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Showing posts with label Vitamin D. Show all posts
Showing posts with label Vitamin D. Show all posts

Wednesday, 8 March 2017

Take your Bumetanide Studies with a Pinch of Salt



This blog does try to be based on evidence, but sometimes you do have to question the validity of what appears in peer reviewed journals.  It might concern what does, or does not cross the blood brain barrier, or what works in vivo versus in vitro.

Two interesting papers were recently brought to my attention regarding Bumetanide.


With a pinch of salt is an English idiom which means
to view something with skepticism 



In Tyler’s paper it was rats with epilepsy showing big improvements when taking Bumetanide. 

In Agnieszka’s paper, involving mice and Chinese hamsters, researchers are making the point that so little Bumetanide crosses into the brain that its therapeutic value is limited. 

So which is true? 

Well it seems that in some humans with autism enough bumetanide crosses the blood brain barrier (BBB) to show a positive effect.  Perhaps if a more penetrative analogue of Bumetanide was developed, it would show even greater effect, otherwise adjunct therapies may be needed (Acetazolamide, potassium bromide, estradiol etc) to gain the full benefit of lowering intracellular chloride. 

In the past I have made the case for bumetanide possibly reducing the incidence of epilepsy developing in autism and that this really would be important. This does not mean that one person with autism might not develop epilepsy around the same time he started taking bumetanide. In the study below the rats with seizures seemed to be protected by bumetanide and the number of harmful neural connections detected in the brain was significantly reduced. 




Abstract

There is accumulating evidence that bumetanide, which has been used over decades as a potent loop diuretic, also exerts effects on brain disorders, including autism, neonatal seizures, and epilepsy, which are not related to its effects on the kidney but rather mediated by inhibition of the neuronal Na-K-Cl cotransporter isoform NKCC1. However, following systemic administration, brain levels of bumetanide are typically below those needed to inhibit NKCC1, which critically limits its clinical use for treating brain disorders. Recently, active efflux transport at the blood-brain barrier (BBB) has been suggested as a process involved in the low brain:plasma ratio of bumetanide, but it is presently not clear which transporters are involved. Understanding the processes explaining the poor brain penetration of bumetanide is needed for developing strategies to improve the brain delivery of this drug. In the present study, we administered probenecid and more selective inhibitors of active transport carriers at the BBB directly into the brain of mice to minimize the contribution of peripheral effects on the brain penetration of bumetanide. Furthermore, in vitro experiments with mouse organic anion transporter 3 (Oat3)-overexpressing Chinese hamster ovary cells were performed to study the interaction of bumetanide, bumetanide derivatives, and several known inhibitors of Oats on Oat3-mediated transport. The in vivo experiments demonstrated that the uptake and efflux of bumetanide at the BBB is much more complex than previously thought. It seems that both restricted passive diffusion and active efflux transport, mediated by Oat3 but also organic anion-transporting polypeptide (Oatp) Oatp1a4 and multidrug resistance protein 4 explain the extremely low brain concentrations that are achieved after systemic administration of bumetanide, limiting the use of this drug for targeting abnormal expression of neuronal NKCC1 in brain diseases.
  

Prolonged epileptic seizures may cause serious problems that will continue for the rest of a patient's life. As a result of a seizure, neural connections of the brain may be rewired in an incorrect way. This may result in seizures that are difficult to control with medication. Mechanisms underlying this phenomenon are not entirely known, which makes current therapies ineffective in some patients.
A study conducted with a rat epilepsy model at the Neuroscience Center of the University of Helsinki showed that a change in the function of gamma-aminobutyric acid (GABA), a main neurotransmitter in the brain, is an underlying cause in the creation of harmful neural connections.
After a prolonged convulsive seizure, instead of the usual inhibitory effect of the transmitter, GABA accelerates brain activity. This, in turn, creates new, harmful neural connections, says Research Director Claudio Rivera.
The accelerating effect of GABA was blocked for three days with a drug called bumetanide given soon after a seizure. Two months after the seizure, the number of harmful connections detected in the brain was significantly lower.
"Most importantly, the number of convulsive seizures diminished markedly," says Claudio Rivera.
In this study, new indications may be found for bumetanide in the treatment of epilepsy. Bumetanide is a diuretic already in clinical use. Extensive clinical studies have already been conducted with bumetanide regarding its ability to reduce the amount of convulsions or prevent them entirely in the acute phase of seizures. This, however, is the first time that bumetanide has been found to have a long-term effect on the neural network structure of the brain.
Further study of the newly found mechanism may eventually help limit the exacerbation of epilepsy and prevent the onset of permanent epilepsy after an individual serious seizure. It may also be possible that a similar mechanism is responsible for the onset of epilepsy after a traumatic brain injury.
"The next step is to study bumetanide both by itself and in combination with other clinically used drugs. We want to find out the ways in which it may offer additional benefits in the treatment of epilepsy in combination with and even in place of currently used antiepileptic drugs," says Claudio Rivera.



