A Technical Bulletin on Nutrient and Contaminant Levels in LabDiet® Certified Laboratory Animal Feeds

Based on an Independent Study Conducted from 1992-1996 By:

Dr. Dorrance Haught
Dr. Dan Hopkins

Published May, 1999
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TABLE OF CONTENTS
Introduction / Key Points
Background / Materials & Methods
Results
References
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Quality Through Consistency and Control
We understand the integrity of your work depends on controlled experimental protocols. The role we play in your work is to provide constant animal nutrition that enables you to isolate, distinguish and define breakthroughs.
Every step of our manufacturing process, from the purchased ingredients we select to the date-coding of our packaged products, is implemented to deliver the most consistent nutritional diets available.
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Ingredients are purchased through approved      suppliers who understand and adhere to the demands of research diets.

Computerized formulation and near infrared spectroscopy allow us to maintain the most stringent feed ingredient standards in the industry. Ingredients are analyzed daily for nutritional content and purity. LabDiet® formulations are then monitored before approval to ensure our high quality standards are met.
The result? Precisely formulated, Constant Nutrition™ that enables you to conduct accurate     research by optimizing the performance of your laboratory animals.
Introduction
Consistent and proper nutritional content is a vital consideration in diets of animals used in laboratory studies.1 To keep experimental protocols controlled, feed manufacturers must ensure all batches of feed meet the approved nutitional profiles.2

“Certified Diets” for laboratory animals are commonly used in toxicological studies when control of contaminants, such as
pesticides and heavy metals, is imperative. Certified LabDiet® products manufactured by PMI Nutrition International (PMI) are analyzed prior to sale and guaranteed not to exceed established maximum concentrations of key contaminants. Certified LabDiet® products control undesired variables that could adversely affect study animals and possibly cause erroneous evaluation of the safety of the test compounds. Additionally, Certified LabDiets meet the requirements of the FDA’s Good Laboratory Practices Program.

 
Key Points
• Results sought in experiments often include biochemical measurements of animal fluid or tissue. Many of these parameters can be altered by the animal’s diet.
• Constant nutrition is an important factor in discovery studies where structurally similar test compounds are compared
and in studies requiring historical data.3
• Investigators conducting safety studies also rely on constant nutrition to maintain physiological behavior when compared to
historical data.4, 5, 6, 7
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Certified LabDiet® products are formulated and manufactured for a variety of laboratory animals. Levels of contaminants are guaranteed to be below established levels. Key nutrients, such as protein, fat, crude fiber, calcium, phosphorus, and vitamin C (in monkey and guinea pig rations only) must also be within established ranges.

This bulletin reports the results of an independent study conducted over a five-year period where the nutritional components and contaminant levels in Certified LabDiet® products were measured in order to demonstrate the variability that can be expected.

 
Background
Results of the five-year study conducted by an independent laboratory* demonstrate that nutritional content and level of contaminants are well controlled in Certified LabDiet® products manufactured by PMI. The program followed by PMI delivers a consistent nutritional package.
Monkey scan.TIF (457564 bytes) While nutritional content of raw ingredients may change due to variables such as geographic location, harvest time and processing practices, minor adjustments of all ingredients can and should be made in order to maintain constant nutrition.
In the future it will become increasingly important to maintain constant levels of isoflavonoids, polyphenolic compounds and other groups of compounds yet to be identified. Steps taken to maintain constant nutrition with minimal changes in ingredient levels should maintain constant levels of those types of compounds.

* Ralston Analytical Laboratories
801 Chouteau Avenue,
St. Louis, MO 63102

 

PRODUCT

 Samples Analyzed for
Nutritional Profile
1992 1993 1994 1995 1996
Certified Rodent Diet, 5002 3 3 2 3 3
Certified Canine Diet, 5007 3 4 2 3 3
Certified Guinea Pig Diet, 5026 2 3 2 3 3
Certified Primate Diet, 5048 2 3 2 3 3
Certified Rabbit Diet, 5322 2 3 2 3 3
Materials & Methods
Constant nutrition is important in laboratory feeds. In order to measure nutritional consistency of the diets, periodic samples of Certified LabDiet® products were taken, starting in the year 1992. A brief description of the products sampled for nutritional profile and the number of samples taken each year is summarized in Table1.

Data were collected from 1992 through 1996 for nutritional profiles. However, due to premature removal from the database, contaminant profiles for the 1992 samples were not included. Therefore, the contaminant profiles were collected from 1993 through 1997, in order to present five full years of analyses.

Representative feed samples were sent to an independent laboratory for analysis.* The samples were analyzed for proximate constants, essential amino acids, vitamins, micro and trace minerals. The analytical procedures employed were those specified by the Association of Analytical Chemists or by procedures that provide equivalent results.

The average analyzed values for nutrients were compared with calculated formulation values. The standard deviation of the analysis for each nutrient and for each product was tabulated.

