Intolerance Test and Dysbiosis: a new insight

Pierluigi Pompei, Pharm.D., Ph.D.

Unit of Pharmacology and Pharmacotherapy University of Camerino, Italy

✔ The Human Microbiome and the Dysbiosis

✔  Intolerance test (Alcat test)

✔ Dysbiosis Test

but before this…………………….

What is the Human Microbiome

and why is it so important lately?

Microbes are all over us

Thousands of different species on the skin alone Some thrive on dry patches of the elbow, others thrive in moist environment of armpit

There are millions of microbes per square inch on our body

It is estimated that there are more microbes in our intestine than there are human cells in our body!

What is the Human Microbiome?

Microbe: tiny living organism, such as bacterium, fungus, protozoan, or virus

Microbiome: collectively all the microbes in the human body; a community of microbes

Biofilm: a community of microbes that live together on a surface

Microbes in the Human Microbiome include species from each major domain

Bacteria

“Extremophile”Archaebacteria

Fungi

Microbes are normally found in and on the human body

The following sites are “hotspots” for microbial life

http://nihroadmap.nih.gov/hmp/

Some microbes are native, normally found in the body

Some microbes are introduced, suddenly arriving at a new residence in the body

THE  HUMAN  INTESTINAL  MICROBIOME  

>3.000.000 Genes

>58.000 Enzymes

>25.000 Chemicals

Metabolomics Proteomics Genomics

Kau et al, Nature 2011 Qin et al, Nature 2011

Human Gut Microbiome

Ü  $107 billion in direct health care expenditures in 1992.

Ü  200 million sick days, 50 million physician visits, 16.9 million school days lost, 10 million hospitalizations, & almost 200,000 deaths per year.

Ü Digestive tract cancers are responsible for 117,000 deaths/yr, & non-cancerous digestive diseases cause 74,000 deaths/yr.

The human gut microbiome is so important as it often causes:

Digestive Problems Nationally

Always pathogen

Lactobacillus

Eubacteria Bifidobacteria

Always beneficial

Ps-Aeruginosa Proteus

Staphylococcus

Veillonellae Clostridium

Enterococcus E. coli

Streptococcus

Batteroides

The gut flora as a forgotten intestinal organ:intestinal flora composition

“Eubiosis” Reduced microbiota

biodiversity and increased instability

Reduction of anti- inflammatory bacteria

Increase in Proteobacteria (pathogenic bacteria)

SPECIFIC ALTERATIONS FOR SPECIFIC DISEASE ….

“Dysbiosis” vs

The Microbiota- Eubiosis (1) Gut bacteria exert a variety of activities such as:

Ü  The correct morphological and functional growth of the digestive system

Ü  The digestive processes

Ü  Metabolic processing of proteins, sugars and lipids

Ü  Metabolic processing of biliar acids and steroid hormones

R. Fornengo , Microbiota and metabolic disease. A brief review J AMD 2016| VOL. 19| N. 3

Ü  The qualitative and quantitative control of gastrointestinal gas and bloating

Ü  Production of vitamines and utilizations of exogenous vitamins

Ü  Drugs metabolization

Ü  Gluthation production

Ü  Inflammatory response modulation via cytokines production.

Sirisinha S. The potential impact of gut microbiota on your health:Current status and future challenges. Asian Pac J Allergy Immunol. 2016 Dec;34(4):249-264. Kullisaar T et al, Complete glutathione system in probiotic Lactobacillus fermentum ME-3, Prikl Biokhim Mikrobiol. 2010 Sep-Oct;46(5):527-31. Borchers AT, Selmi C, Meyers FJ, Keen CL, Gershwin ME. Probiotics and immunity. J Gastroenterol. 2009;44:26–46. M. Appukutty et al, Effect of orally administered soy milk fermented with Lactobacillus plantarum LAB12 and physical exercise on murine immune responses, Beneficial Microbes Feb 2015; DOI: 10.3920/BM2014.0129

.

The Microbiota- Eubiosis (2)

Dysbiosis

Ü Dysbiosis is an intestinal imbalance where pathogenic microorganisms outnumber beneficial bacterial species.

Ü Digestive diseases are

often caused in part by or result in dysbiosis.

Drasar B.S. “Intestinal Microbiology”. American Society for Microbiology, Washington, 1985

Early Development

Dysbiosis

• Decreased immune function • Poor diet — excessive carbohydrates, sugar and trans fat • Drugs — especially antibiotics, oral contraceptives and cortisone-like medications

• Intestinal infection • Decreased intestinal motility (aka constipation) • Stress — including long-term emotional stress • Maldigestion and malabsorbtion

Major Causes

Dysbiosis

• Abdominal pain or cramps • Colon cancer • Constipation or diarrhea • Distention/bloating • Fatigue • Fatigue after eating • Flatulence

• Bad Breath • Body Odor • Food Allergy • Hypoglycemia • Inability to lose weight • Irregular bowel movements

• Irritable bowel syndrome • Itchy anus • Leaky gut syndrome • Poor complexion • Poor digestion • Rheumatoid arthritis • Spastic colon

Common Symptoms

Why should one care?

