and bone health and their review had almost 4 pages of references. I will hit the highlights of what they found:

– Many clinical trials show that adding purified proteins to the diet increases calcium excretion through the urine.

– Phosphorus, in which meat and dairy are rich, counteracts the increase of calcium in the urine between 40 and 65%.

– Findings that older people in Western countries have higher hip fracture rates are confounded by the fact that people in Western countries live longer, protein intakes were not estimated for individuals, and there are ethnic differences in bone structure and lifestyles.

– High protein diets increase acid excretion in the urine, but this can be handled by the body’s acid buffer system without the need for calcium.

– Studies measuring whole-body calcium balance (as distinct from excretion) in relation to high protein diets have been mixed, but this might partly be due to the difficulty in measuring calcium balance and because high protein diets might reduce calcium balance when calcium intakes are particularly low.

– In low-calcium, but not high-calcium diets, higher protein intakes probably increase calcium absorption from the digestive tract causing an increase in calcium excretion in the urine.

– Fruits and vegetables are beneficial to bone health, probably due to their high potassium and magnesium content. This could cause confounding in protein studies because diets high in protein are often low in fruits and vegetables.

– As I describe in my post Protein Intake and Bone Health, Darling et al. (2009) found that a large majority of the cross-sectional surveys and cohort studies have reported either no association or a beneficial association between protein and bone mineral density.

– There is some evidence that a beneficial effect of protein on bones is only seen when calcium intake and vitamin D status is adequate.

– Maintenance of adequate bone strength and density with aging is dependent on adequate muscle mass which is dependent on adequate intake of protein.

– An increase in IGF-1 is most likely the mechanism for increased bone health with higher protein intakes.

They conclude, “Although HP [high protein] diets induce an increase in net acid and urinary calcium excretion, they do not seem to be linked to impaired calcium balance and no clinical data support the hypothesis of a detrimental effect of HP diet on bone health, except in the context of inadequate calcium supply.”

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Reference

1. Calvez J, Poupin N, Chesneau C, Lassale C, Tomé D. Protein intake, calcium balance and health consequences. Eur J Clin Nutr. 2012 Mar;66(3):281-95. | link

Lysine is the limiting amino acid in most vegan diets. It is typically not hard for vegans to get enough lysine, but you have to eat three to four servings of high lysine foods each day. High lysine foods are legumes, seitan, quinoa, and pistachios.

The USDA lists pumpkin seeds in the same entry as other squash seeds and the entry for such seeds has listed high amounts of lysine. But I have not included them among the high lysine foods because it was not clear which seeds they are specifically talking about.

A reader decided to write the USDA Nutrient Data Laboratory and ask them about their entries. It turns out that three of the “pumpkin and squash seed” entries are for pumpkin seeds only.

According to the pumpkin seeds-only entry for roasted kernels (USDA nutrient #12016), a one-quarter cup of pumpkin seeds has 360 mg of lysine, which qualifies it as a high lysine food.

I have updated the Protein article at VeganHealth.org to reflect the addition of pumpkin seeds to the list of high lysine foods.

Awhile back, a reader sent me a copy of a 2009 paper that analyzed the amino acid amounts in wheat gluten protein (1).

The USDA nutrient database does not give amino acid amounts for wheat gluten and it doesn’t include seitan at all. I have long suspected that seitan would be a good source of the amino acid lysine (which is generally the limiting amino acid in vegan diets), but I had no way to verify this until getting the 2009 paper.

Upon doing some calculations I estimate that White Wave seitan has approximately 656 mg of lysine per serving, making it one of the highest sources of lysine among plant foods. I have added this information to Table 3 of Protein at VeganHealth.org. You can see more details about how I came up with this amount in the footnotes of the table, if interested.

There is one disconcerting thing about the 2009 paper – it has no amounts for tryptophan and no explanation as to why. And if you add up the amounts of the other amino acids in mg/g of protein, it comes to almost exactly 1 g, leaving no room for tryptophan. I have written the authors to find out why this might be.

The paper is rather technical and not being a laboratory scientist, I didn’t even understand most of it. If any readers would like to take a look at it, let me know through the contact form.

In any case, I feel confident now that seitan is a good source of lysine and I have added it to my protein recommendations as an option for getting your daily amount of lysine.