Vitamin D and Autism

Two medical readers of this blog highlighted this recent paper showing an apparent universal benefit of vitamin D supplementation in autism.

Is it too good to be true?  Time for the pinch of salt?

One important point to note is that this study was in Egypt and, although sunny, are children there eating food that has already been fortified with vitamin D, like it is in Western countries?

Randomized controlled trial of vitamin D supplementation in children with autism spectrum disorder

Abstract

BACKGROUND:



Autism spectrum disorder (ASD) is a frequent developmental disorder characterized by pervasive deficits in social interaction, impairment in verbal and nonverbal communication, and stereotyped patterns of interests and activities. It has been previously reported that there is vitamin D deficiency in autistic children; however, there is a lack of randomized controlled trials of vitamin D supplementation in ASD children.

METHODS:



This study is a double-blinded, randomized clinical trial (RCT) that was conducted on 109 children with ASD (85 boys and 24 girls; aged 3-10 years). The aim of this study was to assess the effects of vitamin D supplementation on the core symptoms of autism in children. ASD patients were randomized to receive vitamin D3 or placebo for 4 months. The serum levels of 25-hydroxycholecalciferol (25 (OH)D) were measured at the beginning and at the end of the study. The autism severity and social maturity of the children were assessed by the Childhood Autism Rating Scale (CARS), Aberrant Behavior Checklist (ABC), Social Responsiveness Scale (SRS), and the Autism Treatment Evaluation Checklist (ATEC).

RESULTS:



Supplementation of vitamin D was well tolerated by the ASD children. The daily doses used in the therapy group was 300 IU vitamin D3/kg/day, not to exceed 5,000 IU/day. The autism symptoms of the children improved significantly, following 4-month vitamin D3 supplementation, but not in the placebo group. This study demonstrates the efficacy and tolerability of high doses of vitamin D3 in children with ASD.

CONCLUSIONS:



This study is the first double-blinded RCT proving the efficacy of vitamin D3 in ASD patients. Depending on the parameters measured in the study, oral vitamin D supplementation may safely improve signs and symptoms of ASD and could be recommended for children with ASD. At this stage, this study is a single RCT with a small number of patients, and a great deal of additional wide-scale studies are needed to critically validate the efficacy of vitamin D in ASD.

Conclusion
Take your research with a pinch of salt.




Sunday, 3 January 2016

Vitamin A (and ATRA) Upregulate Oxytocin via CD38


 A familiar site to Maja, the confluence of the Sava and the Danube


Today’s post is to document an interesting discovery by Maja, one reader of this blog.  She is just ahead of some Korean researchers, who very recently published a paper in Experimental Neurobiology on the same subject.

Maja noticed that giving a small dose of fish oil produced the same benefits as those often claimed for Oxytocin; she then did some investigation and noted that an enzyme called CD38 upregulates oxytocin in the brain.  The level of CD38 is affected by inflammatory cytokines and certain vitamins.  In particular, all-trans retinoic acid (ATRA) increases CD38. All-trans retinoic acid (ATRA) is made in the body from vitamin A.  ATRA is also called vitamin A acid.

