naturopathic nutrition year 2 lecture 3

Naturopathic Nutrition Year 2

Assessment & Diagnostics /

Functional Testing © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Lecture 3:

Learning Outcomes

In today’s lecture you will learn: • How to use of functional testing in nutritional therapy

• How to interpret the results of a range of anthropometric and functional

tests

• The application of various types of testing available to nutritional therapists

© CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Functional Testing vs Diagnostic

• Testing gives a snap shot of what is happening in the body at the moment in time.

• Physicians use testing to diagnose a condition. As nutritional therapists we are only gaining more information around the function of the body in a snap shot in time, to help you with your working hypothesis. Any worrisome results you find will need a referral to a GP for a diagnosis.

• NO TEST IS INFALLIBLE – if a test has a reliability of 96%, it still means 4 out of 100 people may have false positives or negatives – so this means TREAT THE PERSON, NOT THE TEST


Types of Tests

• Anthropometric testing – Height, weight and body

mass (BMI) – Body composition analysis

(body fat %, water, lean mass, bone)

• Stool – non invasive, can be performed by the client at home. – Used for investigation of digestive

function and detection of yeasts, pathogenic bacteria and parasites

• Saliva – non invasive, can be performed by the client at home. – Can be useful for

hormone, DNA and antibody testing


• Urine - non invasive, can be performed at home. – Hormone activity eg. Thyroid,

oestrogen metabolites. – Intestinal Permeability. – Detox metabolites and toxic metal

clearance. – Mitochondrial metabolites –

organic acids – Bone turnover rate

• Blood – invasive test either via pin-prick or serum. – Basis for most standard medical

tests – Cellular function inc. blood glucose – Allergies & intolerances – Nutritional status – Homocysteine – Lipid & cholesterol levels

Types of Tests


• Hair – non invasive, can be performed by the client at home. – Controversy over reliability &

validity of results – Measures mineral levels – Measures toxic metal levels – Can help highlight metabolic

imbalances

• Over the Counter tests – Blood glucose – Blood cholesterol – Food intolerances (York and Food

Detective) – Prostate Specific Antigen (PSA) – Litmus paper – pH saliva and urine – Pregnancy tests and ovulation tests – H. Pylori

Types of Tests


In Clinic Testing

• You may wish to use some simple ‘testing’ or information gathering in clinic.

• These may include – Weight, height, BMI calculations – Bio-impedance analysis – Urine dipsticks – Pinprick/ blood spot testing – Or any others that you are interested in?

• Remember, make sure you dispose of any biohazards in the way that is

recommended by your council. Do not break someone’s skin if you are not trained in phlebotomy


Anthropometric Testing

Literally meaning “measurement of humans”

• Body Composition: • Skinfold measurement – rarely used now, except in athletes. • Body Mass Index (BMI) • Waist to Hip ratio • Waist Circumference • Bioimpedance Analysis


Body Mass Index (BMI)

• Estimates healthy body composition based on typical healthy weight patterns relative to height.

• Accurate predictor of body fat elevation/insufficiency in adults, except for elderly, pregnant women or very muscular athletes.

• Weight and divide by Height squared [W / (H x H)] – Height -1.82 m - (1.82 * 1.82) = 3.3124. – Weight is 70.5 kg – BMI is 21.3 (70.5 / 3.3124)

• The higher the figure the more ‘overweight’

• Only an indication and other issues such as body

type and shape have a bearing as well e.g. Where is fat/muscle distributed? Bone density?

(Top End Sports, 2009) © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Body Mass Index (BMI)

The BMI weight ranges, as set out by the World Health Organisation (WHO), are outlined below. Less than 18.4 - Underweight Between 18.5 and 24.9 - Ideal weight Between 25 and 29.9 - Over the ideal weight Between 30 and 39.9 - Obese Over 40 - Morbidly obese

• Advantages: simple calculation. Correlated with CVD and Diabetes Type 2 risk.

• Disadvantages: Lean muscular athletes have higher weight relative to height, as do pregnant women. This does not increase risk of disease.

(WHO, 2008) © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Waist Circumference

• Measures waist girth: • Estimate visceral adiposity

risk. • Predictive of CVD and

Diabetes risk when: • > 94cm (> 37”) - White and

Black men • > 90cm (> 35”) for Asian men • > 80cm (> 31.5”) for ALL

women

(WHO, 2008)

RED FLAG: 102 cm in men and 88 cm in women – Significant risk requiring urgent intervention. (Adult Treatment Panel III (ATPIII) definition of the metabolic syndrome). Waist Circumference and Waist-Hip Ratio Report, (WHO, 2008)

• Find the top of the hip bone • Find the bottom of the rib cage • Mid point between • Breathe in and take measurement on

the expiration


Waist:Hip Ratio

Waist to hip ratio • The waist-to-hip ratio is determined by measuring the circumference of the waist

the and circumference of the hips at the greatest protrusion of the buttocks.

• The waist circumference is divided by the hip circumference. - Waist to Hip Ratio (WHR) = Gw / Gh (Gw = waist girth, Gh = hip girth)

• Waist-to-hip ratio is highly predictive of increased risk of cardiovascular disease

and type 2 diabetes as well as some cancers when: – Men > 0.9 – Women > 0.85

(WHO, 2008) © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Waist to Height Ratio

Waist to height ratio • Waist to height ratio (WHtR) is a better

screening tool than BMI or WC for screening adults for cardiometabolic risk factors, and is a strong predictor of mortality risk.

• Keeping waist circumference measurement to less than half your height is ideal.

• Initial research suggest children as young as 5 can be screened in this way.

(Ashwell et al. 2012; Ashwell et al. 2014) © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Bio Impedance Analysis (BIA)

A Bioelectric Impedance Analyser is an instrument, used in research and analysis. • They work by passing an electrical current through the body using electrodes and

monitoring the level of electrical resistance to the current. • Different tissues, eg. muscles, bone, fat will resist the electrical current to different

degrees, depending on the water content and membrane health. • On a budget level, many bathroom type weighing scales are also available with a

Bioelectric Impedance Analyser, which is two built-in footpad electrodes on the base of the scale which the person stands on.

• The results are then analysed by a computer program set across mean ranges to give muscle mass/fat mass/electrolyte/ water readings

• This makes them useful in a range of applications where this information can be used as a monitoring tool (weight loss, athletes, cancer, anti-aging etc)

• The downside is initial set up cost is expensive


In groups, create a list of core tests/markers, which you may need to request from a GP in the event someone is coming to see you with: • Female age 45 - Poor energy, weight gain, loss of libido, hair has thinned and

feels dry.

• Male age 30 - Fatigue, increased itching, alcohol consumed daily, though client does not feel this is an issue, keeps finding bruises on himself with no explanation of where they are from.

• Male age 65 - Often gets shortness of breath. Recently feeling more anxious than normal with palpitations, never has the energy to get through the day, frequent urination and wakes at night.

Group Activity


Obtaining Basic Tests

Conventional tests can be obtained through many routes: • The NHS has it’s own pathology services • Online suppliers like: Thriva or Medichecks, which clients can self-use • Private GP’s and consultants • Once qualified, through different providers such as Functional Dx (using

Weatherby functional reference ranges) or TDL, and some of the functional testing labs provide some of the basic tests

• Please remember, the NHS is not ‘free’, it is paid for with taxpayers money. GP’s are the gatekeepers and have to adhere to a stringent set of rules of what tests they are allowed to ask for, as every test costs money. Asking GP’s for screening or functional tests that are outside of their scope throws us in a bad light. Always check NICE guidelines first before writing a referral letter to ask for testing


https://thriva.co/?gclid=Cj0KCQjwm6HaBRCbARIsAFDNK-h6VAjjRicf2-4NJW1X9gbn9JOQyLiSLRRIF0j7FPt5adczjZzcr7gaAmkTEALw_wcB

https://www.medichecks.com/?gclid=Cj0KCQjwm6HaBRCbARIsAFDNK-jUcilqmTjBt6g318I3v9M8w0W_JnOgXw-ZaXd19aJ_kKQnHXvO8acaAmKlEALw_wcB

https://functionaldx.com/practice-benefits/

https://tdlpathology.com/

Reference Ranges

• Reference ranges are obtained by surveying the ‘normal’ population in comparison to those with symptoms

• They are often revised, and can sometimes vary between laboratories that use different research to inform them

• Some disease states have clear guidelines of what is the absence or presence of disease, some are more looking at a pattern that is occurring

• When evaluating ‘function’ instead of diagnosing it is worth taking into account high and low ‘normal’

• As labs vary often in methodologies, it is best to use the reference ranges that are provided by the laboratory


Full Blood Count

• The test includes: – Mean cell/corpuscular volume (MCV) –

ie. Size of RBC – RBC’s; used to differentiate cause of

anaemia B12/folic acid deficiency (macro) or – Fe or B6 (micro) – Mean cell/corpuscular haemoglobin (MCH) –

average weight of Hb in cell; used to differentiate cause of anaemia; high = B12/Folic acid; low = Fe or B6

– Mean cell/corpuscular haemoglobin concentration (MCHC) – average concentration of Hb in RBCs; used to monitor anaemia therapy

– Platelet count – evaluating bleeding disorder


Full Blood Count

(Royal College of Pathologists Australasia, 2009)

• It directs further investigation for pathology tests. Examples: • Microcytic hypochromic

– small, pale RBCs could indicate iron deficiency anaemia • Macrocytic normochromic

– larger size but normal colour RBCs may indicate folate or Vitamin B12 deficiency


White Cell Count

Leukocyte Count (WCC) • Part of the FBC, this test indicates possible infection, inflammatory disease, bone

marrow failure, haematological or other malignancy Leukocyte Differential Count • Measures neutrophils (phagocytic WBC), monocytes (macrophages-phagocytosis

and presentation of antigens to T cells), eosinophils (high with allergies, parasites), basophils (contains histamine and serotonin) and lymphocytes (T and B cells) present.


