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A Story with Bearing: Cholesterol

A Story with Bearing: Cholesterol

By Michael Pickering

Years ago our friend Peggy told us that her doctor had prescribed a diet devoid in cholesterol, as her blood test indicated worrisome numbers.  The doctor recommended that all of the usual suspects be excluded from her diet, such as egg yolks and butter, but it was all to no avail.  Regardless of how long she maintained the exclusive diet, her blood numbers did not budge.  So, she decided to experiment with her own dietary exclusions.  One of her first experiments was targeted at a food everyone who knows Peggy is well aware that she is addicted to – milk chocolate.  It was only then that her blood cholesterol numbers improved.

While it is true that one’s diet is an important factor in the level of cholesterol in one’s blood, the amount of cholesterol in one’s diet is not germane.  Unlike the essential amino acids and minerals which must be harvested from the diet, the cholesterol in our blood is synthesized inside our bodies from smaller synthons (many acetates, a popular biosynthetic mode, see flavonoids).

So the issue isn’t whether cholesterol is in one’s diet, but rather how cholesterol is behaving in one’s blood.

The key link between the importance of diet and the behavior of cholesterol in one’s blood is the amount and type of fat you ingest.  Highly saturated fats have the most negative impact on the solubility of cholesterol in the blood.

mink coat front

Blood chemistry is necessarily dominated by water soluble processes.  Magnesium, sodium, citrate, and all manner of water soluble nutrients must course around freely.  However, cholesterol is not water soluble, even though it must move as freely through our veins.  The body’s solution is to cloak the cholesterol with a hydrophobic interior (cozy coat) with a hydrophilic exterior (sort of like a 1960’s Bill Blass coat with the mink on the inside and the satin on the outside).mink coat back

These water taxis are called LDL and HDL: low density lipoproteins and high density lipoproteins.  Our body considers the HDL to be better than the LDL because, among other things, it’s easier to void.   Tom Scheve’s description of the reason for this in his article for Discovery Health, entitled “What’s the difference between LDL and HDL Cholesterol” eloquently expresses my own musings:

When the lipoprotein has more protein than cholesterol [HDL], it resembles a Ferrari, gunning through your body without stopping until the cholesterol arrives at your liver, where it’s converted into bile acids.  […]  When the lipoprotein has more cholesterol than protein [LDL], however, this makes for a rickety    ride, and that jalopy doesn’t get too far.  Cells have special receptors that bind tightly to these lipoproteins as they pass.  This LDL sputters down the road, careening off the arteries, running into things and leaving bits all over the place.  While the HDL Ferrari sees a pileup and nimbly speeds around it, the LDL jalopy crashes right into it, adding to the jumble of tangled fenders and tailpipes (or platelets and plaque).

The overall solubility of cholesterol in the blood is governed by a ternary phase diagram. ternary phase diagramMaintaining these three components in the proper ratio crates a zone of solubility in the triangle.  If the diet (the source of phospholipids and fats) biases the ratio out of the soluble zone, the cholesterol precipitates with the fenders and tailpipes.  And like all solids in a moving fluid, they deposit in the zones of slowest flow.  In a vascular system the slowest flow is in the arteries.

The lipids (fats) in our diet can be broadly sorted into two categories: 1) naturally occurring, and 2) man-made.  Obviously the naturally occurring fats and oils are derived from plants and animals.  The man-made fats are partially hydrogenated vegetable oils.  The saturation level determines the melting point and viscosity regardless of the source.  So highly unsaturated lipids like sesame oil have a low melting point and viscosity and so are inappropriate for frying, whereas poly-saturated lard and butter have a high melting point and viscosity, and are well suited for frying.  Similarly, an award-winning pie crust can be made with lard or butter, but not with unsaturated oil.  Partial hydrogenation thus controls the melting point of the fat and establishes its suitability for any particular application.

However, a side reaction also occurs during the hydrogenation: isomerization.  Natural unsaturation tends to be cis-configuration but hydrogenation isomerizes the bonds to trans-configuration.

cis trans fat structuresWhile the hydrogenation controls the melting point precisely (which is essential for processed foods), the resulting fat is not recognized by the body as food.

Our wild type diet is clearly designed around whole grains as the staple, a source uniquely rich in unsaturated (cis-) fats, phospholipids, and protein.  The goal is to manage the trace chemistry in our blood, the hydrophobic components.  So the lesson is: eat as little saturated (mostly animal) fat as you can tolerate, eat whole grains and exclude all partially hydrogenated vegetable oil.  Read the label!