 
Results
Protein/Amino Acids
The observed protein levels are well above the calculated levels (Table 2A). Table 2B indicates the consistency in protein levels for 5002 during the same period. PMI is conservative in assigning protein levels to ingredients in order to avoid manufacturing rations below the level guaranteed by the tag. This means a constant bias exists that ensures the actual protein level is greater than the guaranteed level.

Proteins are composed of amino acids. The animals which consume the diets presented in this booklet have specific dietary requirements for numerous amino acids such as arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. All of these must be provided in the proper level in the diet or the animal cannot grow, reproduce or maintain its body properly. Lysine and methionine are the first limiting amino acids in many instances, so their levels are shown in Tables 2C and 2D as indicators of the tight regulation of the proper amino acid composition of the diets.

 

Diet

 # of Samples Calculated Value % 5 Yr. Average
Table 2A.
Protein Levels
in Certified
LabDiet
Products over
5 Year Period
Certified Rodent Diet, 5002 14 20.1 21.2% +/- 0.41
Certified Canine Diet, 5007 15 25.5 26.9% +/- 0.6
Certified Guinea Pig Diet, 5026 13 18.5 20.0% +/- 0.5
Certified Primate Diet, 5048 13 25.0 27.1% +/- 0.6
Certified Rabbit Diet, 5322 13 16.2 17.5% +/- 0.4
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Hamster & beaker.TIF (920000 bytes) Fat
Fat in the study was analyzed by two procedures: ether extract and acid hydrolysis. The ether extract procedure measures neutral fat and is an appropriate procedure for most feeds. However, fat in extruded feeds, such as canine and primate diets, must be hydrolyzed with dilute acid to free the fat which is then extracted and measured. The acid hydrolysis procedure also removes bound fat from ingredients, along with some other types of lipids, such as phospholipids. Consequently, fat measured by acid hydrolysis will
usually be one or two percentage units greater than fat measured by ether extract. The calculated levels in Table 3 of fat measured by ether extract in the study do not take into account fat bound by the extrusion process. Thus, the measured ether extract of extruded diets (Lab Canine, Lab Feline and Lab Primate diets) is much lower than the calculated values. Fat in Certified Rodent and Primate diets continue to be measured by ether extract and acid hydrolysis. Table 3 illustrates findings for Certified Rodent Diet 5002 and Certified Primate Diet 5048.
Table 3.
Levels in Certified Rodent Diet 5002 and Certified Primate Diet 5048
  # Samples Analysis Calc. 1992 1993 1994 1995 1996 5 Year Ave.
5002 14 E.E.2 4.5 3.9+/-1.0 4.2+/-0.1 4.5+/-0.3 4.5+/-0.0 4.5+/-0.01 4.3+/-0.5
    A.H.3 5.1 5.9+/-0.5 5.9+/-0.2 5.9+/-0.2 5.9+/-0.2 5.7+/-0.2 5.8+/-0.2
5048 13 E.E.2 4.7 3.1+/-0.6 2.6+/-0.0 3.7+/-0.4 5.9+/-4.6 4.0+/-0.5 3.8+/-2.0
    A.H.3 5.6 6.0+/-0.5 6.6+/-0.5 6.3+/-0.5 6.4+/-0.8 6.4+/-0.7 6.4+/-0.7
1 Standard Deviation

2 Ether Extract

  

3 Acid Hydrolysis

  

Minerals-Calcium/Phosphorus
Observed mineral levels were near the calculated formulation levels. (Tables 4A &4B)

Two of the primary macrominerals are calcium and phosphorus. They are ubiquitous in mammalians and are important in maintaining bone, teeth, nerve function, energy metabolism, milk production and serve as enzyme co-factors. In addition to the importance of being in the proper dietary level, it is also important that the calcium/ phosphorus ratio be maintained at a value of greater than 1 to 1.

 
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Vitamins
Vitamin levels generally checked well with calculated levels. Observed levels of riboflavin, pantothenic acid, and vitamin B12 were higher than calculated levels, and were extremely constant within given diets over the years.

Calculated levels have been purposely assigned lower dietary requirements to provide a safety margin for formulated diets and still meet specific vitamin levels. Therefore, riboflavin, pantothenic acid and B12 levels in LabDiet® products meet the nutritional needs of the animals.

 

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 Riboflavin - PPM Pantothenic Acid - PPM B12 - MCG/KG
Table 5.
Riboflavin, Pantothenic Acid, and B12 Levels in Certified LabDiet® Products over 5 Year Period
Calc. Value 5 Yr. Av. Calc. Value 5 Yr. Av. Calc. Value 5 Yr. Av.
5002 8.0 8.5+/-2.0 17 19.0+/-1.6 19.8 30.7+/-10.81
5007 4.5 5.0+/-0.9 20 22.0+/-1.4 26.9 41.0+/-8.1
5026 6.0 7.9+/-2.4 19 23.0+/-0.9 13.2 22.2+/-7.5
5048 8.5 8.6+/-2.2 50 57.0+/-2.9 46.0 51.8+/-8.6
5322 5.0 7.8+/-1.9 19 22.0+/-1.7 6.6 17.7+/-5.7

1 Standard Deviation
Contaminant Analysis
The possible contamination of diets with heavy metals and/or chemicals is a serious matter which must be closely monitored.
The Certified LabDiet® profile has a maximum allowable concentration for each compound measured. As shown in the tables below, minimum detection levels are set at much lower than the maximum allowable concentration. It is the intent of PMI to maintain the contaminants as close to the minimum detection level as possible. In fact, many of the contaminants measured were not found above the detection levels.