WBC - Functional Considerations • Reference range - 4 - 11.0 X109/L • Optimal range – 5 - 7.5 X 109/L • Alarm range - <3.0 or >13 X 109/L High levels: >7.5 X 109/L - High Adrenaline levels (Stress) - High refined food intakes - Intestinal parasites, asthma and adrenal dysfunction. Low levels: <5.5 X 109/L

- Raw food diets - Multiple food allergies - B12, B6 and folate anaemia - Chronic intestinal parasite infection

(Weatherby & Ferguson, 2002) © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Ferritin • Most of the iron stored in the body is attached to ferritin therefore serum ferritin is

one of the most sensitive lab test for iron deficiency anaemia. • Raised in:

– iron overload disorders such as haemochromatosis and porphyria, acute malnourishment (e.g. anorexia nervosa) and as a marker for inflammation.

• A normal C-reactive protein can be used to exclude elevated ferritin caused by inflammation.

• Normal range is 10-120ug/L although some doctors suggest that this range should be increased to 50-120ug/L for a more accurate assessment.

• N.B. Iron deficiency Anaemia is probable if levels drop below 33ug/L and serum iron drops below 50umol/dL, with low FBC results. B12 and folate deficiency anaemia can be present with elevated levels though.

• High Ferritin levels are associated with increased risk of CVD, cancer and oxidative stress (Royal College of Pathologists Australasia, 2009)


Transferrin or Total Iron Binding Capacity (TIBC)

• Iron is transported in the blood bound to a protein called transferrin. Transferrin transports the iron in the body from the iron storage sites to where it is needed and back to the storage sites when not required.

44.8 – 62.7 umol/L May indicate: • Low – haemochromatosis (with increased serum Fe, % transferrin saturation

and ferritin) • High - iron deficiency anaemia. N.B. OCP medication can falsely increase levels. Hyperthyroidism can lower levels, as can many inflammatory conditions.



B12 Testing

• Serum B12 testing is often used to confirm B12 deficiency anaemia because a full blood count alone cannot easily differentiate between Folate (B9) deficiency and B12 deficiency.

• Laboratories vary significantly in their reference ranges in the UK. • Typically, anything from 110ng/L up to 900ng/L. • 140ng/L is the most typical lower reference range. In London some labs use a

lower reference range of 290ng/L. • There is an increased risk of clinical symptoms of insufficiency below 300ng/L. • Serum B12 testing can be confounded by analogues, such as those

found in Spirulina and many seaweeds.


Functional B12 Testing

• Many people experience signs of B12 deficiency with normal B12 serum. • MethylMalonic Acid (MMA) – available in serum or urinary testing – gives an

indication of how B12 is used as a coenzyme in cell metabolism. • High MMA levels indicate low B12 status, since MMA-CoA is converted to

Succinyl-CoA when there is sufficient B12. • Serum Reference range: 0.07 - 0.27 µmol/l • Urinary Reference range: <3.60 mmol/mol creatinine


HoloTranscobalamin (Active B12) • The gold standard in B12 testing is HoloTranscobalamin. This is a transport

protein, also known as Transcobalamin II, which is used to uptake B12 to the tissues.

• This has the highest predictability of B12 of all tests to date. • Only 10-30% of all B12 in the body is bound to HoloTC however it is the only way

B12 that can actually be up-taken by cells. Hence it is often called Active B12. • Whilst it is technically available to some (not all) labs in the UK, it is very

expensive and many have not heard of it or do not know how to interpret the test. • Reference range: >30pmol/L


Homocysteine

• Homocysteine is a sulphur-containing amino acid and is an intermediate metabolite of methionine metabolism.

• Metabolised by two pathways; trans-methylation and trans-sulphation pathways. • Regulation of homocysteine depends on certain key nutrients, including vitamin

B12, vitamin B6 and folic acid. • High homocysteine levels are an important biomarker in risk assessment for

CVD, strokes and other inflammatory diseases. • Functional biomarker for low B6, B9 and B12; also excess methionine. • May also indicate effect of genetic variances (SNPs) on methylation (see Nutrigenomics lecture).


C-Reactive Protein (CRP)

• A general marker for inflammation and infection. • Since many things can cause elevated CRP, this is not a specific prognostic

indicator. • Reference interval - <5 mg/L.

– For high sensitivity (hsCRP) - 0.2 to 3 mg/L • Nevertheless, a level above 2.4 mg/l has been associated with a doubled risk of a coronary event compared to levels below 1 mg/l. • From a functional testing perspective it is used to rule out inflammation as a driver



Erythrocyte Sedimentation Rate (ESR)

• Sedimentation occurs when the erythrocytes clump together. • The ESR is the rate at which erythrocytes settle out of anti-coagulated blood in 1

hour. • Non-specific indicator of inflammatory and neoplastic disease • Raised in pregnancy, inflammatory conditions (e.g. rheumatoid arthritis, OA, SLE,

gout, nephritis, endocarditis), chronic infections and neoplastic disease (carcinoma, lymphoma, neoplasms)

• Decreased in polycythemia, sickle cell anaemia and congestive heart disease



Blood Sugar Markers

• Fasting Blood Glucose or Oral Glucose Tolerance Tests • Detects hyper & hypoglycaemia and is useful as a screening test for diabetes

mellitus and gestational diabetes.

• Oral glucose tolerance tests

NICE recommended target blood glucose level ranges

Target Levelsby Type Upon waking Before meals (pre prandial)

At least 90 minutes after meals(post prandial)

Non-diabetic* 4.0 to 5.9 mmol/L under 7.8 mmol/L Type 2 diabetes 4 to 7 mmol/L under 8.5 mmol/L Type 1 diabetes 5 to 7 mmol/L 4 to 7 mmol/L 5 to 9 mmol/L Children w/ type 1 diabetes

4 to 7 mmol/L 4 to 7 mmol/L 5 to 9 mmol/L

(Diabetes UK, 2018) © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Blood Sugar Markers

Haemoglobin A1C/glycated haemoglobin • This test reflects blood glucose levels over the past 3-4

months. • In diabetes mellitus it monitors how well glucose levels are

controlled.

(Diabetes UK, 2018) © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Blood Sugar Markers

• Insulin • Serum levels used in conjunction with GTT or glucose to ascertain insulin

resistance. • Reference interval

– <5 mU/L during hypoglycaemia – 4-10 mU/L after 8 hour fast, and with a normal plasma glucose


Auto Antibodies

• Auto-antibodies are immunoglobulins towards self that are found in high states in auto immune diseases

– DNA - confirms diagnosis and monitors patients with SLE – ANA (antinuclear) antibodies - SLE and other rheumatological conditions – Gastric parietal cell - useful for diagnosis of gastric atrophy in pernicious anaemia

due to lack of intrinsic factor – Intrinsic factor - Used for the diagnosis of pernicious anaemia – Smooth muscle - this test is useful in the diagnosis of autoimmune chronic hepatitis – Thyroid microsomal/ peroxidase and thyroglobulin antibodies – Hashimotos

thyroiditis and/ or Graves’ disease – Endomysial/transglutaminase antibodies - diagnosis of coeliac disease and

dermatitis herpetiformis (Royal College of Pathologists Australasia, 2009)


Liver Function Test (LFT)

• A LFT gives an indication of how much the liver is inflamed, damaged or functioning below par- it is not a marker of detoxification abilities

• ALT and AST levels indicate the degree of inflammation – The aminotransferases are enzymes that are present in the hepatocytes. They

leak into the blood stream when the liver cells are damaged. – These values are usually high in hepatitis – possibly 20-50 times higher than

normal. – The ALT value is more specific to the liver than the AST value. – The AST value may also give an indication of muscle damage elsewhere in the

body – esp. cardiovascular – Low levels of both can indicate low B6, early fatty liver disease and

alcoholism. (Royal College of Pathologists Australasia, 2009)



• ALT and AST levels indicate the degree of inflammation – Ratios of these enzymes can be helpful in Nonalcoholic Steatohepatitis (NASH)

and alcohol-related liver disease. – > 1 – alcoholic liver disease – < 1 – NASH

Liver Function Test Range Alanine aminotransferase (ALT) 0 - 45 U/L

Aspartarte aminotransferase (AST)

40 <U/L

Alkaline phosphatase (ALP)

25-120 U/L

Gamma glutamyl transferase (GGT)

1- 70 U/L

Lactate Dehydrogenase (LDH)

1-240 U/L




• Alkaline phosphatase (ALP)- an enzyme found mainly in the bile ducts of the liver.

• Low levels may be associated with zinc deficiency. • Increases in ALP and another liver enzyme called Gamma GT (GGT) can

indicate obstructive or cholestatic liver disease, where bile is not properly transported from the liver because of obstruction of the bile duct.

– GGT is tested with ALP to make sure that ALP increases are coming from the liver. – The GGT is also a potential indicator of alcohol usage and is increased with certain medications eg. Phenytoin.




Gamma GT (GGT) – marker of oxidation • The primary role of GGT is to metabolise and transport extracellular reduced

glutathione (GSH), allowing for precursor amino acids to be assimilated and reutilised for intracellular GSH synthesis.

• As the need for glutathione production increases so does the production of GGT to facilitate GSH production.

• Serum GGT correlates with F2-isoprostanes (an oxidative product of arachidonic acid), fibrinogen, and C-reactive protein—all markers of inflammation.

• While oxidative stress and inflammation are not the same, they are highly correlated. • From a functional perspective, GGT over 40 may be an indication of high

oxidation. • Low levels may suggest increased Magnesium and B6 requirements



Lactate Dehydrogenase (LD) • Apart from detecting liver disease this test is useful in myocardial disease, skeletal

muscle disorders and anaemia where haemolysis is present due to levels being elevated with tissue breakdown.

