Dept. of Environmental and Infectious Disease Science

Michael King

•In 2007, there were thousands of cases of kidney- related illness in pets

across North America. The cause of illness was traced back to certain lots of

wet pet foods.

•Hundreds of cats and dogs died of acute renal failure. Water insoluble

crystals in the kidneys were found to be the cause.

•Extensive research determined melamine to be one of the main

contaminants. The contamination was traced back to a Chinese factory, which

used melamine-tainted wheat gluten. (The protein content in food is measured

in nitrogen levels, not actual protein content, hence the term “apparent protein

levels.” Food with higher protein levels can be sold for a higher price.)

Raw melamine

Melamine itself is not very toxic, so researchers hypothesized the existence of a second contaminant that would combine with the melamine to form insoluble crystals. Cyanuric acid was found to be the second contaminant.

In 2008, there were widespread reports of kidney illness in young children in China. The children affected were found to be consuming melamine-tainted baby formula.

Over 300,000 children became ill; however, 4-10 deaths have been reported.

Uric acid theory-A group of researchers working in China reported that the melamine in baby

formula bonded with uric acid (a naturally occurring chemical in the urinary system) to form water insoluble crystals.

Melamine/cyanuric acid crystal structure

-It has already been established that cyanuric acid will bond with melamine to

form insoluble crystals, but it seemed unlikely that the same thing would

happen with uric acid and melamine. We wanted to test if uric acid would

form crystals with melamine, and if they did we sought to obtain IR spectra

from these crystals for future reference to be able to identify the crystals in

patients.

-We wanted to examine the melamine-cyanuric acid crystals to see if an excess

of either compound would change the crystal composition. We planned to

use GC-MS to evaluate the crystal composition.

Uric acid Cyanuric acidMelamine

-Smiths Illuminat IR II (IR spectrometer)

-IR Microsystems molecular analysis system (IR spectrometer)

-Agilent Technologies 6890N GC system (Gas chromatography device)

-Agilent Technologies 5975 mass selective detector (Mass

spectrometer)

-Agilent Technologies 7683 series autosampler

-IEC centra CC2 centrifuge

-Eppendorf research pipettes [200 micro liters, 1000 micro liters]

-Fisherbrand 5 ¾ “ disposable Pasteur pipettes

-TurboVap LV evaporator

-Olympus BX51 (Light microscope)

-S-3400N Hitachi Scanning Electron Microscope

• Mixed different concentrations of melamine and cyanuric acid (at room temperature)

• Mixed melamine and uric acid (at room temperature)

• Mixed melamine and uric acid (at 37˚C , body temperature)

• Mixed melamine and uric acid (at pH 6 and pH 10)

• Mixed melamine and uric acid, then added cyanuric acid (at room temperature)

• Analyzed the solutions with IR spectroscopy and/or GC-MS

Every solution of melamine and cyanuric acid formed

new crystals. All of the new crystals were collected and

scanned using IR spectroscopy. The IR spectra all looked

similar and the GC-MS data suggested a nearly constant

ratio.

None of the melamine/uric acid solutions formed new

crystals. Any crystals present were found by IR

spectroscopy to be uric acid only.

It was found that uric acid is insoluble in water, unless the

solution is at basic pH levels.

Example of a melamine/cyanuric acid crystal chromatogram

Ratio (ca/m) CA average area Mel. average area ReRatio (CA/M)

*90/10* 689758 1203297 0.573223402

*70/30* 2671438 3603430 0.74135976

*50/50* 6544769.5 7778200.5 0.841424633

*30/70* 2594576.5 5109277 0.507816762

*10/90* 904972 1487489.5 0.608388832

•Ratios are similar

Cyanuric acid Melamine

Cyanuric acid mass spectrum

Melamine mass spectrum

•Melamine and cyanuric acid are water soluble

•When mixed they form water insoluble crystals (shown in bottom spectrum)

•Bottom spectrum matches spectra from pet kidney tissue

•Solid collected after mixing melamine and uric acid

was found to be uric acid

• The results of this project point to cyanuric acid being the

agent which bonded with the melamine in pet food to form

water insoluble crystals.

• The results also show it is unlikely that melamine from the

baby formula bonded with uric acid to form insoluble crystals

in the babies’ kidneys.

• Bearing this in mind, the reported composition of uric

acid/melamine crystals is likely erroneous.

Why should we care?

-Increasing amounts of imported food

-Processed food has bulk ingredients

Many brands could be tainted

-Food tainting is a common problem

-Despite the efforts of the FDA inspections, contamination may happen

again

How does knowing help?

”A pound of cure is worth an ounce of prevention”

-Doctors know what they’re dealing with (Better, faster treatment)

-FDA officials and Investigators know what to look for

I give many thanks to the following people;

Dr. Mullick- For making this program possible

Dr. Kalasinsky- For being an excellent mentor, for showing me the way

Dr. Lewin-Smith- For being another excellent mentor, for the ride and for

heading the slide conferences

Ms. Laura Burry- For being a great teacher, for helping me in the lab

Ms. Marie Jenkins- For her kindness, for letting me use her centrifuge, and

for her stories

Ms. Lynn Blubaugh- For being another great teacher, for showing me how

to use the SEM

Ms. Leslie Middleton- For organizing everything, and for your patience,

time, and reminders

Ms. Stacy Strausborger- For teaching me how to do molecular calculations

The Environmental department- For their kindness, patience and positive

attitude