So while the amount of cholesterol present in a milk chocolate bar (24 g) is comparable to that of a tablespoon of butter (30 g), the two had very different effects on Peggy’s blood work.  Cocoa fat is among the most saturated of the vegetable oils.  The melting point is so high that the bars are wax-like at room temperature.  The chocolate was in effect creating an excess of LDL jalopy wreckage in Peggy’s blood stream, by causing the LDL and HDL levels to get out of whack.

The cholesterol had no bearing.

 

Editor’s Note: Always consult a physician first. The views presented herein are strictly editorial in nature.

Chromatography Quiz No.14

Chromatography Quiz #13 Results

We would like to congratulate our grand prize winners of our last newsletter’s Carbamate Analysis Chromatography Quiz: Irene Taylor from Orange County Utilities, Jim Balk from DHHS Public Health Environmental Laboratory, Narjes Ghafoori from LA County Environmental Toxicology Laboratory, Helene Lachance from Shur-Gain Nutreco, and Jamie Palmer and Matthew Hartz from Underwriters Laboratories!!!

laser chess 1They have each won and will be receiving: a Laser Board Game from laser chess 2Sharper Image!  (This strategy-based board game is similar to chess and features an 80-square Egyptian themed grid.  The goal is to protect your game pieces while eliminating your opponent’s by bouncing eye-safe lasers at them through the maze of mirrors you’ve constructed.)

We would like to thank all of you for your submissions!

The correct answer for the modified Carbamate chromatogram: Reagent #2 (CB910, OPA, and Thiofluor) was partially oxidized.  The peak heights of Carbaryl and 1-Naphthol have significant diagnostic importance in regard to the OPA reagent system. Since 1-Naphthol naturally fluoresces, we can eliminate the fluorescence detector as the problem.  Though smaller peak sizes can reflect problems with Reagent #1, since the Carbaryl peak is full-sized, we can deduce that hydrolysis has occurred (Carbaryl naturally fluoresces after hydrolysis).

Particularly astute observation yields additional details about our troubleshooting chromatogram – slightly shifting retention times towards the end of the run could indicate a problem with HPLC hardware, such as a proportioning valve.

Thank you!

Pickering Labs

 Chromatography Quiz #14:

Complete the cross word puzzle below and win a prize!  Simply email your answer (copy of completed puzzle or list answers with clues) as well as your full contact information to Rebecca at rlsmith@pickeringlabs.com by December 15, 2013 in order to win.  You will receive email confirmation that your submission has been received.  The answer to the puzzle and winner congratulations will be published in the next issue (to be anonymous, please notify Rebecca in submission).

Carbamate/Glyphosate Analysis: Cross Word Puzzle

crossword

34th America’s Cup

By Wendy Rasmussen

OTUSA and ETNZ

Before the America’s Cup came to San Francisco, I never paid much attention to the event. It doesn’t get much press here in the US, and being a native of a land-locked state, sailing was not something I grew up with.

But after living in San Francisco for many years, I learned to sail and gradually my awareness of the America’s Cup increased, and I gained an appreciation of the event and the hard work that goes into designing and sailing the boats.The Cup

Those who wish to race for the cup become Challengers.  If multiple teams are challenging, they must duel it out to decide who will then face the Defender in the America’s Cup Finals.  This series of races became known as the Luis Vuitton Cup. From the America’s Cup Website:

In 1970, more than one yacht club interested in challenging for the America’s Cup, so for the first time, a competition was staged to determine the single Challenger that would face the Defender, the New York Yacht Club.

The French malletier Louis Vuitton became involved with the America’s Cup in 1983, supporting the Challenger Selection Series that came to be known as the Louis Vuitton Cup. The idea was twofold; to develop and identify the strongest possible challenger for the America’s Cup and ensure that they were sufficiently battle-tested through tough competition to beat the Defender.

(Be sure to check out www.americascup.com for information, videos, history, race results and more).

This year, Emirates Team New Zealand (ETNZ) ETNZ low reswon the Luis Vuitton Cup to become the Challenger, sailing against the Defender, Oracle Team USA (OTUSA).

Living in San Francisco, I had the wonderful opportunity to watch several of the races, to meet many of the sailors, and to watch the teams ready themselves for the “big day”. The teams were in town for several months before the regattas to practice. The AC72s were (are) an amazing site to see, both from land and from the water.OTUSA They are truly a sight to behold, especially when foiling at speeds of up to 45kts*

The 34th America’s Cup finals proved to be the longest in history due to several postponements due to wind conditions.  In fact, the official end date for the regatta was September 21; the final race was on September 25th. But perhaps the most amazing feat of this regatta was the comeback made by OTUSA:

In order to win the Cup, a team must earn nine points. Ordinarily, this would mean winning 9 races (1pt per win). But before the regatta began, Oracle were penalized two points due to an infraction in an earlier series. And so while ETNZ needed to win 9 races, OTUSA had to 11. 