Maja suggested this paper:-



Deficits in social behavior in mice lacking the CD38 gene have been attributed to impaired secretion of oxytocin. In humans, similar deficits in social behavior are associated with autistic spectrum disorder (ASD), for which genetic variants of CD38 have been pinpointed as provisional risk factors. We sought to explore, in an in vitro model, the feasibility of the theory that restoring the level of CD38 in ASD patients could be of potential clinical benefit. CD38 transcription is highly sensitive to several cytokines and vitamins. One of these, all-trans retinoic acid (ATRA), a known inducer of CD38, was added during cell culture and tested on a large sample of N = 120 lymphoblastoid cell (LBC) lines from ASD patients and their parents. Analysis of CD38 mRNA levels shows that ATRA has an upmodulatory potential on LBC derived from ASD patients as well as from their parents. The next crucial issue addressed in our study was the relationship between levels of CD38 expression and psychological parameters. The results obtained indicate a positive correlation between CD38 expression levels and patient scores on the Vineland Adaptive Behavior Scale. In addition, analysis of the role of genetic polymorphisms in the dynamics of the molecule revealed that the genotype of a single-nucleotide polymorphism (rs6449182; C>G variation) in the CpG island of intron 1, harboring the retinoic-acid response element, exerts differential roles in CD38 expression in ASD and in parental LBC. In conclusion, our results provide an empirical basis for the development of a pharmacological ASD treatment strategy based on retinoids.


In December some Korean researchers also suggested that ATRA might be used therapeutically to increase Oxytocin.  Maja discovered that vitamin A can also be used, which makes sense.

The Korean paper reviews the existing literature and clinical trials on oxytocin in autism, and I suggest those interested should read it.

Some people clearly benefit from oxytocin, some do not and some suffer side effects.

In those that benefit from oxytocin, it might be simpler to upregulate the body’s own oxytocin via ATRA, or vitamin A.


Is this proof?

Of course there are other explanations possible for what Maja has noted.  She was using fish oil as a source of vitamin A, so it could be related to the other constituents.

However, I for one think it is highly plausible and does fit nicely with the ideas put forward by the Korean researchers and the earlier paper.


Vitamin A for all?

We know that autism genes include many for oxytocin, oxytocin receptors and indeed CD38, so anyone with those genes dysfunctional might benefit.

However, as we saw with biotin, more people may be affected to a lesser degree.

CD38 affects oxytocin secretion in the brain and CD38 is affected by inflammatory cytokines, so at times of elevated cytokine expression, CD38 and oxytocin might be reduced in people with no relevant genetic dysfunction.

You can have too much vitamin A, this is called Hypervitaminosis A.  You cannot suffer this condition by eating fruit and vegetables, but you can by eating too much preformed vitamin A from foods (such as fish or animal liver), supplements, or prescription medications; it can be prevented by ingesting no more than the recommended daily amount.

High intake of provitamin carotenoids (such as beta carotene) from vegetables and fruits does not cause Hypervitaminosis A, as conversion from carotenoids to the active form of vitamin A is regulated by the body to maintain an optimum level of the vitamin. Carotenoids themselves cannot produce toxicity.

So, too much cod liver oil can be bad for you, but you can eat carrots like Bugs Bunny and do no harm.  If you really overdo it, your skin may change colour to orange, something called carotenosis

You can buy vitamin A supplements as the preformed vitamin or as beta carotene.


Too much of a good thing?

In times gone by, children used to be given a tablespoon of cod liver oil daily, as a good source of vitamin D and vitamin A.  These days that amount of both vitamins would be seen as excessive.  Excess of both vitamins is bad for you, but easy to achieve, by accident, while trying to do a good thing.


Maja’s Dose

Maja achieved her positive results with a modest dose of fish oil (using 40% of one capsule) giving 3-4000 IU of vitamin A.

This is actually quite a high dose of vitamin A, if you look at the maximum safe dose.

I think many people are giving kids with autism much larger doses of fish oil and thus far too much vitamin A and D.  This has been raised as an issue by Seth, another reader of this blog.


CD38

CD38 has many other functions other than regulating oxytocin. In people who have an oxytocin dysfunction due to an upstrean CD38 dysfunction, correcting the lack of CD38 might be particularly beneficial.   

CD38 is used as a prognostic biomarker for leukemia.  This is a complex area of science.  In essence, it is an accepted fact that increased CD38 expression is associated with favorable prognosis in adult acute leukemia.

Leukemia is associated with Down Syndrome. 

Not surprisingly, both vitamin A and ATRA can be beneficial in treating leukemia.
ATRA (All Trans-Retinoic Acid) for acute myeloid leukaemia (AML)


CD38 expression is apparently easy to measure.

Perhaps in those numerous oxytocin trials for autism, they might want to bother measuring CD38?