Addendum of November 7, 2012:

I have communicated with one of the paper’s authors, Dr. Ine Rombouts who told me that the protein in wheat is 80% gluten and that gluten is very low in tryptophan – that it only has negligible amounts. The other 20% of wheat protein is made up of albumins and globulins which have more tryptophan. This is why wheat has tryphtophan but wheat gluten does not.

Reference

1. Rombouts I, Lamberts L, Celus I, Lagrain B, Brijs K, Delcour JA. Wheat gluten amino acid composition analysis by high-performance anion-exchange chromatography with integrated pulsed amperometric detection. J Chromatogr A. 2009 Jul 17;1216(29):5557-62. Epub 2009 Jun 3. | link

Good article from Christine Kasum Sexton of the Vegetarian Resource Group on preventing age-related muscle loss:

Can a vegan diet provide enough protein to slow down age-related muscle loss?

Just added to the Protein page at VeganHealth.org:

“A study out of Boston published in 2011 but performed using data collected during the 1980s, found that vegan and non-vegan, middle-aged women had similar levels of muscle mass despite differences in protein intake of 1.0 g/kg/day for vegans and 1.3 g/kg/day for omnivores (14). However, the muscle mass was not measured directly – rather it was estimated using formulas based on creatinine clearance (a byproduct of muscle metabolism). The researchers believed the formulas to be accurate, but since they have not been validated on vegans it should be viewed with some uncertainty. At 30 mg/kg/day, the vegan women did not meet the RDA for lysine which
is 38 mg/kg/day. However, the study showed the vegan women to be consuming only 1511 kcal/day vs. 1866 kcal/day for the omnivores, yet their body mass indexes were very similar at 20.0 and 20.7 respectively. This could indicate that food intake for the vegans was underestimated, possibly due to a lack of data on vegan foods.”

More commentary: This is one of only two studies that have looked at protein status in actual vegans, the other study being one that measured serum albumin levels (12). While not conclusive, it provides evidence that vegans generally have adequate protein status.

Reference

12. Haddad EH, Berk LS, Kettering JD, Hubbard RW, Peters WR. Dietary intake and biochemical, hematologic, and immune status of vegans compared with nonvegetarians. Am J Clin Nutr. 1999 Sep;70(3 Suppl):586S-593S. (Link)

14. Andrich DE, Filion ME, Woods M, Dwyer JT, Gorbach SL, Goldin BR, Adlercreutz H, Aubertin-Leheudre M. Relationship between essential amino acids and muscle mass, independent of habitual diets, in pre- and post-menopausal US women. Int J Food Sci Nutr. 2011 Nov;62(7):719-24. Epub 2011 May 16. (Link)

I see that you advocate higher intakes of proteins, but what about renal function? In the ADA Position I read:

This is the reference: ncbi.nlm.nih.gov/pubmed/17383270.1

Answer:

This is a legitimate concern.

Someone with kidney disease should be careful about protein intake. My recommendations for protein (Table 1 in the article Protein) are generally lower than what the population normally consumes, though higher than what many vegans might consume, and should be safe for someone without kidney problems, especially since some studies have shown vegetable protein to be less harmful than animal protein.

My recommendations for older people are a range from .8 to 1.3 g/kg/day. If someone has kidney disease, they might want to stick to the lower end of the range, or even lower than that if indicated during monitoring by their physician. And vegans should focus on high lysine protein foods to make sure they get enough of the amino acid lysine.

Warning: Lots of biochemistry discussed below. Hopefully the general ideas of the article will make sense even if you skim over the more technical parts.

A study just came out with a scary title, “Vegetarianism produces subclinical malnutrition, hyperhomocysteinemia and atherogenesis.” The abstract shows that the cross-sectional study measured homocysteine levels (among other disease markers) of a rural population of men living in Chad who were supposedly vegetarian (1). They found homocysteine levels to be elevated and therefore concluded that the men might be at risk for cardiovascular disease. Except for the provocative title, there was nothing particularly interesting in the abstract, and I decided not to pursue it further as there is a steady flow of cross-sectional studies on semi-vegetarians from developed countries and their cardiovascular disease markers.

But a number of people contacted me about it and so I changed my mind. I’m glad I did, as the paper was much more interesting than the abstract, though still not very relevant to vegetarians in developed countries.