Ü  Chemical farming and pesticides have lead to sterilization of soil and water (chlorination).

Ü  Antibiotics, hormones, and other drugs are given to chickens and cattle used for food.

Ü  Stress, medications, and typical American diet all promote dysbiosis.

Dysbiosis Test Clinical Significance The test is able to monitor, via urine morning sample testing, two markers of the tryptophan metabolites, “INDICANO” and “SCATOLO”. Tryptophan, regularly ingested with food, undergoes a metabolization process caused by specific gut bacteria such as Peptostreptococcus Indolicus- Bacteroides Putredinis – Clostridium Sporogenes, leading to loss of some side chains with the production of the Indole. Indole is absorbed by the intestinal mucosa and, via the liver-enteric circulation, is shifted to liver where is being detoxified (phase 1 and 2). INDICANO is screened through a Colorimetric Analysis, SCATOLO is screened through HPLC.

Lab diagnosis

Dysbiosis severity degree

Mild Dysbiosis: bloating, aerophagia, diarrhea and constipation. Medium Dysbiosis: may affect the urogenital tract, with intestinal mycosis, vaginal candidiosis, chronic cystitis and prostatitis. Severe Dysbiosis: spread dissemination of most gut and urogenital organs, weakness of the immune system, cutaneous diseases, cephalic aureas, skin aging, chronic weakness, anxiety, depression, bone pain.

Hawrelak JA et Al, The cause of intestinal dysbiosis: a review. Altern Med Rev 2004; 9: 180-197 Guarner F et Al, Gut flora in health and disease. Lancet 2003; 360: 512-519

………..Introducing the Intolerance test (ALCAT Test) In order to evaluate the sensitivity to sugars (fructose, sugar cane and sugar beet) the ALCAT test has been used. The ALCAT test is not a diagnostic tool for food allergies, as these are IgE mediated. It is currently considered a medical device that offers informations upon putative food intolerances and/or chemicals.

The Alcat test (acronime of antigen leukocyte cellular antibody test) is a lab test through which it is possible to diagnose a Food Intolerance. The patient blood, after venous withdrawal, is exposed to a range of suspected foods. The test highlights an adverse reaction to food allergens. These allergens are able to induce a modification either in the number or the size of of blood cells, particularly the granulocyte neutrophils. When this reaction occurs it yields an adverse reaction to the tested food.

TESTING PROCEDURE

The ALCAT®

Diagnostic System

by AMTL Corporation

A Technical Overview

The ALCAT® Diagnostic System

Basic Theory

Reagent Cassette

Reagent

Preparation

Instrument

Overview

Technical

Characteristics

Sample results

Basic Theory

Ü Combine blood sample with food, chemical and/or drug reagent

Ü Allow time for any immunologic, toxic or pharmacologic reaction to occur

Ü Measure white blood cells to indirectly detect mediator release by size and/or volume changes

Ü Compare volume distribution curve of reacted sample against baseline

Ü Report results grouped by reaction strength

Reagent Cassette

Ü Vacuum sealed kit provides six months shelf life

Ü Kit includes blood diluting reagent, RBC lysing agent, standards and food, chemical or drug reagent

Reagent Cassette Preparation

Ü Keyed cassette with 50 reagent & 5 control (standards) wells.

Ü 80 microliters of diluted blood is dispensed into each well then the cassette is incubated.

Instrument Overview

The ROBOCat instrument is an automated liquid handling system designed to measure blood cells using the electronic

principle of particle counting and sizing (measuring changes in electrical resistance produced by a blood cell suspended in a

conductive liquid traversing a small aperture).

ROBOCat II Instrument

Technical Characteristics Linear power supply, 110/220 Volts

Acrylic aperture chamber (reading area)

Main controller board that communicates with the front end PC via the RS232 port

Capacity is 3 reagent cassettes and analysis time is 20 minutes each cassette

Unit is approximately 38cm x 38cm x 38cm and weights 15kg.

CE Mark

ROBOCat II Advantages

Fast operation: 50 item test completed in 20 minutes. Less consumables: only 200mL of electrolyte necessary. Better software: The Robocat II has an easy to use & user-friendly interface to handle multiple tests. Easy to use: Walk away system with audible alert alarms.

ROBOCat II Advantages

Ü Better liquid handling: Robocat II uses air driven technology for liquid flow rather than mechanical flow providing for less mechanical failures and more reliable operation.

Ü Easier to maintain: Robocat II incorporates an automatic self cleaning routine.