The Recent Korean Paper


Here is what the Koreans have to say about Oxytocin:-




CD38 is a transmembrane antigen that has been studied as a negative prognostic marker for chronic lymphocytic leukemia [72]. CD38 participates in the oxytocin secretion in the brain and affects maternal nurturing and social behavior [73]. Plasma levels of oxytocin are strongly reduced in CD38 knockout mice (CD38-/-mice) and subcutaneous oxytocin injection or lentiviralvector-mediated delivery of human CD38 into the hypothalamus rescued social memory and maternal care in these mice [73].

CD38 transcription is highly sensitive to cytokines and vitamins, including all-trans retinoic acid (ATRA), a known inducer of CD38 [75]. In a study on lymphoblastoid cell lines in patients with ASD and their parents, ATRA exhibited an upmodulatory potential on CD38 mRNA [75]. Although there have been almost no follow up studies on ATRA and ASD treatment, there is a possibility that substances affecting CD38 expression, such as ATRA, may be potential therapeutic candidates














Friday, 28 February 2014

Vitamin D in Autism – too much or too little?


Reader’s of this blog will be aware that serotonin plays a major role in autism, and also in many other mental health conditions, like depression.

Vitamin D also regularly raises its head in discussions about autism.  You may recall the Somali autism clusters in Sweden and Minneapolis; researchers suggested that the Somali immigrants were not getting enough sun and therefore lacked vitamin D and so produced children with autism.  I did point out that another large Somali autism cluster exists in sun-drenched San Diego.
Even Martha Herbert talks about vitamin D deficiency and autism.

A while back we had a guest blogger, Seth Bittker, present his opposing view, that too much vitamin D added to food in the American diet may be contributing to the rise in autism there.
In same week that Seth has published his paper on this subject, yet another paper has appeared with the opposing view.  So who is right?

The case for (even) more Vitamin D

The first paper is:- 

 
The authors make the following case:-
Serotonin and vitamin D have been proposed to play a role in autism, however, no causal mechanism has been established. Now, researchers show that serotonin, oxytocin, and vasopressin, three brain hormones that affect social behavior related to autism, are all activated by vitamin D hormone. Supplementation with vitamin D and tryptophan would be a practical and affordable solution to help prevent autism and possibly ameliorate some symptoms of the disorder.

After absorbing L-tryptophan from food, our bodies convert it to 5-HTP (5-hyrdoxytryptophan), and then to serotonin.
The supplements L-tryptophan and 5-HTP are widely available and have been used in ADHD and autism but there is no evidence that they are effective.  All that has been shown is that too little tryptophan is bad; there is nothing to show that abnormally large amounts do any good.
If you read the full paper there is an excellent explanation of the role of serotonin in autism.  It is beyond doubt that in many kids with ASD there is high blood serotonin, but low brain serotonin.
To fully treat autism, one thing to be done is to raise brain serotonin levels, without any nasty side effects.  SSRI drugs like Prozac, used to treat depression, do raise brain serotonin but often cause dependence and side effects (like suicidal thought).
It would be great if some vitamin D and tryptophan could do the job.

If you read the older literature, you will see that there is nothing new about the idea to supplement with Tryptophan in autism.  The results to date have been nothing special.
Here is a paper by Paul Whiteley and Paul Shattock:-
 

“It has been shown that a diet depleted of tryptophan is not beneficial for children with ASDs and that some symptoms are exacerbated. Presumably, the existing lack of available serotonin (and other tryptophan derivatives) was exacerbated under these circumstances. Supplementation with tryptophan would probably not be helpful in the majority of cases because the conversions along the important pathways are inhibited and tryptophan is likely to be converted along the IAG route, which would be unhelpful. Anecdotal clinical reports suggest that some children show benefits and others may get worse but no formal studies have been reported.

For this reason, and because tryptophan is a prescription-only drug*, we have looked at other methodologies. The active transmitter, serotonin, does not cross the blood brain barrier and so would be ineffectual in this respect. However, the precursor molecule 5-HTP does cross the blood brain barrier and reach the appropriate target areas. Some parents have reported impressive consequences, particularly with regard to sleep patterns; some physicians have been able to reduce the doses of e.g. risperidone (an anti-psychotic drug) by supplementing 5-HTP but, on the whole, the results have been less useful than would have been predicted.”