• Can be associated with B12/Folate deficiency anaemia. • Low levels can be present with reactive hypoglycaemia. • Other disorders indicated by elevated LD include cancer, meningitis, encephalitis

and HIV, viral infections and tissue inflammation. • Reference Range: 1-240 U/L.




• Other pertinent tests in assessing progression of liver disease: albumin, bilirubin, clotting studies: prothrombin time (PT) or international normalised ratio (INR), if the prothrombin time (clotting ability) is prolonged this may indicate worsening chronic liver disease.

• Elevated Bilirubin can be a functional marker of impaired glucuronidation conjugation (phase 2 liver detoxification), increased intestinal beta-glucuronidase (and therefore enterohepatic recirculation), as well as haemolytic anaemia and gall stones.

• Raised urea and creatinine indicates that the kidneys are also affected.



Cholesterol • Chylomicrons - carry triglycerides from the intestines

to the liver, skeletal muscle, and to adipose tissue. • Very low density lipoproteins (VLDL) - carry (newly synthesised) triglycerides from the liver to adipose tissue.

• Low density lipoproteins (LDL) - carry cholesterol from the liver to cells of the body.

– "bad cholesterol" lipoprotein.

• High density lipoproteins (HDL) - collects cholesterol from the body's tissues, and brings it back to the liver.

– "good cholesterol" lipoprotein.


Cholesterol Hyperlipidaemia

– high lipid levels in the blood – relates to total cholesterol vs. individual components

Dyslipidemia

– disorder of lipoprotein metabolism including lipoprotein overproduction or deficiency

– elevation of plasma cholesterol, triglycerides (TGs), or both, or a low high density lipoprotein level

(Goldberg, 2018) © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Triglycerides

Conditions that may cause high triglycerides include: • Obesity • Poorly controlled diabetes • An underactive thyroid (hypothyroidism) • PCOS • Kidney disease • Regularly eating more calories than you burn • High sugar consumption • Drinking excess alcohol • Certain medicines may also raise triglycerides

e.g. hormonal medication and steroids • Certain types of high cholesterol and high triglycerides are genetic


Cholesterol levels

NICE guidelines suggest that measuring non-HDL cholesterol is more practical (it is non-fasting), cost effective and accurate at predicting CVD risk.

Lipid Marker Acceptable Optimal Total Cholesterol <5mmol/L <4.2mmol/L Non-HDL (Total minus HDL) <4mmol/L <3.37mmol/L LDL-C <3mmol/L <2.1mmol/L HDL >1.2mmol/L >1.6mmol/L Fasting Triglycerides <2mmol/L <1.7mmol/L

(NICE, 2018) © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Newer CVD Biomarkers

Biomarker What is measured? Reference Ranges LDL-P Number of particles of LDL 1000 nmol/L HDL-P Number of particles of HDL >34.9 μmol/L LDL-Size Size of the LDL protein Large (Pattern A): 23.0 - 20.6

(preferred)Small (Pattern B): 20.5 - 19.0 (harmful form)

Lipoprotein(a) Lipoprotein, like LDL, however it can also increase LDL uptake and inhibit clotting.

< 1.07 μmol/L

Apo-A1 Protein which activates enzymes in HDL to take up cholesterol

1.17 - 2.10 g/L

Apo-B100 Protein which activates enzymes in LDL and receptors needed for cellular uptake of cholesterol.

0.55 - 1.32 g/L

Homocysteine Inflammatory marker; functional indicator of excess methionine, insufficient B6, B9 or B12.

3 - 10 μmol/L


TG/HDL - IR

• Triglycerides and high-density lipoprotein (HDL) ratio are good surrogate markers for identifying insulin resistance in overweight patients, according to the results of a cross-sectional study published in the Nov. 18 issue of the Annals of Internal Medicine.

> 1.8 in SI units • The TG/HDL-C concentration ratio was used because the higher the value, the greater the risk of CHD, and the more likely the individual is to be insulin

resistant hyperinsulinemic, with the additional abnormalities associated with this defect in insulin action.

(Medscape Medical News, 2003)


Female Reproductive Hormones

FSH - (follicle stimulating hormone) • Used for investigation of gonadal hypofunction, suspected pituitary

tumour. Levels are increased when pituitary secretion occurs in the absence of feedback inhibition by gonadal androgens or oestrogens (primary gonadal hypofunction, post-menopausal state, castration).

• High FSH - PCOS, menopause, ovarian failure • Female: 1.0-8.0 U/L

Post-menopausal female: >18.0 U/L • Adult male: 1.0-5.0 U/L (age related).



Female Reproductive Hormones

LH - (Luteinising Hormone) • Investigation of male and female infertility to distinguish primary gonadal failure from

pituitary/hypothalamic failure • Identification of ovulation in the investigation of menstrual cycle disturbances and female

infertility – High levels are found in primary gonadal failure – Low levels occur with hypothalamic failure – LH peak occurs just prior to, and identifies, ovulation – High FSH/LH confirms PCOS (normally 1:1ish)

• LH secretion is pulsatile and single results may be misleading • Adult male: 2-10 U/L

Adult female: 2-15 U/L Post-menopausal: 15-100 U/L (Royal College of Pathologists Australasia, 2009)


Female/Male Reproductive Hormones

Testosterone • Assessment of hirsutism or virilisation in females • Investigation of testicular failure or precocious puberty in males

– Increased levels may be found in women with hirsutism or virilisation – Levels are increased in males with precocious puberty – Decreased levels are found in males with testicular failure – In plasma, testosterone is bound to SHBG, so abnormal levels of SHBG may cause a

discrepancy between free and total testosterone



Oestradiol

• Diagnosis of precocious puberty. • Monitoring of oestrogen therapy is only possible if the drug being administered is

oestradiol. • Monitoring ovulation induction in in vitro fertilisation. • Assessment of women with suspected hypothalamic or pituitary disease. • Female

Early follicular phase: 100-200 pmol/L Preovulatory phase: 500-1700 pmol/L Luteal phase: 500-900 pmol/L Postmenopausal: 70-200 pmol/L.



Progesterone

• Investigation of infertility. – Failure of progesterone levels to increase in the latter part of the menstrual

cycle indicates an anovulatory cycle or corpus luteum inadequacy.

• Method dependent, typically: Follicular phase: 2.0-4.5 nmol/L Luteal phase: 7.0-70.0 nmol/L


Prostate Specific Antigen (PSA)

• There is no specific blood level. In the past <4ng/ml was normal, however it is now evaluated based on a number of factors.

• < 2.83 ng/mL is considered an optimal target, with zero after prostatectomy being ideal.

• Levels may increase with: – prostatitis – benign prostatic hypertrophy (BPH) – After a biopsy – prostate cancer

• the test is not recommended as a stand-alone screening test for carcinoma of the prostate; use with digital rectal examination and other clinical findings.


IGF-1

• Other biomarkers for prostate risk, as well as many other cancers or disorders of disrupted cell cycle function, as well as longevity, include IGF1.

• IGF1 is a growth factor which activates mitosis, promoting cell proliferation. It is upregulated in a number of cancers as well as BPH.

• Lower levels associated with increased longevity, slower cell-turnover and enhanced DNA repair.

Age Reference Range 21 - 30 128-315 ng/mL 31 - 40 114-289 ng/mL 41 - 50 86-249 ng/mL 51 - 60 87-215 ng/mL 61 - 70 75-230 ng/m

70 + 53-205 ng/mL © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Thyroid Hormones

Thyroid Stimulating Hormone (TSH) • Levels are increased in hypothyroidism

and decreased in hyperthyroidism, also used to monitor thyroid replacement therapy (Levothyroxine) in hypothyroid (or Hashimoto’s) patients.

Thyroxine (T4) & Triiodothyronine (T3) • Elevated in hyperthyroidism and

decreased in hypothyroidism. • If T4/T3 levels are low and TSH is not

elevated, the pituitary gland is more likely to be the cause of hypothyroidism.


• © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Reverse T3

Reverse T3 (functional testing only) • Inactive isomer of T3 • Same chemical formula, but one Iodine binds to a different carbon. • Created by 5’ Deiodinase Type III (Active T3 uses type I and II). • Upregulated by:

- Selenium deficiency - Excess cortisol and adrenalin (Stress) - Low cortisol (Adrenal fatigue) - Inflammatory cytokines - Free Radicals (ROS) - Fasting and Ketosis

• Can cause the symptoms of hypothyroidism and slow metabolism © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Thyroid Antibodies

Anti-Thyroid Peroxidase (TPO) Antibodies • Most common antibody test performed via GP • 90% of Hashimoto’s patients will test positive. • 75% of Grave’s Disease patients test positive.

Anti-ThyroGlobulin (TG) Antibodies • Not routinely tested via GP where Thyroiditis is suspected. • Present in 60% of cases of Hashimoto's and 30% of cases of Graves

Anti-Thyrotropin Receptor (TRAbs) Antibodies • Least common test – rarely performed privately or via GP. • Usually only if Graves suspected – more than 70% incidence.


Reference Ranges

Biomarker Reference Range Optimal Range TSH (Thyroid Stimulating Hormone) 0.4-4.0miU/L 1.0-2.0miU/L

FT4 (Free T4) 10-22pmol/L 12-20pmol/L TT4 (Total T4) 58-154nmol/L 77-150nmol/L FT3 (Free T3) 2.8-6.5pmol/L 3.4-6.0pmol/L TT3 (Total T3) 0.9-2.5pmol/L n/a

RT3 (Reverse T3) 0.14-0.54pmol/L 0.14-0.40pmol/L Anti-TPO Antibodies (Thyroid

Peroxidase) 0-35IU/mL 0-30IU/mL

Anti-TG Antibodies (Thyroglobulin) 0-40 IU/mL 0-35IU/mL

See Example Sample Report: https://www.gdx.net/uk/core-uk/sample-reports-uk/Thyroid-Plus-Sample-Report-END27.pdf


Thyroid Testing

• Serum analysis is the gold standard • T3 and RT3 are specialist tests, as are TPO and TG antibodies. Occasionally they

are offered by the GP as standard but this is rare. • NHS will normally prefer to test for TSH and FT4 as the base line

– Cheaper and provides a good baseline comparison – Abnormalities may then be further investigated – Some traditional GP’s may only order TSH – There are private testing companies which offer more comprehensive tests if GP does not investigate further. Functional Dx Medi Checks Thriva


https://functionaldx.com

https://www.medichecks.com

https://thriva.co

Subclinical Hypothyroidism

• A reading of TSH and T4 may fall in the normal range but the person still exhibits symptoms of hypothyroidism

• High TSH with normal T4 suggests probably subclinical hypothyroidism and should trigger comprehensive testing esp. antibody testing.