The comeback began on Sept 19, 2013. ETNZ were ONE race away from winning the Cup. OTUSA still need to win EIGHT.  For days on end, we all said, “Well, this is it. Kiwi’s are going to take home the cup.” But then OTUSA continuously surprised us all. In the end, they won an unprecedented EIGHT RACES IN A ROW! in order to come back and win the regatta. Click here for the Results.

I won’t speculate here as to why OTUSA made such an amazing comeback, or why ETNZ lost in the end. I will only say that it was so exiting to be a part of that historic moment, in an amazing city with an amazing natural venue for watching the races. And most importantly for me, the members of both teams handled themselves with an incredible amount of dignity and sportsmanship. The members of both teams were all under an incredible amount of pressure, and yet they still managed to greet and sign a few autographs for their fans  — yes, this fan included.

*45kts = 51.8mph. By comparison, our sailboat at it’s fastest will do about 12kts, or 13.8mph

Calculation of Whey Protein Fraction in Milk-Based Infant Formula

A method for the calculation of whey protein fraction in milk-based formula products was developed and validated by a group of researchers from Abbott Nutrition, Covance Laboratories and AOAC International. The method is based on calculating the ratio of (Asx + Ala)/(Pro+Phe) determined from the amino acid profile of the hydrolyzed sample.

The method was approved as AOAC Official First Action Method 2012.07 and is appropriate for accessing compliance with whey content requirements of GB 10765-2010 (National Food Safety Standard Infant Formula)*. It is applicable to finished products containing both intact and partially hydrolyzed whey proteins.

According to this method, the Amino Acid Profile of hydrolyzed samples is determined using cation-exchange chromatography with post-column derivatization and UV/Vis detection. The researchers used Pickering Laboratories post-column derivatization system, our patented Trione Ninhydrin reagent and Pickering Sodium analytical column and buffers.

The method performance was evaluated using non-fat dry milk and products with whey levels from 32% to 63%, including NIST Infant Formula Standard Reference Material. The repeatability ranged between 0.3 and 2.5 %, while intermediate precision were between 2.6 and 3.4%. Average recoveries ranged from 97 to 100%.

The unmatched accuracy and precision of amino acid analysis by HPLC with post-column derivatization allowed the researches to develop a robust mathematical model for calculating whey protein fraction that can be successfully applied to a wide range of products. This proved once again that post-column derivatization analysis of amino acids is still a method of choice for laboratories that require highly accurate results.

Pickering Laboratories offers a total solution for amino acids analysis, including post-column derivatization systems, reagents, columns, buffers and standards.

The reference to the published study is below. The First Action Official Method 2012.07 can also be found online at http://www.eoma.aoac.org/.

Calculation of Whey Protein Fraction in Milk-Based Infant Formula: First Action 2012.07

Authors: Wesley Jacobs, Paul Johns, Phillip Haselberger, Joseph J. Thompson, Darryl Sullivan, Steve Baugh.

Source: Journal of AOAC International, Volume 96, No. 3, 2013, pp. 502-507

 

(*) this is a Chinese Standard

Amino Acid Analysis of Cell Culture Media

We have a new Method Abstract! MA371, Amino Acid Analysis of Cell Culture Media

Cell cultures are widely used to produce biopharmaceuticals and other biologically active compounds. The composition of the cell culture media affects the yield and structure of the desired products and must be carefully optimized. Cell culture media is typically composed of mixtures of amino acids, vitamins, carbohydrates, inorganic salts as well as different peptides, proteins and other compounds. As the cells grow, they consume nutrients and release target biopharmaceuticals as well as waste products.

Amino Acids serve as the building blocks of proteins, as well as intermediates in many metabolic pathways. Amino Acids are typically added to cell culture media to provide nutritional requirements for the cells. Monitoring and adjusting Amino Acid composition is an essential part of optimizing the manufacturing process to ensure high quality and optimum yield of the final product.

Amino Acid Analysis using cation-exchange chromatography with post-column Ninhydrin derivatization allows for easy determination of Amino Acid concentrations in many complex matrices, including cell culture media. The post-column method is very sensitive, reproducible and rugged. It has been and continues to be a method of choice for laboratories running biological samples, protein, peptides and foods analysis. Most chemical compounds present in the media do not interfere with analysis, so the majority of samples only need diluting with citric buffer and filtering before analysis. If serum is added to the media, then the proteins need to be precipitated using either Seraprep™ solution or ultrafiltration.