Vitamin D and Children with ASD
Children with autism are probably amongst the most “vitamin-supplemented” of any, since parents tend to give copious amounts of multi-vitamins and also vitamin D rich omega 3 fish oil.  It is hard to imagine that any of these children are deficient in vitamin D.
 

The case for too much vitamin D
In his paper, Bittker seeks to correlate the increase in vitamin D fortification in America with the rise in autism; he highlights groups that do not have vitamin D fortified food and where autism is far less prevalent.

 

Conclusion
So who is right?  Well for sure too little tryptophan or vitamin D is bad for you; but are abnormally high levels good or bad?  In the case of tryptophan, plenty of people have tried supplementation in autism and ADHD and we would probably have heard if it produced a great effect.

Do large amounts of vitamin D help with autism? I very much doubt it, but it would be very easy to do a trial, assuming you found some parents who had not read the Bittker paper.
The good thing is that raising the low level of brain serotonin seems agreed by everyone as a prime target of any autism intervention. For me, vitamin D and tryptophan is not the answer.

 

Sunday, 8 December 2013

A Hypothesis: Vitamin D, Calcium, Milk, and the Autism Epidemic



by Seth Bittker

My name is Seth Bittker, and I am the father of a boy with ASD.  My son’s development was slow up until about 2 1/2 years of age.  Around this time, his milk consumption increased, and it began crowding out other sources of sustenance.  Within a few months he regressed, and we received an autism diagnosis soon after this.  Stopping his milk consumption was the first step in helping him get better.
Like some other parents of those with ASD children, I noticed that his behavior varies significantly with what he eats.  When he consumes large amounts of calcium fortified foods or beverages, his behavior gets worse: he becomes more autistic in a behavioral sense.  Since milk has a lot of calcium, and calcium fortified foods do as well, it seemed like there might be some connection to calcium and his symptoms.  Later based on a doctor’s recommendation we gave him supplemental vitamin D.  We did not notice much initially, but a few weeks later he developed hives, and he regressed.  Since supplementation with vitamin D increases absorption of calcium, it appeared that the bad reaction to calcium and vitamin D might be connected.  Based on these experiences and others, I eventually inferred that we should keep him on a relatively low-calcium diet and avoid vitamin D supplementation or fortification (milk as well as many other foods in the US are fortified with vitamin D).
I also concluded that my son was different from most others affected with autism as some have suggested that vitamin D deficiency is involved in inducing autism,[1] and he evidently did not have a deficiency based on the vitamin D trial even though vitamin D levels in his blood were relatively low.
But is he really different than others with autism?  If one examines the evidence from a number of genetic syndromes that are comorbid with autism, in many cases the cause is over-active calcium channels (effectively too much intra-cellular calcium).[2]  You can see this by looking at syndromes such as Timothy syndrome, Williams syndrome, and Sotos syndrome, which have high comorbidity with autism and seem to be caused by too much intra-cellular calcium.  In Williams syndrome the connection is through a gene which upregulates vitamin D.[3]  In some of these syndromes is it well known that supplementation with calcium and vitamin D are contraindicated.[4]
In addition there are certain biochemical markers that are typical in autism, and my son seems to have this same biochemical gestalt.  For example two types of immune system cells released by the thymus are called “Th1” cells and “Th2” cells, and Th2 cell levels relative to Th1 cells are much higher in the blood of those with autism than in controls.[5]  As Th2 is associated with extra-cellular immunity, this suggests that those with autism will be prone to allergies,[6] which fits with anecdotal observation.  In addition with autism we see elevated markers for oxidative stress and endothelial damage.[7]  Also those with autism typically have functional deficiencies of magnesium[8] and potassium.  By functional we mean the levels may not be low in the blood relative to controls, but there is a biochemical need for greater consumption.  To see the latter you can give somebody with autism a small dose of supplemental potassium, and the result is generally a reduction in autistic symptoms.  For more on this, see Peter’s work: http://epiphanyasd.blogspot.com/2013/08/potassium-may-play-important-role-in.html.
It turns out that vitamin D skews the immune system to produce elevated levels of Th2 cells.[9]  One can infer that it also produces functional deficiencies of potassium and magnesium as these minerals offset the effect of calcium on calcium channels and vitamin D increases the absorption of calcium.  In addition in high doses vitamin D causes oxidative stress and endothelial damage as we see in autism.[10]

Oral supplementation with vitamin D can cause other consequences as well.  Supplementation with significant doses of vitamin D early in life, results in more cases of allergies, asthma, and dermatitis later.[11]  Asthma, allergies, and dermatitis all feature high levels of Th2 cells like autism.[12]  Is it too much of a stretch to suggest that supplementation with vitamin D early in life, might result in more cases of autism later as well?