• Full thyroid analysis panels can determine a number of things: – Poor conversion of T4 to T3 (due to Se deficiency) – Presence of antibodies towards the thyroid gland – Increased ROS (free radical) inhibition


Analyte High Low Thyroid stimulating hormone (TSH)

Good indicator of primary hypothyroidism. An indicator that the pituitary is working overtime to stimulate the thyroid into action,

Can still be normal with secondary or sub-clinical hypothyroidism. Suppressed levels can indicate hyperthyroidism

Free T4 May indicate hyperthyroidism. Oral contraceptives, exogenous oestrogen and pregnancy will increase values

Indicative of primary hypothyroidism, iodine deficiency, steroid use.

Total T4 May indicate hyperthyroidism. Oral contraceptives, exogenous oestrogen and pregnancy will increase values

Indicative of primary hypothyroidism, iodine deficiency, steroid use.

Free T3 May indicate hyperthyroidism May indicate hypothyroidism, selenium deficiency. In Wilson’s syndrome T3 is low but TSH is normal

Reverse T3 Impaired peripheral conversion of T4 to T3. May be indicative of Wilson’s syndrome, high levels of cortisol or selenium deficiency *

N/a

Peroxidase antibodies

Indicative of auto-immune activity which usually precedes deterioration of thyroid function

No evidence of auto-immune activity

Thyroglobulin antibodies

Indicative of auto-immune activity which usually precedes deterioration of thyroid function

No evidence of auto-immune activity

Subclinical Hypothyroidism


Immunoglobulins High Low

IgG 700-1600 mg/dL

Can indicate acute immune activation against antigen or remaining immunity after antigen contact

Chemotherapy (up to 2 months after), radiation, chronic distress, cortisone medication, Diabetes, kidney problems. smoking, allergies

IgM 40-230 mg/dL

With first contact with antigen, also in relapses, reliable indicator for any new infection, no matter where in the body; present with liver disease

Chemotherapy, immune suppressive medication, cachexia (eg. Cancer, HIV/AIDS), muscle wasting disorders.

IgA 70-400 mg/dL

Multiple Myeloma; Chronic Infections; Chronic Liver Disease; SLE (Lupus), RA (Rheumatoid Arthritis), Sarcoidosis, Thymic Aplasia, Leukaemia

Chemotherapy (up to 2 months after), radiation, chronic distress, allergies, surgery, genetics, SLE (Lupus – high or low), Cirrhosis.

IgE 150-300mg/dL

Originally acute parasitic Today main diagnostic parameter for acute allergic reactions

N/A

NB: These are general, total serum levels. However a doctor might test for specific antibodies eg. Allergens, autoimmunity.


RAST (Radioallergosorbent Test)/ IgE

• RAST testing uses blood screens for specific IgE antibodies to diagnose allergies to specific allergens.

• RAST testing is VERY expensive and therefore tests usually involve only one to four allergens screened at a time.

• As specific antibodies to specific antigens are being measured, you must have some idea of which allergens are most strongly suspected.

• You cannot test for multiple random allergens. • This is second line testing for allergies – the first line testing is the Skin Prick

Allergy Test.


Skin Prick Allergy Testing

• 1st line of allergy testing available via primary healthcare. • Cheaper than RAST testing and allows for a wider range of allergens to be tested

simultaneously. • Skin is pricked with a needle or pin containing microdose of an antigen (allergen). • Localised IgE mediated histamine release by mast cells causes “wheal and flare”

reaction – red, swollen, itchy skin (urticaria). • Limitations:

- Decreased sensitivity of the skin in infants and elderly. - Different body tissues show different types of mast cell responses therefore it is less reliable for non-urticarial reaction e.g. reactions in intestines.

• Does not assess for delayed onset food allergies.


Skin Prick Testing

Specific indications for Skin Prick Testing: • Severe skin disease (eg. Eczema) • Non-reactive skin • Hyper-reactive skin (urticaria) • NB: Skin test might be hazardous

– e.g. penicillin, bee or wasp venom sensitivity Other less invasive alternatives are sometimes used, such as Patch testing, which uses patches containing antigens rather than needles. This is preferred with infants and young children, though skin prick testing is still widely used in infants.


Common IgE Allergens

• Aspergillus • Grass mix • Bee venom horse hair • Blue mussel • Latex • Cat dander • Egg • Wasp venom • Fish • Wheat

• Mould mix • Cheese • Peanut • Cow’s milk • Penicillin • Dog fur • Shrimp • Dust mite • Soya bean • Gluten • Yeast


IgE Testing: Cautionary Note

Interpretation • A positive result on any form of IgE testing does not always mean the patient will

suffer an allergic illness when exposed to that allergen – IgG4 levels can block an IgE reaction, so it is best if they are tested in conjunction

• A negative result does not entirely exclude sensitivity. – False negatives are more common in children under the age of 5.

• The food tests are the least reliable. • A negative result does not exclude sensitivity.


Vitamin D

25-OH-Vitamin D • Most nutritionally relevant form of Vitamin D testing. • Another test (1-25-OH-Vitamin D) is less useful so always specify. • Available by serum (via GP) or pin prick (Better You or Vitamindtest.org.uk) • Low levels associated with increased inflammation, autoimmunity, cancer

mortality, infections. • U-Curve trend has been noted: Long term high levels are as harmful as long term

low levels.


Reference Ranges

• Labs vary dramatically in their interpretation of what is a appropriate reference range.

• Below 25nmol/L is almost universally agreed to be deficient. • Many labs believe that below 50nmol/L is inadequate. • Most U-Curve research shows that between 70-80nmol/L is associated with the

lowest all cause mortality longer term with 75nmol/L considered optimal. • Many labs now use the reference range 75-125nmol/L (some variance). • Research on autoimmune diseases eg. SLE has found that increasing vitamin D

to 85-125nmol/L improved T-reg cells. It isn’t clear what impact this has long term however.

(Terrier et al. 2012) © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Autoimmunity

• Studies tend to show lower levels of vitamin D in those with autoimmune • conditions such as RA and SLE. (Lopes Marques, 2010) • Unclear if this is a cause or effect • Increased risk of VDR polymorphisms in some autoimmune conditions. • Some research shows increased incidence of Th2 dominance (allergies) with long

term high doses (Hyppönen et al. 2004) • Other hypotheses suggest potential risk of infection with long term high vitamin D

in autoimmunity: cross-reactivity and premature T-cell maturation in the thymus could worsen or cause additional autoimmune diseases, due to Th1 suppression (Albert, Proal & Marshall, 2009)


How Sure is the U-Curve? • An increasing amount of research suggests vitamin D has a golden zone of 70-80nmol/L

or even 75-80nmol/L. • Above or below this risk increases for a range of diseases including cancer, CVD,

mortality. • A recent review described some of the limitations and unanswered questions regarding

the U-curve trend, including failure to account for confounding variables, activation of Calcitriol (inc. kidney dysfunction or increased breakdown of Calcitriol), prior vitamin D status and supplemental exposure, secondary nutrients. Read here (Grant et al. 2016)

• It’s important to note that this review was not a systematic review (prone to cherry picking of research) and the lead author received funding from 2 vitamin D councils and one nutraceutical company which specialises in high dose vitamin D supplements.


Vitamin & Mineral Assays

The Additional medical assays performed: – A - 0.7-2.8 µmol/L – C - Serum: 30-80 µmol/L; Leukocytes: 1.1-3.0 µmol/109 leukocytes.

• They do not necessarily always indicate a vitamin deficiency • Strategy is based on assumption that circulating concentrations reflect organ and

tissue content • Micronutrients are directed to circulation, storage and tissue pools • Physiological stress, recent dietary intake and acute phase response alter

plasma levels of micronutrients. CRP might be a useful co-indicator

(Royal College of Pathologists Australasia, 2009; Sarris & Wardle, 2010)


Functional vs Diagnostic Testing Pathology/Diagnostic Laboratory Biomarkers Functional Laboratory Biomarkers

Often associated with specific pathologies Not always associated with specific pathologies. May be confirm/negate formal diagnosis Not always directly to formal diagnosis confirmation

Focus on disease states and diagnostic criteria Focus on functional imbalances Not always sensitive to subclinical compromise in

function Aim to identify potential subclinical variations away

from optimal function Not always directly focused on nutritional applications Many biomarkers have specific links to nutritional

applications/pathways. Often have a strong evidence base Often have controversial or weak evidence base

Easier to research and gather evidence for due to simplistic diagnostic targets/focus

Harder to research and gather high quality evidence due to relevance/application in a broader range of

circumstances not linked to formal diagnosis.

May be sufficient as standalone markers to draw conclusions

Usually only relevant when part of a wider consideration of multiple factors (S, Sx, Biomarkers)

Taken seriously by medical profession Often considered “quackery” or “irrelevant” if conventional biomarkers are within normal range.


What is Functional Testing?

• Instead of focusing on disease pathology, functional testing highlights the functioning capability of the body, so that the practitioner can steer their client back towards optimum homeostatic functioning.