Pickering Laboratories, Inc. offers the complete solution for Amino Acid Analysis, including post-column derivatization instruments, columns, eluants, reagents and standards. The Pinnacle PCX derivatization system has a programmable column oven to allow for shorter run times and easy method optimization.

YChromatograms of Cell Culture Mediaou can download this application note, and many others from our website: www.pickeringlabs.com

METHOD
Analytical conditions
Column: High-efficiency Lithium cation-exchange column, 4.6 x 75 mm, Catalog Number 0354675T
Flow Rate: 0.55 mL/min
Mobile Phase: See method in Table 1

Post-Column Conditions
Post-column System: Pinnacle PCX
Reactor Volume: 0.5 mL
Reactor Temperature: 130 °C
Flow Rate: 0.3 mL/min
Detection: UV/VIS 570 nm for primary amino acids, 440 nm for secondary amino acids
Injection Volume: 10-50 uL

gradient table for AAA

Polyether Antibiotics in Animal Feed

Our updated Method Abstract 264 describes the analysis of Monensin, Salinomycin, Narasin, & Lasalocid in Animal Feeds.

Polyether Antibiotics are commonly used for preventing coccidiosis and other infections in poultry and for improving feed efficiency for beef cattle and swine. The use of Polyether Antibiotics is strictly regulated, with only specific ionophores approved for use in feeds intended for different animals.

Analysis of Polyether Antibiotics by HPLC with post-column derivatization and UV/Vis detection has been proven to successfully identify and quantify Monensin, Narasin and Salinomycin in medicated feeds, supplements and premixes as well as to determine trace contamination levels in non-medicated feeds [1, 2].

Post-column derivatization of Polyether Antibiotics is done using highly acidic Vanillin or DMAB reagents. The Pinnacle PCX derivatization system (Pickering Laboratories, Inc.) has an inert flow path and automated system wash capabilities that make it uniquely suitable for handling corrosive reagents. The two-pump system is recommended to extend reagent stability, but the single-pump system for this application is also available.

Adding a Fluorescence detector to the instrumentation allows for using the same extraction procedure and HPLC conditions to also determine Lasalocid, which doesn’t require post-column derivatization.

Chromatograms of Polyether AntibioticsMETHOD
Sample Preparation
To 25 g of finely ground feed sample, add 100 mL of extraction solution (90% Methanol – 10% water). Shake for 1 hour at high speed using a mechanical shaker. Let the solids settle and filter an aliquot of the extract for injection. Dilute with extraction solution if needed to fit the calibration curve. Use a 2.5 g portion when testing premixes.

Analytical conditions
Analytical Column: Polyether Column, C18, 4.6 x 250 mm, Catalog No 2381750
Temperature: 40 ºC
Flow Rate: 0.7 mL/min
Mobile Phase: 90% Methanol, 10% of 5% Acetic Acid solution in water, isocratic
Injection volume: 20 μL

Post-Column Conditions
Post-column System: Pinnacle PCX
Reactor Volume: 1.4 mL
Reactor Temperature: 90 °C
Reagent 1: Concentrated Sulfuric acid / Methanol (4:96 v/v)
Reagent 2: 60 g of Vanillin in 950 mL of Methanol
Reagent Flow Rate: 0.3 mL/min
Detection: UV/VIS 570 nm (for Lasalocid – FLD, Ex. 322 nm, Em. 370 nm)

Conclusion
Analysis of Polyether Antibiotics by HPLC with Post-column derivatization is a robust and sensitive method that utilizes standard equipment and could easily be adopted by testing laboratories. It allows for testing of different ionophores at wide range of concentrations,
including at trace levels. Using Pinnacle PCX post-column derivatization system, factory configured for the analysis, guarantees stable and reproducible results.

The full application note as well as our complete Product Catalog can be downloaded from our website: www.pickeringlabs.com

table for polyether antibiotics

NEMC 2013

Pickering Laboratories participated in the National Environmental Monitoring Conference in San Antonio, Texas from August 4-8.

niosita low res
Old San Antonio

Rebecca and Wendy were on-hand to share Pickering’s products with the environmental laboratories involved with the conference.  On display were the new FREESTYLE sample clean-up system and the Pinnacle PCX.

Some highlights of the conference included a new product by InnovaPrep, which will rapidly concentrate pathogens in liquid samples, and the 2-D GCMS analysis of POPs in human milk. There were also several interesting discussions about pesticide extraction and analysis as well as the use of Hydrogen as a carrier gas in GC (instead of Helium, for which there is currently a shortage).

The meeting was held at the Hyatt in downtown San Antonio on the Riverwalk. This area of town is full of history and interesting architecture. We were also located directly across the plaza from the Alamo.

alamo low res
Remember the Alamo

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