Here is a summary. Twenty-four apparently healthy men from a rural part of Chad, a country in Africa, were compared to 15 men from a nearby urban part of Chad. The rural men ate very little animal products and less than their urban counterparts. There is no indication that this was due to any sort of “vegetarianism,” but rather simply because of the food available to them in their area.

Protein intakes for the rural men were an average of 50 g per day compared to 63 g per day for the urban men. The RDA for meat-eating men of that height would be 51 g and the recommended protein intake for vegan men that height would be 57 g. That would put these rural men at a “probably adequate” protein level, in my opinion. However, their average intake of the sulfur amino acids methionine and cysteine was only 10.4 mg/kg while the RDA is 19 mg/kg.

The rural men were marginally suffering from an indicator of protein malnutrition, known as prealbumin (aka as transthyretin), which was at an average level of 176 mg/l (compared to 292 mg/l for the urban men). The lower limit of a healthy prealbumin is listed by most sources as 180 mg/l, while the upper limit of healthy is listed as anywhere from 300 to 400 mg/l.

The B12 levels of the rural vs. urban men were 174 vs. 269 pmol/l. Homocysteine levels of the rural vs. urban men were 19 vs. 11 µmol/l.

Some background: Homocysteine is a byproduct of methionine metabolism and is considered to be a risk factor for heart disease and stroke (among other diseases). Generally, homocysteine is raised by either vitamin B12, folate, or vitamin B6 deficiency. Vegetarians and vegans who do not supplement with vitamin B12 typically have elevated homocysteine levels. A level of 8 µmol/l or below is ideal, whereas greater than about 12 µmol/l is associated with increased risk of disease.

In the past, some people have thought that elevated homocysteine was caused by high levels of methionine in the diet, although this was put to rest some years ago.

Now, here is the interesting part of this study (if you happen to find the folate/methionine cycle interesting). Because the rural men were not technically deficient in vitamin B12, but were marginally protein malnourished, the researchers thought it was not vitamin B12 deficiency that was causing the elevated homocysteine but rather marginal intakes of the amino acid methionine. Their theory is that when you are deficient in methionine, the body produces excess homocysteine from cysteine so that it can then create methionine and, in turn, s-adenosylmethionine (SAMe) which is an important methyl donor for many reactions throughout the body.

My take on this is somewhat different. Although the vitamin B12 levels in the rural men were technically not in the deficiency range, they were not ideal for homocysteine levels. Selhub (2) suggests a minimum vitamin B12 level of 300 pmol/l for minimizing homocysteine levels and this is born out in the current study in that even the urban men with a B12 level of 269 pmol/l had a slightly elevated homocysteine of 10.8 µmol/l while getting plenty of protein.

Despite the title of the study saying that vegetarianism produces atherogenesis, there was no mention of this in the paper. In fact, the cholesterol levels of the rural and urban men were at relatively low levels of 154 and 166 mg/dl respectively (which is not a direct measure of atherogenesis, but low cholesterol levels are often associated with low atherogenesis).

A press release published in SFGate.com did a write-up on the study (which is what caught some people’s attention): Vegetarian Diet Might Increase the Risk of Cardiovascular Diseases, According to a Recent Study, Says Nutri-Med Logic Corp. While I would not describe their release as terribly inaccurate, they fail to mention that the study was of semi-vegetarians in Chad who were arguably malnourished. Their suggestion of supplementing with alpha-lipoic acid to combat what is either B12 or protein malnutrition is a stretch.

The take home message from this study is: People who limit animal product consumption need a regular source of vitamin B12. People who follow a vegetarian diet due to a lack of food in an area with low amounts of available plant protein could become protein malnourished and this could possibly exacerbate elevated homocysteine levels. Vegans in developed countries can easily avoid these problems by supplementing with vitamin B12 and getting enough protein.