• Functional biochemical tests may involve the measurement of a metabolic product or intermediary by-product in the blood/urine which can indicate a lack of a nutrient dependent enzyme.

• It helps to ascertains the person’s specific needs and helps you the practitioner to tailor your dietary and nutraceutical choices specifically.

(Sarris & Wardle 2010) © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Testing Suppliers

UK Providers of functional testing: – Genova Diagnostics – Invivo Clinical – Lifecode Gx – Cambridge Nutritional Sciences (CNS) – Healthpath – Yorktest Laboratories – BioLab Medical Unit – The Candida Test – Regenerus Laboratories – Functional Dx

You can start an account with these labs, and use them in student clinic under supervision until you are fully qualified.

– Also online test suppliers such as Thriva and Medichecks can be used straight from consumer


https://www.gdx.net/uk/

https://www.invivoclinical.co.uk/

https://www.lifecodegx.com/

https://camnutri.com/

https://www.yorktest.com/

https://www.biolab.co.uk/

https://www.candidatest.co.uk/

http://regeneruslabs.com/page/homepage

https://functionaldx.com

https://thriva.co/?gclid=Cj0KCQjwm6HaBRCbARIsAFDNK-h6jIOr4dCSVAG9_cnKZ9ecDifrQH5l_jH4g8TIcXrL6VysZjsvpG0aAogEEALw_wcB

https://www.medichecks.com/?gclid=Cj0KCQjwm6HaBRCbARIsAFDNK-gmdFL3CvAPpmzn3UXdNhjYnhr6xECP1DUFMK-Z2fHiRe_Ir5v9jmUaAvB5EALw_wcB

Group Activity

Susie, 34yo, has come to see you in clinics. She has been desperately struggling with a number of niggling symptoms occasional headaches, tender joints, digestive issues, chronic skin issues such as acne, recent changes in her cycle. She hasn’t been able to identify any particular patterns. She is single and works full time but has a low income. Her dad has offered to pay for tests she wants, to help her get well, but he is retired and sceptical. He wants you to outline objective rationale for your choices. To help you be really objective in prioritising your choices, what criteria would you use to help you decide which testing is most useful? What ethical considerations do you think you would need to consider?


Considerations for Functional Testing

• What rationale from the case history do you have to consider the use of the test? (Naturopathic “Rule of Three”).

• Have you ruled out red flags, conventional tests or referral considerations that might indicate something more significant needs investigating?

• What mechanisms might underpin the symptoms the client is experiencing? • Do you already have a working hypothesis of what you may want to do? • If so, how specifically would the test help to change what you might otherwise do?

(think of three ways) • Have you already tried making diet or lifestyle changes first? If not, why? Would it be

unsafe or unethical to make recommendations in this instance without testing? • How do you know that the specific test you have chosen will be the best value for

money? (Think of 3 ways that you can be sure about this). • What is the quality of evidence available to support the use of the test?


Functional Testing: Organic Acid Testing

• Fairly easy to do (requires certain foods or supplements to be avoided during the process of collection) and non-invasive

• Tested via urine as the organic acids are not largely reabsorbed in the kidneys • Provides a metabolic snapshot of a client’s overall health, these may not always be

extensive but allows us to determine the client’s digestive state, neurotransmitter health, some nutrient markers, mitochondrial/cellular health and their detoxification ability

• The test can be used with complex cases to determine if further extensive testing is required in a specific system

• Again it is important to look at the full picture and assess why a client’s body is showing suboptimal markers and how can we support the client’s body so it can shift into an optimal range

• See sample report here: https://www.biolab.co.uk/index.php/cmsid__biolab_test/Organic_Acids_Profile


https://www.biolab.co.uk/index.php/cmsid__biolab_test/Organic_Acids_Profile

Genetic Testing

• Can enable the practitioner to analyse client’s genetic code to help determine what may be contributing to their poor health or how to optimise it

• Remember though: Every one has hundreds to thousands of SNP’s, these are not necessarily being expressed. The role of epigenetics is far more important and this is the part as practitioners how we can help clients, empower them giving the tools to support this (see nutrigenomics lecture for further detail)

• The testing process - • Sampling - Usually collected via saliva sample or cheek swab, making it

simple for client to collect at home or in clinic. • Sequencing - Decoding, or reading, the DNA to generate raw data • Interpretation - Explaining the meaning of the data in context


Genetic Testing - Sequencing

• Sequencing is the decoding, or ‘reading' of DNA, generating ‘raw data’.

• Whole Genome Sequencing (WGS) - full (almost) genome, about 3 billion base pairs; and Whole Exome Sequencing (WES) - examine protein coding variants, about 1% of the genome

• WGS and WES may be used to identify rare variants that can have significant health impacts, and diagnose rare diseases. Used within NHS.

• SNP Genotyping - between 10 to 500,000 data points, < 0.02% of the genome • Can be a cost effective way to identify common variants (SNPs) that can have a range of

impacts but can usually be compensated for by nutrition and lifestyle. Used in Nutritional Therapy, FxMed and Direct To Consumer (DTC)

• Consider - scope of the test. More is not necessarily better - risk of lower accuracy, and exclusion of the most clinically relevant SNPs (23andMe V5*)


Genetic Testing - Interpretation

• Interpretation is the explanation of the meaning of the raw data -

• Scope - Does it report clinically relevant SNPs. Or is it too limited or too generic (does not differentiate between high and low/no impact SNPs).

• Transparency - Does it report gene name, SNP ID (e.g. rs1234567 or C677T) and genotype result (e.g AG)

• Explanation - Is the potential impact of the SNP explained in plain English, and is it clear how the potential can be impacted by nutrition and lifestyle

• Context - is the information presented in a clinically relevant and useful way

• Evidence - does the report provide research references, and ideally links

• It is vital that the genetic data is analysed in the context of the person including family history, health history, symptoms, functional test results, and nutrition and lifestyle analysis, hence the role of the therapist.


Genetic Testing - Companies

• Direct To Consumer Testing & Interpretation • 23andMe or Ancestry - 500k base pairs, but excludes some clinically important ones. Low cost. Accuracy issues have

been reported with rare SNPs. Data is shared for research purposes (opt out is possible).

• DNA Fit - about 30 SNPs in total, limited scope. Low cost. Interpretation may not be aligned with NT principles.

• Interpretation only (with 23andMe data) • Direct to Consumer - StrateGene MTHFR Support.com NutraHacker • Practitioner - Opus 23

• Practitioner Testing & Interpretation

• Lifecode Gx - about 150 clinically relevant SNPs. Mid cost. Data is not shared. UK lab. Specialist panels including Methylation, Detoxification, Oestrogen, Thyroid, Histamine, APOE, Nutrient Core and Athlete. Genotype results presented as biochemical pathways in context of nutrition and lifestyle.

• DNA Life (Nordic Labs) - about 100 clinically relevant SNPs. Mid cost. Denmark base. Genotype results presented as a list. Specialist panels include DNA Diet, Health, Mind, Skin, Sport, Oestrogen.

• Genova Diagnostics - about 40 SNPs. Expensive. Specialist panels include DetoxiGenomic, EstroGenomic, ImmunoGenomic, NeuroGenomic, CardioGenomic. Panels are relatively limited in number of SNPs.


https://www.23andme.com

https://www.ancestry.co.uk

https://www.dnafit.com

http://go.strategene.org/genetic-analysis

https://mthfrsupport.com

https://www.nutrahacker.com

http://datapunk.net/opus23/

http://datapunk.net/opus23/

https://www.lifecodegx.com/

http://nordiclabs.com/page-DNA_Life-587.html

https://www.gdx.net/uk

Genetic Testing - Ethics

• Informed consent is a legal requirement. Parental consent is required for under 16’s. Test companies may have additional constraints.

• Data protection, storage and security policy. Some test companies share data with 3rd parties by default (there should be an opt out clause).

• Uploading raw data files to online web pages or apps may (is likely to) be insecure. The client may choose to do this at their own risk. Client data requires protection & confidentiality as rules apply to DNA data.

• Make sure you understand the scope and potential implications of a test before you recommend it, and brief your client accordingly.


Food Intolerances: Types of Sensitivity Reactions

IgE mediated allergies • “True” allergies – they result in degranulation of mast cells • More consistent immunological reaction, with memory. Tests likely repeatable. • Reactions may worsen or get more life-threatening with repeated antigenic exposure. • These may be lifelong issues, even though desensitisation is possible

IgG mediated INTOLERANCES • Though antibodies may be involved they are not considered a true immunologically

mediated allergy – they are an intolerance. • Immunological memory is not strongly evidenced and hence not life-threatening and

not always life-long. • Test results may not be repeatable within a few months of avoidance.


Non-Antibody Mediated Sensitivity

Biogenic Amine Intolerance • Histamine and Tyramine are the most common • Dose Dependent • No overt antibody mediation • Multi-factorial (e.g. Bacterial synthesis, hypomethylation, SNPs, DAO

insufficiency, stress, dietary load – amine containing/releasing foods) • Can test for indirect markers but no specific food tests available. • Not life long as no memory involvement – but can be complex to overcome due to

multiple influences. • Symptoms can mimic IgE mediated reactions inc. anaphylaxis in rare cases.


Polyphenol Intolerance • Salicylates and other naturally occurring phenols, occur in plant foods and are

also found in many medications and cosmetics. • Some people can develop symptoms similar to other intolerances due to poor

metabolism. • No immunological influence at all. • Often linked to multiple factors (eg. which metabolites of the phenols are created

by bacteria in the gut; hypomethylation; dose; SNP’s). • There are no tests available, though occasionally organic acid tests may show

some metabolites in urine which can sometimes be caused by bacterial • metabolism of phenols – but this hasn’t actually been linked to intolerance in

research at all.