Ü Easier to service: Robocat II has modular components and a complete diagnostic routines to quickly alert the operator of any failures.

Rotation Diet Guide

Ü  Custom 4 day rotation diet plan.

Ü  Integrated with the test results.

Ü  Balanced and categorized diet.

Ü  Additional list of associated foods to avoid.

Sample Results

Ü  Multi-lingual format available

Ü  Additional software to generate rotation diet and foods to avoid list

Ü  Graphical output for raw data available

Ü  Windows 95/98/2000/XP compatible

The degree of reactivity is determined by comparing a baseline distribution curve (of the white cells) against the distribution curve generated by the analysis of each test agent/blood sample, and calculating the absolute differences between the curves and the standard deviation (SD).

Ü  ALCAT test is effective in improving body mass index (BMI) and/or scale weight. Studies have reported that participants who followed the ALCAT plan achieved rather dramatic changes in their body composition, showing that 98% of the subjects following the ALCAT plan either lost scale weight or improved their body composition. Kaats, Gilbert. Parker, Larry. Pullin, Dennis. The Short Term Efficacy of The ALCAT Test of Food Sensitivities to Facilitate Changes in Body Composition and Self-reported Disease Symptoms: A Randomized Controlled Study. The Bariatrician; 1996:18-23

Ü  Other studies reported that iso-caloric food elimination diets, based on ALCAT test results, promoted enhanced weight loss, implemented reduction of adipose tissue, rather than muscle mass, as determined by DEXA studies in a population of refractory weight loss subjects. Cabo-Soler, J.R. Comments on Diets in Esthetic Medicine. In The 14th Mediterranean Day of Esthetical Medicine & Dermatological Surgery. Venice, Italy; 1995:22-23.

Ü  A reproducibility of 92.07% of ALCAT test was reported by Neetling et. al. at the University of the Free Orange State, also in South Africa, which makes it an acceptable screening model for intolerance testing in humans.

Neetling WML, Kachelhoffer AM. Reproducibility of the Antigen Leucocyte Cellular Antibody Test (ALCAT). Study conducted by the University of the Orange Free State in Bloemfontein, South Africa, Jan – April, 1998. In Publication.

Ü  In Addition, a Norwegian study reported the ALCAT test to be >90% reproducible.

Fuglerud P. Study of the ALCAT Test in 10 subjects. Final Statistical Report. Statistical Report: 99072 conducted by Parexel Medstat, Lillestrøm, Norway. November 24, 1999.

The results of the ALCAT test are displayed using the “code color”. Test results: four distinct areas. RED: foods listed indicate a severe reaction and should be strictly avoided at all for a minimum of 6 months; ORANGE: foods listed indicate a moderate reaction and should be strictly avoided for a minimum of 3 to 6 months. YELLOW: foods listed indicate a mild reaction, should be avoided if possible, for 3 months, especially if there are few red and orange foods. GREEN: foods listed indicate no reaction and can be eaten freely on a rotation basis, no more than every other day.

Aim of this study: The aim of the study was to evaluate whether food intolerance is associated with an intestinal dysbiosis.

Dysbiosis test were performed through urine sample collections via Scatolo and Indicano marker use, whereas intolerance food test has been processed through ALCAT test.

Methods 177 patients (122 females, 54 males, median age 36.5 years) positive for dysbiosis test were tested by ALCAT. Dysbiosis test is considered positive when at least one of the parameters considered (Indicano and Scatolo, both tryptophan metabolites) exceed the 10 µg/L (mild dysbiosis).

The dysbiosis diagnosis ranges used were: a)  between 10 and 20 µg/L (mild dysbiosis), b)  between 20 and 40 µg/L (medium dysbiosis), c)  greater than 40 µg/L (severe dysbiosis).

Subjects with positive dysbiosis due to the INDICANO parameter (mild small intestine inflammation)

35% of the subjects showed at least one food intolerance in the red zone

(severe neutrophil degranulation) è yeast.

Results

On the other hand, only the 18% of the subjects whose positivity is due to either SCATOLO or INDICANO higher than 40 (severe large intestine

inflammation) show food intolerance in the red zone (severe intolerance)

p-value<0.001

For the comparisons between the two selected types of patients, a Chi-squared test (X-squared = 15.415, df = 2) was performed.

Rejecting the null that population probabilities of N-L equal those in G-G, there is a statistical evidence of differences between the two types.

Conclusions

-  A mild dysbiosis given by the INDICANO may hide severe intolerance, that, in absence of a clear diagnosis, may worsen clinical symptoms.

-  The fact that only the 18% of the patients with permeability alteration show a severe food intolerance may reflect other factors leading to inflammation (life-style, drugs, smoking, alcohol…).

This study provides evidence that in presence of a limited alteration of the intestinal permeability, a reaction to food antigens (via ALCAT test) can be more severe in patients with greater intestinal permeability condition.