On looking at the data we can see that my son as well as most other babies in much of the civilized world received, and in many cases continue to receive, large oral doses of vitamin D starting from birth.  As a baby we gave him vitamin D drops, and he also occasionally received some formula.  Starting at one year of age he consumed increasing amounts of milk (fortified with vitamin D in many countries) as well as children’s multivitamins, which also contained vitamin D.   Here is a graph showing vitamin D content of various beverages:



 
We are giving babies today much more oral vitamin D than they would consume from human milk alone and also much more than they would have consumed in past decades through formula and food sources when vitamin D fortification was more restrained.  In fact based on nutrition data and caloric intake, a baby just out of the womb consuming Similac baby formula today will receive approximately 855 IU per day of vitamin D.[13]  I am 190 pounds.  If we assume that dose should be proportional to weight, which is a good baseline assumption for most vitamins, I should consume 16,245 IU per day of vitamin D.  This is a huge dose, and based on past experience I know it would have a devastating effect on my health.  Why should it not be the same for some subset of babies?
When and where vitamin D consumption by the young is high, is where autism rates are high.  For example, in the United States autism rates are high relative to much of the rest of the world and they rose gradually starting around 1980 as much of the population transitioned to lower fat milks, which increased consumption of vitamin D.[14]  Later rates rose again in the early 1990s as the amount of vitamin D in milk was increased significantly with no change in the label.[15]  Rates continued rising with the popularization of the Sippy Cup starting in the 1995.[16]  They rose further during the 2000s as the Institute of Medicine increased the supplementation recommendations for babies in 2003 and again in 2008.[17]

When looking internationally, the evidence also points to a role for oral consumption of vitamin D in inducing autism.  Cuba for example has extraordinarily low rates of autism.[18]  The Cuban Health Service does not supply vitamin D drops to babies.  Nor do they fortify milk or other foods with vitamin D in Cuba.[19]  The Amish also have very low rates of autism as well.  They do not provide vitamin D drops to babies, and the milk they consume comes straight from cows: no vitamin D is added.[20]  In the United Kingdom rates of autism are lower than in the United States.[21]  Fortification and supplementation are common in the United Kingdom, but at somewhat lower levels than in the United States.[22]
Vitamin D is also used as a rodenticide, and experiments in rats show that it is more deadly to male rats than female rats.[23]  Is it any coincidence that autism strikes many more males than females?  Babies who get most of their sustenance from formula also receive more vitamin D than those who receive vitamin D from human milk.  Not surprisingly autism rates are higher among those who are formula fed.[24]
Vitamin D’s involvement also explains why some with autism benefit from a “casein” free diet as such diets avoid milk, which has high calcium content and is fortified with vitamin D in many countries.  It also explains why low-oxalate diets are beneficial as well as ketogenic diets.  Oxalates cause excess calcium salts to precipitate in the body and ketones effectively remove excess calcium due to acidity.[25]  Both lower the calcium load.  Oral supplementation with vitamin D also explains why some with autism have a favorable reaction to chelation.  Chelation agents remove excess calcium at the same time as they remove other metals.[26]  So they also lower the calcium load.
So the evidence suggests that over-consumption of vitamin D by babies and toddlers may induce autism in a genetically susceptible subset of the population.  This explains the biochemistry of autism as well as the nature of the autism epidemic.  There are a number of other data points that fit with this hypothesis.  If you are interested in this topic, here is an ebook with a lot more detail: http://www.amazon.com/dp/B00GVB46ES.

I want to thank Peter for allowing me to be a guest blogger here, and I also want to thank you for reading this. How do you view this hypothesis?  Do you have observations that are relevant to it?  Are you aware of existing data that would help confirm it or refute it?




[2] For more on calcium channels and autism, please see http://www.autismcalciumchannelopathy.com/Genetic_Factors.html.
[10] http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3052582/ and Gross, David Ross, Animal Models in Cardiovascular Research, p. 316.
[15] FDA, M-I-92-13.