Food intolerance - IgG • IgG testing is the most common form of food sensitivity testing offered by nutritional

therapists. • If a client comes to you in clinics reporting they have food allergies or intolerances,

ask them how they were informed of this (e.g. Trial and error, elimination diet, IgE mediated testing, Bio-energetic Stress Testing, or IgG testing).

• IgG intolerance is thought to be the presence of an adverse reaction to a specific food or food ingredient

• Not necessarily associated with clearly defined immune reactions in the same way as food allergy, and may involve a number of different reactions

• Research is available on IgG testing for dietary exclusion – though the quality, methodology, results and repeatability/relevance are highly controversial.

• Many practitioners believe there is at least good anecdotal evidence for its use- it is often a good place to start creating a grand elimination diet (gold standard) – though there could be multiple influences here to consider.


Confounders in Anecdotal Outcomes • Before looking at some of the research on IgG testing, let us consider the idea that anecdotal

evidence may justify the use of expensive clinical testing. • What factors might influence anecdotal outcomes of test results following use? • Placebo/Nocebo • Observational-Selectional Bias: Noticing patterns we hadn’t identified before, believing they

have increased in frequency. • Confirmation Bias: The tendency to look for or pay attention to information that supports our

current beliefs, expectations or habits, whilst ignoring the significance of information that threatens it.

• Overall dietary quality improvements in general may reduce symptoms, with or without the tests.

• Focusing on specific foods to exclude may encourage wider variety of foods to be consumed increasing nutrients

• Without first making lifestyle improvements it’s hard to be certain if any improvements that may have arisen from testing would have occurred anyway.


Alternative Explanations

• It’s also important to remember the possibility that someone may test positive for an IgG food, yet show improvements upon avoidance for another reason that is not linked to IgG antibodies.

• Eg. Dairy – IgE allergy, histamine or tyramine intolerance, lactose intolerance due to lack of lactase.

• If one of these more complex factors may be influencing symptoms, the true causes of these may not be addressed. e.g. If it’s a histamine intolerance, methylation issues/SNP’s, slow DAO enzyme, bacterial synthesis, SIBO etc. may go unidentified.

• Be sure the full picture is clear before jumping to testing.


IgE: A Closer Look • IgE mediated allergies indicate an atypical picture: Antibodies have been formed to

an antigen which in most people is not considered harmful. • IgE antibodies form complexes with mast cells when they bind to receptors,

triggering mast cell degranulation and histamine release. • People will manifest predictable clinical symptoms and will usually test IgE positive

even if they have not recently been exposed to the antigen. • Whilst the tests are far from perfectly consistent, they do offer a good level of

predictability and are based on larger bodies of evidence.


IgG: A Closer Look

• IgG food intolerance tests can look at IgG or a more specific IgG4 antibody presence.

• IgG4 antibodies are unique amongst the IgG antibodies in that they do not activate the complement inflammatory cascade but are present in only small amounts as a proportion of IgG (about 5%) in the blood.

• IgG4 antibody responses increase when there has been regular and recent exposure to food antigens.

• They are not associated with the same mast cell degranulation responses and hence do not show the same types of responses as true allergies.

• IgG4 can actually block IgE mediated reactions (this is how desensitisation therapy works, by raising IgG4)


IgG Testing – A Basic Introduction

• Proponents of IgG testing state that regular exposure to an unvaried diet, along with other factors such as leaky gut, can increase sensitivity to foods.

• It is has been found that with food allergens specifically (IgE), intestinal permeability is an important mediating factor and that repeated exposure exacerbates the symptoms. Removal of the allergen improves intestinal permeability and allows tight junctions to repair (Yu, 2012).

• Many practitioners believe that the same is true with IgG reactions and that by supporting gut repair and reducing exposure to IgG-positive foods, it reduces the overall antigenic load and reduces IgG levels, improving symptoms.

• By keeping the diet well rotated, it is possible to avoid further intolerances developing. • Many people find that within a few months of avoidance, their food intolerances can be

retested and show changes.


IgG4 Correlations

• IgG4 antibodies have been found to be elevated with gastrointestinal and immune issues.

• E.g. A study in 2016 looking 200 children, comparing 50 healthy vs. 50 with wheat allergy (IgE), 50 with Coeliacs and 50 with H. Pylori found looked at IgG and IgG4 response to wheat and rice, when eliminated and consumed: - No correlations with IgG/IgG4 in healthy children – any levels which were present with consumption were reduced with elimination of food. - IgG and IgG4 were elevated for wheat and rice in all the GI disorder groups, even after elimination. - IgG4 present with wheat allergy group for both rice and wheat even though asymptomatic with rice.

• Conclusion: IgG presence in GI groups seemed correlated to dysfunction of the gut and immune system, rather than to wheat per se.

Read Full Text here: (Czaja-Bulsa, Bulsa & Gebala, 2016)


IgG and IBS

• One of the most frequently cited studies on the benefits of IgG testing was a an RCT on IBS improvements. It is cited on websites such as York Laboratories, who offer IgG testing.

• This study published in GUT, a well respected BMJ journal, used 150 patients with IBS. They were assigned either a protocol based on their own IgG food antibody responses or a sham diet for 12 weeks.

• Results: -10% greater reduction in symptoms scores compared to the sham diet in those with moderate compliance - 26% reduction in those with full compliance. - 24% worsening of symptoms in those with “relaxation” of compliance after a while, in those on the IgG diet compared to the Sham diet.

• Conclusion: IgG testing was more predictive of improvements than placebo. Or is it….?

Read Full Text: (Atkinson et al. 2004) © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Study Design: A Closer Look

• 84% of subjects in the study tested positive for yeast IgG • In other RCT studies looking at elimination of yeast in IBS, between 5-12% of the

subjects actually showed symptomatic reactions to yeast. • It is unlikely that 84% in one study of similar population sizes, would all have reactivity

to yeast yet not in others. • 84% of subjects in the active study group tested positive for milk and 49% for wheat –

two of the most common triggers for IBS. • By contrast, in the sham diet group, only 1.3% avoided milk and 8% avoided wheat,

when given advice by the nutritional consultant, despite the frequency of these common IBS triggers.

• This suggests the blinding of the study was likely compromised and the advice was weighted in favour of the IgG study group outcome.


Statistical Review: NNT

• The global rating (the outcome of the whole active group) had an NNT of 9. • NNT is a statistical term standing for Number Needed to Treat • This means that on average, 9 people would need to be treated with an IgG test in

order for 1 adverse outcome to be avoided (IBS symptoms). • Generally, high quality studies show that between 1.5-2.2 people would need to be

treated using effective elimination diets to avoid adverse outcomes. • To be fair, the study did report an NNT of 2.5 for the fully compliant group, but

because this is calculated using questionable sham comparisons this number becomes less reliable.

• A more comprehensive critique is published here in GUT: (Hunter, 2005)


IgG Criticisms

• Whilst IgG proponents often acknowledge that IgG antibodies indicates persistent exposure to a food antigen, directional causal relationship between antigens and symptoms have not been shown in research and require high level of consistency in causal direction in clinical research. – only recent exposure.

• IgG, especially IgG4 antibodies are often associated with improved tolerance to IgE allergies

(Skripak et al. 2008; Jones et al. 2009; Savilahti et al. 2009; Tomicˇic et al. 2009)

• A recent review of mechanisms underpinning ways by which IgG4 may reduce IgE reactions proposed: - Blocking IgE receptor activation of mast cell degranulation - Stimulation of an inhibitory receptor, FcγRIIb, reducing immunologic response.

Read Full text: (James & Till, 2016)


Causal Relationships

• Correlation does not equal causation • Causal relationships can ONLY be shown by high level clinical evidence. • These studies must include large population sizes, be well designed and show

repeatable results. • Without this, we cannot be sure whether the correlation has a meaningful

explanation – and if it does, what that explanation is. • Much of the IgG – gut – hypersensitivity hypotheses have been extrapolated from

our understanding of IgE allergens and gut immunity • Conclusions have been drawn prematurely and claims of causal relationships

used to justify the use of tests. • At present the quality of most studies are low and are not consistent in supporting

the current hypothesis.


Available Intolerance Tests

• York and Cambridge Nutritional Science – pin prick blood sample which tests for IgG reactions to food

• Genova– serum IgE and IgG tests • Invivo offer the Dunwoody full antigen profile -

IgG C3D IgE IgG4 • Regenerus labs offers the Cyrex profiles (cross

reactivity panels) and the ALCAT (cytokines) • Full elimination & challenge test

– requires dedication on the part of the client


Group Work

• Let’s go back to Susie, our 34yo client we met earlier.

• Consider that the prices for IgG tests range anything from £80 to £450, would you consider the use of IgG testing to support her symptoms based on the current level of evidence?

• Why/Why Not?


Food Intolerances Consider WHY clients may be reacting to foods: • Lack of chewing or eating too quickly • Low HCl and/or enzymes • ‘Leaky gut’ / poor mucosal integrity • Candida or parasite overgrowth • Lack of beneficial bacteria and prebiotics • Inflammatory processes

– increased cytokine, leukotriene, prostaglandin and chemokine synthesis


Digestive Function & Parasitology: Intestinal Permeability

• One of the common issues with the gastrointestinal tract is assumed to be increased permeability or “leaky gut”

• Linked to: IgE mediated reactions, inflammation, alterations in the blood brain barrier, autoimmunity and other systemic hypersensitivity reactions

• Often permeability is just assumed, in clinical practice due to symptomology • To evaluate intestinal permeability there are a variety of tests available.