References

1. Ingenbleek Y, McCully KS. Vegetarianism produces subclinical malnutrition, hyperhomocysteinemia and atherogenesis. Nutrition. 2011 Aug 26. [Epub ahead of print] Link

2. Selhub J, Jacques PF, Dallal G, Choumenkovitch S, Rogers G. The use of blood concentrations of vitamins and their respective functional indicators to define folate and vitamin B12 status. Food Nutr Bull. 2008 Jun;29(2 Suppl):S67-73. Review. Link

I’ve been reviewing the scientific literature on protein and bone health. As many readers know, there has been a theory that animal protein, by way of increased sulfur amino acids, causes calcium excretion leading to osteoporosis. This theory was always on shaky ground and more recent evidence has contradicted the theory. I just read a meta-analysis on the subject and added the following to the article, Bones, Vitamin D, and Calcium, on VeganHealth.org:

“Another 2009 meta-analysis found that among five well-designed studies measuring calcium balance, net acid excretion was not associated with either decreased calcium balance or a marker of bone deterioration (48).”

The paper had an interesting list of cohort studies in the discussion:

“First, during 8 yr of follow-up, fracture risk was not reduced among a cohort of 36,217 postmenopausal women who consumed either lower protein or lower NAE [net acid excretion] diets (37). Second, wrist fracture risk was highest among 1865 peri- and postmenopausal women who consumed the lowest protein intakes over 25 yr of follow-up (38). As well, a recent 2-yr trial in 276 postmenopausal women either supplemented with potassium citrate (expected to neutralize the acid of the Western diet) or encouraged to consume increased fruit and vegetables showed that these interventions did not reduce bone turnover or decrease bone loss (36).”

The take home message, which I’ve written about before, is that not eating animal protein does not protect you from osteoporosis. Make sure you get enough calcium, vitamin D, and even (plant) protein.

References

36. Macdonald HM, Black AJ, Aucott L, Duthie G, Duthie S, Sandison R, Hardcastle AC, Lanham New SA, Fraser WD, Reid DM. Effect of potassium citrate supplementation or increased fruit and vegetable intake on bone metabolism in healthy postmenopausal women: a randomized controlled trial. Am J Clin Nutr. 2008 Aug;88(2):465-74. (Link) Free article available.

37. Dargent-Molina P, Sabia S, Touvier M, Kesse E, Bréart G, Clavel Chapelon F, Boutron-Ruault MC. Proteins, dietary acid load, and calcium and risk of postmenopausal fractures in the E3N French women prospective study. J Bone Miner Res. 2008 Dec;23(12):1915-22. (Link) Free article available.

38. Thorpe DL, Knutsen SF, Beeson WL, Rajaram S, Fraser GE. Effects of meat consumption and vegetarian diet on risk of wrist fracture over 25 years in a cohort of peri- and postmenopausal women. Public Health Nutr. 2008 Jun;11(6):564-72. Epub 2007 Aug 9. (Link) Free article available.

48. Fenton TR, Lyon AW, Eliasziw M, Tough SC, Hanley DA. Meta-analysis of the effect of the acid-ash hypothesis of osteoporosis on calcium balance. J Bone Miner Res. 2009 Nov;24(11):1835-40. (Link)

A study was published in the Journal of the American Dietetic Association’s August issue doing an analysis of protein and weight gain. The odd thing about it was that they used data from a cohort of male employees of the Chicago Western Electric Company — from the 1950s!

In any case, they found that, after 7 years of follow-up, animal protein intake was associated with being overweight and obese, but vegetable protein was the opposite — inversely associated with being overweight. This would be a no-brainer except that they found this independent of calories, fat, and carbohydrate. The findings were highly significant.

The researchers theorized that the different amino acid composition of the animal vs. vegetable protein could account for the difference.

It would have been interesting to see a cohort study done like this in which fat-free mass was included as a variable. In other words, did the people eating more animal protein have a higher muscle mass leading to a higher body mass index (the measure of overweight and obesity)? But even if it did, I’d be surprised if it could make up for the high level of statistical significance. Further research is needed!

Citation

Bujnowski D, Xun P, Daviglus ML, Van Horn L, He K, Stamler J. Longitudinal Association between Animal and Vegetable Protein Intake and Obesity among Men in the United States: The Chicago Western Electric Study. J Am Diet Assoc. 2011 Aug;111(8):1150-1155. (Abstract)

Good news from Germany:

Germany’s Strongest Man Is A Vegetarian

Excerpt:

Now I have proven finally, that being vegetarian makes you a better athlete!

It certainly proves that being vegetarian does not prevent someone from being extremely strong.