Peptides and protein intolerance

Multiple studies have shown that children with autism may have elevated levels of peptides in their urine, which may be the result of incomplete breakdown of gluten and casein. This leads to the formation of peptides with opioid (morphin-like) characteristics, and there is evidence suggesting that this kind of peptides worsen the symptoms in these patient.

Opioid peptides derived from casein are called casomorphins, while those derived from gluten are called gluten exorphins and gliadorphin. Opioid peptides have also been discovered in other foods, but these are still not well known.

The cause of the elevated levels of peptides in autistic children has not yet been established. Several theories are discussed:

- Reduced level of peptidases, the enzymes responsible for breaking down peptides, may be part of the explaination.

- Inflammation of the gut increases the permeability for peptides (leaky gut), so that they are more easily absorbed into the bloodstream and reach the brain. That will show up as elevated levels of peptides in the urine. Several research groups have in recent years found that autistic children have a particular type of intestinal inflammation, and a large percentage of autistic children have gastrointestinal problems.

- It has also been shown that patients with gastrointestinal problems often suffer from mental disorders.

A diet excluding milk and/or gluten is shown to give significant improvement in the symptoms of autistic children. This is true both when it comes to gastrointestinal problems and behavior.

Neurozym's urinary peptide analysis is often used in cases of autism and ADHD, but also in other mental disorders and when protein intolerance is suspected, especially when combined with gastrointestinal problems.

The peptide analysis is used to give an indication of the usefullness of a casein-free, gluten-free diet or other treatment aiming to eliminate harmful peptides. It’s also possible to use the test to uncover diet breaches. A diet breach is not necessarily intentional. It is often caused by gluten and casein in foods where they are not expected.


HPLC-analysis of peptides in urine

With Karl Ludvig Reichelt’s method as a basis, Neurozym has implemented a HPLC-method to measure the amount of opioid food peptides in urine.

With an HPLC-method (High Pressure Liquid Chromatography), the sample is pumped through a narrow steel cylinder which contains a porous, tightly packed material. The peptides will attach to the particles of the porous material. Then a new solution with a different composition is pumped through the cylinder, which leads to the detachment of the peptides from the porous material. Because the peptides are bound to the porous material with different strength, they are released from the cylinder at different times. As the substance leaves the cylinder, it is registered by a photocell which measures the quantity of the substance. This results in a diagram (chromatogram) with several peaks. Each peak represents a different substance. The size of the peak reflects the quantity of the substance. The position of the peak tells us which peptid it (most likely) is. In each series of analyses, we also run a peptide standard containing the opioid peptides. This shows us the exact location of the individual opioid peptides, and we can use this to locate peptides in the patient's urine. This standard peptide solution now contains:

Peptides from casein

1. Casomorphin 5

2. Casomorphin 7

3. Casomorphin 8

4. Casomorphin 1-3

5. Casomorphin 1-4, amide

Peptides from gluten

6. Gliadorphin

7. Gluten exorphin A5

8. Gluten exorphin B5

9. Gluten exorphin C


10. Dermorphin

Dermorphin is a peptide which has been found in some autistics, but is of unknown origin. It has been suggested that it is made by gut bacteria or fungi.

Below are examples of chromatograms.

Figure 1. HPLC-chromatogram of urine with few peptides.

The upper curve in figure 1 shows substances which absorb light at 215 nm wavelength, and this is the wavelength where peptides are found. Each substance passing by the photocell is registered as a peak. The peak marked with an arrow is most likely casomorphin 7 because it is located at 53,2 minutes, which is the primary characteristic of this peptide. Compared to the chromatogram in figure 2, the peaks here are small and few.

Figure 2. Chromatogram with lots of peptides.

Figure 2 shows an HPLC-chromatogram from a urine sample with many peptides. Compared to figure 1, we see both bigger and more numerous peaks. In this sample we see that the peak corresponding to casomorphin 7 is far larger that the corresponding peak in figure 1. The figure also shows that there can be a lot of peaks in a chromatogram like this, and that they partially float together.

How is the analysis interpreted?

For each series of analysis, a standard mixture of known opioid peptides is run. It shows us the exact position of each peptide and is used to identify these peptides in the urine samples. Peptides and other substances are shown as peaks in a chromatogram. The peaks in the chromatogram are evaluated regarding position and size. This gives us a basis to assess the amount of the opioid peptides in the urine sample.

Normally, the opioid food peptides consitute a minor fraction of the peaks in the chromatogram. The other peaks may be “innocent” peptides and other substances, such as pharmaceuticals. Interpretation of the analysis is necessary to separate the opioid food peptides from other substances. The total peptide level may be high even though the level of opioid peptides is low.

Sometimes, the peaks are very close together, almost on top of each other. I such cases it’s often difficult to decide which are the peptide and which are other substances. It’s therefore important to know if the pasient is on medication, uses herbal medicine etc. The entire analysis is based on the assumption that the sample is morning urine taken before breakfast. After a meal, one usually finds smaller amounts of these peptides, even in healthy individuals. Information about a casein-free, gluten-free diet is of course important.

With basis in the chromatogram, the position and size of the peptide peaks, we decide on the amount. For the individual peptides the amount are given as:

Not found:

When no dairy products or gluten are consumed, they will not be found in the urine. Persons with very efficient breakdown mechanisms may also fall into this category.


After consuming casein or gluten containing food, small amounts of peptides are usually found. This is also true in healthy individuals without protein intolerance.

Slightly elevated:

Such a find may in some cases indicate protein intolerance. A diet might be beneficial. Slightly elevated levels also occur in healthy individuals after considerable intake of casein/gluten.

Significantly elevated:

Levels of peptides clearly above normal. This is seldom seen in healthy individuals. Diet is often beneficial.

Strongly elevated:

Massive amounts of peptides are found in the urine. This indicates accumulation of peptides due to reduced breakdown and/or increased uptake (leaky gut). In such cases a diet should be tried.


The effects of diets are best documented in child autism. A significant proportion of the autistic children experience some kind of improvement when glutenfree and/or caseinfree diets are tried, especially among those with gastrointestinal problems. Other reports also suggest similar connections in other diagnosis, such as ADHD, schizophrenia and depression. Whether diet or other treatment should be initiated also depends on the total clinical situation, assessed by a physician. We who perform the analysis can only give general advice regarding this, since the physician/therapist has the total overwiev and responsibility for the treatment.

Other conditions to consider

Medication may influence the breakdown of peptides. Neuroleptica induce peptidases and thereby increase the breakdown of opioid peptides. Anti-depressants reduce the peptide level in urine, but the mechanisms behind this are unknown. It should be pointed out that patients must NOT stop their medication before the sample is taken.

Sometimes we find opioid peptides in patients who are already on a diet. There can be several explainations for this:

1. Diet breach. Often caused by casein/gluten in products the patient thought was gluten/casein free.

2. Other substances, for example pharmaceuticals, may take the peptide’s exact place in the chromatogram. In such cases, this method can’t distinguish peptides and the unknown substance.

3. In theory, these peptides may have additional origins other than casein and gluten.


© 2006 Neurozym Biotech AS