– Sugar absorption and urinary excretion tests – Zonulin tests – both in stool or serum – Lipopolysaccharides in serum tests


Sugar Absorption Tests

• Urine collection tests • First a baseline reading is collected • Patient then drinks a solution containing an low digestible disaccharide solution –

then urine is collected after ingestion to see the presence of these sugar molecules in the urine, therefore giving a marker of intestinal permeability

• Different labs offer different sugars, such as mannitol, lactulose, Polyethylene Glycol – and due to the different sizes they are used for slightly different reasons


Other Markers for Raised Intestinal Permeability

• Zonulin- is a protein that opens the gap junctions in response to infection, so larger molecules can be taken across to the immune system to deal with, it was discovered in 2000 by Alessio Fasano and his team. It has high links with coeliacs disease and diabetes type 2, and changes often occur a few years before disease process starts. It can be tested in the serum or the stool as a marker of intestinal permeability

• Lipopolysaccharides (LPS and LOS) are the outer cell walls of gram negative bacteria, which are highly activating for the immune system. High blood levels of LPS correlate with endotoxemia, systemic inflammation and autoimmune conditions. Raised intestinal permeability allows higher levels of LPS into the blood stream, so measuring them in serum is a helpful tool


Sample of Tests Available

• GI Mapp (Invivo) and Biovis by (Healthpath) - measures faecal zonulin, bacteria, worms, yeasts, parasites, as well as intestinal immune health markers

• Genova offer stool or serum Zonulin by itself • Advanced Intestinal Barrier Assessment by Invivo – measures serum LPS,

Histamine, DAO and zonulin by Dunwoody labs • Biolab offers PEG intestinal permeability testing • Regenerus offers the Cyrex intestinal antigenic permeability screen - measures

antibodies to actomysin, zonulin, LPS


SIBO

• Small Intestinal Bacterial Overgrowth is a complex issue in clinics. • It occurs when specifically there is an overgrowth of bacteria in the small

intestines. • It’s important to note that the bacteria present here are not necessarily pathogenic

bacteria – often they are normal commensal flora or even bacteria from the large intestines which have transmigrated or growth out of balance in the small intestines.

• It is recognised as an issue in conventional medical practice and is often treated with specific antibiotics

• SIBO can lead to malabsorption of fats, starches and proteins and cause significant discomfort as well as histamine related symptoms.


When to Test for SIBO

• Bloating, excessive burping, halitosis (esp. sulphur smelling) and flatulence. • Symptoms get worse with high sulphur foods (eg. Garlic, crucifers, animal protein,

pulses). • Greasy, malodorous stools not related to Coeliacs or biliary/gall bladder issues. • Stool tests show normal inflammatory markers and pancreatic elastase markers but

there appears to be malabsorption of protein and fat. • Diarrhoea • Constipation which worsens with fibre consumption • Histamine, salicylate or tyramine related issues which show only mild improvement on

elimination of trigger foods. • Symptoms worsen with probiotics even after 1 month. • Symptoms may sometimes improve with antibiotics temporarily. • FODMAPS or SCD diets are not improving symptoms significantly.


Hydrogen/Methane Breath Test

• The standard test for SIBO is a breath test to detect hydrogen/methane levels after a lactulose challenge test.

• Lactulose undergoes bacterial fermentation in the large intestines and so will usually decompose within a predictable time period.

• Normally there are only a small number of bacteria present in the small intestines and these do not usually ferment lactulose.

• When bacteria proliferate in the small intestine that are normally in the large intestine, they will begin to ferment lactulose too.

• The waste products of this fermentation are hydrogen and methane. • These are collected in a bag, transferred into a tube, at regular intervals after lactulose

consumption and the amount of hydrogen and methane present indicates whether SIBO is present and where bacteria are most likely to be dominant.

• See sample reports on website


Stool Testing • Use of stool testing has always been controversial, as earlier methods have not always

been the most accurate • Stool composition changes quickly in response to diet, infection and immunity • Stool testing is helpful in cases of digestive complaints, but as we begin to learn more about

the impact of the microbiome on systemic health, it is often employed in the case of complex, chronic systemic disorders

• May be helpful in: – identifying parasitology and presence of worms – confirming the presence or absence of both beneficial and pathological bacteria – assessing mycology – evaluating and assessing digestive markers and function – evaluating inflammatory markers – evaluating immune response at GIT wall


Stool Testing Methodology

• Stool testing used to mainly be done with culturing and screening technology, where there can be many false readings due to bacteria dying on route to the lab, or not being able to be cultured in the environment of the petri dish.

• With the advent of DNA testing, we can now test the DNA of the microbiome through Polymerase Chain Reaction testing (PCR) which is highly sensitive, and now quantifiable.

• Stool testing is available through: • Genova does multiple panels, but their best seller is the GI-Effects profile, it

mainly uses culture methods with some DNA • Doctorsdata, Healthpath and Invivo offer stool tests which use the full PCR

methodology.


Hormone Testing: Female Reproductive Hormones

• When working with female reproductive issues it is often important to get testing done

• Some laboratories offer salivary analysis of major female sex hormones

– either a one day panel or monthly panel – offers a snap shot of what is going on in the

body in that period of time • The disadvantage of this type of testing is that it

can be somewhat inaccurate due to a misreading • The medical profession will not use it for their

evaluation


Salivary Full Hormone Profile • Measurement from a single sample is of little use in pre-menopausal women as

the hormones fluctuate throughout the menstrual cycle

• Need several collection points throughout the month - Most tests use 12 saliva samples

• Also looks at melatonin, DHEA and cortisol levels (see Stress and Fatigue lecture

re: Pregnenolone Steal for more information).

• See Sample Report: https://www.gdx.net/uk/core-uk/sample-reports-uk/Rhythm-Plus-Comp-Female-Hormone-Sample-Report-END11.pdf


https://www.gdx.net/uk/core-uk/sample-reports-uk/Rhythm-Plus-Comp-Female-Hormone-Sample-Report-END11.pdf

https://www.gdx.net/uk/core-uk/sample-reports-uk/Rhythm-Plus-Comp-Female-Hormone-Sample-Report-END11.pdf

Serum Hormonal Testing

• Progesterone, oestradiol, testosterone, LH, FSH can all be tested via serum – this is the test of choice for the medical profession

• Can be difficult to interpret • Must know what day of the cycle the tests were taken on to read them accurately • You can pass on these test results to specialists


Hormonal Urine Analysis

• Urine testing can be performed to see ovulation peak – normally LH

• Dipsticks and machines can be used for this also eg. Persona • Useful for attempting to achieve or avoid conception • Accuracy is reduced in women with high FSH levels or cycles longer than 32 days • Pregnancy tests are a hormonal test

– they test for levels of human chorionic gonadotrophin (hCG) in the urine – a hormone made by the fertilised zygote


• Able to give an overview of sex and adrenal hormones alongside their metabolites.

• Easy for clients to collect. • Must be used within day 19-22 of menstrual cycle • If using Lab Precision Analytics DUTCH test via Regenerus, tests also look at

CAR (cortisol awakening response), some organic acid markers, melatonin and oestrogen methylation pathways.

• See Sample Report: http://regeneruslabs.com/shop/product/dutch-complete-hormone-hor30-784

Dried Urine Analysis


http://regeneruslabs.com/shop/product/dutch-complete-hormone-hor30-784

2/16 Ratio of Hydroxyoestrone

• 2-hydroxyoestrone (2-OHE1) - tends to inhibit cancer growth • 16α-hydroxyoestrone (16α-OHE1) - encourages tumour development • Urinary assessment of these oestrogen metabolites can help to identify

biochemical metabolic individualities and guide dietary advise and supplements


Commonly Used Serum Female Hormonal Profiles

• PCOS profile – generally includes FSH, LH, oestradiol, insulin resistance and cholesterol test

• Menopause profiles

– generally FSH and oestrogens

• Infertility profile – generally all hormones and test for antibodies as well


Adrenal Hormone Testing

• Cortisol and DHEA are our primary glucocorticoids required to maintain our resistance to stress.

• These hormones are not released constantly throughout the day, but are secreted in a cycle called the circadian rhythm, with the highest values being in the morning and the lowest at night.

• Sometimes in periods of long-term or profound mental or physical stress due to poor nutritional status, smoking, work, eating disorders, divorce, death, intense exercise, alcohol consumption, severe slimming diets, major illness or surgery, these glands can go into overdrive but later become exhausted.

• When this happens the hormonal levels of cortisol, DHEA in the blood stream become imbalanced.


Adrenal Stress Index

• The Adrenal Stress Index (ASI) test enables a detailed assessment of the adrenal hormone cycles by measuring both saliva cortisol and DHEA levels and their ratios throughout then day.

• The ASI is a simple, non-invasive test that uses four saliva samples to measure the adrenal rhythm and obtain a DHEA to cortisol correlation.

Salivary testing of hormones unlike serum testing, measures the free un-bound fraction of the hormone that is available to exert its effect on the target tissue.


Vitamin & Mineral Profiles: Hair Mineral Analysis

• Easy to do and non-invasive and relatively inexpensive • Provides an indication of mineral status and toxic metal accumulation following

long term or even acute hair analysis provides a reading of mineral deposition in hair over a 2-3 month period.

• The test does not provide an assessment of mineral content of other body tissues. It is not known to what degree, if any, these correlate with body tissue mineral or toxic element usage at present.

• Hair analysis is not a diagnostic test.


• The US Environmental Protection Agency has long accepted the method of using hair as a medium for biological monitoring of heavy metals – however this has been used as a research marker, not as a diagnostic marker.

• Hair is often claimed to help assess nutritional status, but the evidence base is very mixed here.

• Results from different laboratories are reported differently and can cause confusion in interpretation.

• If you ever do choose to use hair testing, use one testing house for first and subsequent tests on the same client

Hair Mineral Analysis


• Analytes – calcium, magnesium, sodium, potassium, copper, zinc, iron, selenium,

chromium, manganese, molybdenum, phosphorus, cobalt and boron. – the test claims to tell whether the levels are low, normal or high, as well as

whether they are in balance. • Also detects presence trace heavy metals such as lead, mercury and cadmium.

Hair Analysis


Hair & Nutritional Status?

• Laboratories that test for hair minerals such as mineralcheck.co.uk claim that they are a good way to evaluate nutritional status.