No aflatoxin was detected in any of the products. This is a reassuring finding and indicates that the program for controlling mycotoxin contamination of ingredients is successful.

 
Summary
The cumulative results show that the level of nutrients in the Certified LabDiet® products are remarkably consistent. Levels of the amino acids lysine and methionine, which are the limiting amino acids in most diets, were constant.

Levels of fat are also constant. Fat is important because it has a large influence on the energy content of the diet which, in turn, will affect feed consumption. Fat level is particularly important in the diet of lactating animals to ensure adequate energy intake.

The constant level and appropriate ratio of calcium and phosphorus in LabDiet® products are important for the maintenance of animal health. Vitamin levels were at appropriate and consistent concentrations in LabDiet® products over the 5 year period.

Results outlined in the study demonstrate that nutritional content and contaminant
levels are well controlled in Certified LabDiet® products manufactured by PMI.
  Max. Conc. Levels Detect. Levels 1993 1994 1995 1996 1997 Table 6.
Arsenic Cencentrations in Certified LabDiet® Products over Test Period of 93-97
5002 1.0 PPM 0.2 0.3+/-0.1 0.3+/-0.1 0.3+/-0.1 0.3+/-0.1 0.3+/-0.01
5007 1.0 PPM 0.2 0.4+/-0.1 0.3+/-0.1 0.4+/-0.1 0.4+/-0.1 0.3+/-0.0
5026 1.0 PPM 0.2 0.3+/-0.2 0.3+/-0.1 0.3+/-0.1 0.3+/-0.1 0.3+/-0.0
5048 1.0 PPM 0.2 0.3+/-0.1 0.3+/-0.1 0.3+/-0.1 0.3+/-0.1 0.2+/-0.0
5322 1.0 PPM 0.2 0.2+/-0.2 0.3+/-0.1 0.3+/-0.1 0.3+/-0.1 0.3+/-0.0

1 Standard Deviation
  Max. Conc. Levels Detect. Levels 1993 1994 1995 1996 1997 Table 7.
Malathion Cencentrations in Certified LabDiet® Products over Test Period of 93-97
5002 0.5 PPM 0.2 0.1+/-0.1 0.1+/-0.1 0.1+/-0.0 0.1+/-0.0 0.1+/-0.11
5007 0.5 PPM 0.2 0.0+/-0.1 0.1+/-0.1 0.0+/-0.0 0.0+/-0.0 0.1+/-0.1
5026 0.5 PPM 0.2 0.0+/-0.0 0.1+/-0.0 0.0+/-0.0 0.0+/-0.0 0.1+/-0.1
5048 0.5 PPM 0.2 0.1+/-0.1 0.1+/-0.1 0.0+/-0.0 0.0+/-0.1 0.1+/-0.1
5322 0.5 PPM 0.2 0.0+/-0.0 0.1+/-0.1 0.0+/-0.0 0.0+/-0.0 0.1+/-0.1

1 Standard Deviation
References
1 Cohen, L.A.; Epstein, V. S.; Meschter, C.; and Zang, E. Interactions between 4-HPR and diet in NMU-induced mammary tumorigenesis. Nutr. Cancer 21:271-283, 1994.

2 National Research Council. “Nutrient Requirements of Laboratory Animals,” National Academy Press, Washington, DC, pp 112-113,1995.

3 Verchere, C. B.; D’Alessio, D. A.; Palmiter, R. D.; Weir, G. C.; Bonner-Weir, S.; Baskin, D.G.; and Kahn, S. E. Islet amyloid formation associated with hyperglycemia in transgenic mice with pancreatic beta cell expression of human islet amyloid polypeptide. Proc. Natl. Acad. Sci. USA, V. 93: 3492-3496, April, 1996.

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4 Peterson, C. A.; Baker, D. H.; and Erdman, Jr., J.W. Diet-induced nephrocalcinosis in female rats is irreversible and is induced primarily before the completion of adolescence. J. Nutr. 126:259-265, 1996.

5 Ritskes-Hoitinga, J.; Lemmens, A.G.; Danse, L.H.J.C.; and Beynen, A.C. . Phosphorus-induced nephrocalcinosis
and kidney function in female rats. J. Nutr. 119:1423-1431, 1989.

6 Stonard, M.D.; Samuels, D.M.; and Lock, E.A. The pathogenesis and effect on renal function of nephrocalcinosis induced by different diets in female rats. Food Chem. Toxicol. 22:139-146, 1984.

7 Anand, C. R.; and Linkswiler, H. M. Effect of protein intake on calcium balance of young men given 500 mg calcium daily.
J. Nutr. 104:695-700, 1974.

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