• There have been some correlations identified with minerals and serum levels, though not consistently.

• However it is key to note that there is no established “normal” or reference range for hair minerals in the average population.

• Without a baseline for comparison, it leaves the laboratories with responsibility freedom for setting the reference range without sufficient evidence.

• This means the inferences we can make and the interpretations the laboratories offer about the meaning of the results are questionable.


Toxic Elements?

• Some toxic elements have been found to correlate reasonably well, such as lead, when compared with serum analysis or urinary clearance.

• Some are trickier. For example, Mercury comes in many different forms, eg. Methyl, inorganic, ethyl.

• Methyl mercury comes primarily from fish and has a strong affinity for sulfhydryl bonding to proteins in the body. It has been found that mercury in this form seems to be well correlated between hair and urinary excretion as well as serum testing.

• Inorganic mercury has been found to be less consistent.

(Kempson & Lombi, 2011) © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

References Albert, P.J. Proal, A.D. Marshall, T.G. (2009). ‘Vitamin D: The alternative hypothesis’, Autoimmunity Reviews, 8(8), pp.639–644. Amon, P. & Sanderson, I. (2017). ‘What is the microbiome?’, Archives of Disease in Childhood: Education and Practice Edition, 102(5), pp.258–261. Ashwell, M. Mayhew, L. Richardson, J. et al. (2014) ‘Waist-to-Height Ratio Is More Predictive of Years of Life Lost than Body Mass Index’, PLOS one, 9(9), e103483. Ashwell, M. Gunn, P. Gibson, S. (2012) ‘Waist-to-height ratio is a better screening tool than waist circumference and BMI for adult cardiometabolic risk factors: systematic review and meta-analysis’, Obesity Reviews. 13(3), pp. 275-286. Atkinson, W. Sheldon, T.A. Shaath, N. et al. (2004). ‘Food elimination based on IgG antibodies in irritable bowel syndrome: A randomised controlled trial’, Gut, 53(10), pp.1459–1464. Barrett, S. (1985). ‘Commercial Hair Analysis Science or Scam?’, JAMA 1985(254), pp.1041-1045 Czaja-Bulsa, G. Bulsa, M. & Gebala, A. (2016). ‘Food IgG4 antibodies are elevated not only in children with wheat allergy but also in children with gastrointestinal diseases’, BMC Gastroenterology, 16(1), pp.1–8. Diabetes U.K. (2018) Blood Sugar Level Ranges. [Online]. Available at: www.diabetes.co.uk/diabetes_care/blood-sugar-level-ranges.html (Accessed: 14 July 2018) Goldberg, A. (2018). Dyslipidemia. [Online]. Available at: https://www.msdmanuals.com/en-gb/professional/endocrine-and-metabolic-disorders/lipid-disorders/dyslipidemia (Accessed: 14 July 2018). Grant, W.B. Karras, S.N. Bischoff-Ferrari, H.A. et al. (2016). ‘Do studies reporting ‘U’-shaped serum 25-hydroxyvitamin D–health outcome relationships reflect adverse effects?’, Dermato-Endocrinology, 8(1), e1187349. Hunter, J.O. (2005). ‘Food elimination in IBS: the case for IgG testing remains doubtful’, Gut, 54(8), pp.1203. Hyppönen, E., Sovio, U. Wjst, M. et al. (2004). ‘Infant vitamin d supplementation and allergic conditions in adulthood: northern Finland birth cohort 1966’, Immunology of Diabetes III, 1037(1), pp.84-95. Kempson, I.M. & Lombi, E. (2011). ‘Hair analysis as a biomonitor for toxicology, disease and health status’, Chemical Society Reviews, 40(7), pp.3915–3940. Lopes Marques, C.(2010). ‘The Importance of vitamin D levels in autoimmune disease’, Brazilian Journal of Rheumatology, 50(1), pp.67–80. Mikulewicz, M. Chojnacka, K. Gedrange, T. et al. (2013). ‘Reference values of elements in human hair: A systematic review’, Environmental Toxicology and Pharmacology, 36(3), pp.1077–1086.


References Namkoong, S. Hong, S.P. Kim, M.H.et al. (2013). ‘Reliability on intra-laboratory and inter-laboratory data of hair mineral analysis comparing with blood analysis’, Annals of Dermatology, 25(1), pp.67–72. National Institute of Care and Excellence. (2015). Lipid modification - CVD prevention. [Online]. Available at: http://cks.nice.org.uk/lipid-modification-cvd-prevention#!backgroundsub (Accessed: 14 July 2018). James, L.K. & Till, S.J. (2016). ‘Potential Mechanisms for IgG4 Inhibition of Immediate Hypersensitivity Reactions’, Current Allergy and Asthma Reports, 16(3), pp.1–7. Jones, S.M. Pons, L. Roberts, J.L. et al. (2009). ‘NIH Public Access’, Journal of Allergy and Clinical Immunology, 124(2), pp.1–20. Royal College of Pathologists of Australasia (2009) Pathology Tests. [Online]. Available at: https://www.rcpa.edu.au/Library/Practising-Pathology/RCPA-Manual/Items/Pathology-Tests (Accessed: 9 September 2009). Sarris & Wardle. (2010) Clinical Naturopathy: an evidence based guide to practice. Sydney: Elsevier. Savilahti, E.M. Rantanen, V. Lin, J.S. et al. (2009). ‘Early recovery from cow's milk allergy is associated with decreasing IgE and increasing IgG4 binding to cow's milk epitopes’, Journal of Allergy & Clinical Immunology, 125(6), pp.1315–1321. Skripak, J.M. Nash, S.D. Rowley, H. (2008). ‘A randomized, double-blind, placebo-controlled study of milk oral immunotherapy for cow’s milk allergy’, Journal of Allergy and Clinical Immunology, 122(6), pp.1154–1160. Terrier, B. Derian, N. Schoindre, Y. (2012). ‘Restoration of regulatory and effector T cell balance and B cell homeostasis in systemic lupus erythematosus patients through vitamin D supplementation’, Arthritis Research & Therapy, 14(5), R221. Tomicic, S. Norrman, G. Filth-Magnusson, K. (2009). ‘High levels of IgG4 antibodies to foods during infancy are associated with tolerance to corresponding foods later in life’, Pediatric Allergy and Immunology, 20(1), pp.35–41. Top End Sports (2009) Fitness testing. [Online]. Available at http://www.topendsports.com/testing/tests/skinfolds.htm (Accessed: 23 September 2009). WHO. (2008). Waist circumference and waist-hip ratio. [Online]. Available at: http://apps.who.int/iris/bitstream/10665/44583/1/9789241501491_eng.pdf (Accessed: 9 September 2009). Weatherby, D. & Ferguson, S. (2002). Blood chemistry and CBC analysis – Clinical laboratory testing from a functional perspective. USA: Bear Mountain Publishing. Yu, L.C.H. (2012). ‘Intestinal Epithelial Barrier Dysfunction in Food Hypersensitivity’, Journal of Allergy, 2012(596081), pp.1–11.


Further Reading: Research On Hair Minerals

• In 1985, a study in JAMA looked at hair samples from 2 healthy individuals to 13 different laboratories.

• The results showed significant inconsistencies in reference ranges, levels of minerals and toxic elements present in the sample, interpretation of these, and recommendations for supplements/doses.

• The study criticised the lack of standardisation amongst these tests (Barrett, 1985)


How Have Things Changed?

• There have been improvements in standardisation but there have still been issues noted.

• In 2013 a meta analysis was published looking at reference ranges based on population data from several studies. 5 studies were included in the final review after filtering.

• They found that whilst there was a stronger weight of evidence than in the past, the clinical utility of hair mineral analysis was still limited by the lack of: - Standardisation of sample preparation (size, location, washing methods etc) - Standardisation of technology used to analyse - Standardisation of Certified Reference Materials (CRM) used by labs in the

absence of clear reference ranges.

• Read full text here: (Mikulewicz et al. 2013) © CNM: Nutrition Year 2 – Assessment & Diagnostics/ Functional Testing. EB.

Intra-Lab Variation

• Most criticisms centre around variations between different laboratories and limitations include the technology used and sample preparation requirements (size, location etc) as well as reference range.

• In 2013, split samples were sent from one healthy volunteer to 3 different labs. Split sampling means that the same samples were sent twice, as though they were two different patients.

• All 3 labs used the same quantitative methods. • The results were compared against serum tests. • 2 of the 3 laboratories showed replicable results with both samples. • 1 of the laboratories showed consistent correlations between blood test and hair

tests, but the other did not.


Discrepancy In Results

• One laboratory (B) found that in one sample there was an inconsistency in mineral identification between the two samples they were sent.

• One showed low zinc and the other, low phosphorus. • This suggests that this laboratory specifically, may have poorer quality control

standards. • Due to the small sample size (one) it’s not known if this was an anomaly or

something which would be repeated.


Blood vs. Hair

• Although Laboratory A showed consistency with the split samples, it did not show complete correlation with blood test analytes.

• Laboratory B showed the least reliable results when compared with blood tests. • This suggests that the reference ranges these two labs are using may not be

appropriate. To Read the Study in Full: (Namkoong et al. 2013)


Further Reading Bass, D.A. Hickock, D. Quig, D. and Urek, K. (2001). ‘Trace element analysis in hair: factors determining accuracy, precision, and reliability’. Alternative Medicine Reviews. 6(5), pp.472–481. Kempson, I.M. and Lombi, E. (2011). ‘Hair analysis as a biomonitor for toxicology, disease and health status’. Chemical Society Reviews, 40(7), pp.3915–3940. Watts, D.L. and Ph, D. (n.d.). Hair Tissue Mineral Analysis : Fact and Fiction. Director. Available at: http://www.nutritionalbalancing.org/center/htma/science/_p/_file_dwnld.php?index=8. (Accessed: 14th July 2018).


naturopathic nutrition year 2 lecture 3

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