What are organic acids? What do they tell us about our metabolic processes? What can we do with that information?

1. Building the Organix Profile Into Clinical Practice An educational module for users of the Metametrix Urinary Organic Acid (Organix) Profile Welcome to “Building the Organix into Clincal Practice”.

11. Stages of Energy Extraction From Food Food CO 2 + H 2 O + Urea In a very healthy state, the body operates in a similar fashion to a finely tuned vehicle. Pure air and food in: Pure carbon dioxide, water, and urea out. © 2005 Metametrix Clinical Laboratory, Inc. This chart shows the pathways of the cellular “engine” of the body. Macronutrients from food serve as the raw fuel – carbohydrate, fat, and protein energy resources that the cell uses to build ATP. In a sub-optimal state, the “engine” of the body may not run so cleanly. Kinks in metabolic pathways can lead to accumulations of various biochemical intermediates. Several of these GLYCOLYTIC and KREB’S CYCLE intermediates (in red) may be high on an Organix Test when the cell metabolism isn’t running smoothly. Succinate a-Ketoglutarate Fumarate Malate Isocitrate cis-Aconitate Citrate Pyruvate Lactate = step requiring vitamin cofactor (shaded areas)

12. C D Functional Indicators of Nutrient Deficiency A B B E 1 Most of the analytes found on the Organix operate with the following concept in mind: they are biochemical intermediates that accumulate as a result of some form of deficiency. This example shows a simple reaction, starting with SUBSTRATE “A”. The end PRODUCT, later indicated as “D” is the result of several connected intermediate biochemical reactions that use COENZYMES derived from VITAMINs or MINERAL COFACTORS – essential components of a successful reaction. Be mindful of the green, yellow, and red lights – as they indicate the likely progress of a chemical reaction. The presence (or absence) of enzyme cofactors and coenzymes may determine the rate at which a given enzyme works – and the color of the light. The enzyme (in this case E1) is a critical component of any relevant biochemical reaction represented by the Organix test. Some enzymes need no cofactors to ensure a smooth reaction (such as this case, where E1 converts “A” to “B”). © 2005 Metametrix Clinical Laboratory, Inc. Or, an enzyme may rely on the assistance of a vitamin or mineral to make a biochemical reaction proceed. In the case of E2, a VITAMIN coenzyme is required. Let us suppose that INTERMEDIATE “B” is an analyte found in the Organix test, much like METHYLMALONATE, as cited in earlier examples. Vitamin B12 is an important cofactor that helps the intermediate, METHYLMALONATE, change into another biochemical – one that can eventually be recycled and reused. Little METHYLMALONATE will appear in the urine when B12 levels are healthy and sufficient. The reaction from “C” to “D” requires the presence of an enzyme with a MINERAL cofactor. Without this cofactor, the reaction would proceed very slowly, or not proceed at all. A deficiency state of vitamin B12 would slow the forward progress of the “B” to “C” reaction considerably. Leaving “B” to accumulate. Soon, this excess would appear excreted in the urine, and show as an elevation of “C” (METHYLMALONATE) on the Organix Test. Observe the following brief animation… An even greater deficit of a vitamin cofactor (in this example, B12) would further slow the progress of this series of reactions. Urine C D C D B A Intermediary metabolite E n Enzyme Coenzyme (derived from vitamins) Cofactor (mineral ion) E 2 E 3

14. 6 Step Organix Evaluation Antioxidants Detoxification Dysbiosis B-Complex Vitamins Cellular Metabolism Neurotransmitters (Middle Page 1) (Bottom Page 1) (Top Page 2) (Middle Page 2) (Middle Page 2) (Bottom Page 2) © 2005 Metametrix Clinical Laboratory, Inc.

17. Carnitine resides in the mitochondrial membrane. It serves as an intermembrane transport facilitator for fatty acids. Fatty acid Metabolism markers in the Organix give us clues about how the body utilizes fatty acids. Carnitine Fattyacylcarnitine CO 2 + H 2 O + ATP When carnitine is abundant inside the mitochondrial membrane, fatty acids will bind to it – forming fattyacylcarnitine. In turn, carnitine will ‘flip’ the fatty acid through the membrane to the inside of the mitochondria. Here, abundant enzymes reside, capable of oxidizing fat into ATP, carbon dioxide, and water. Should carnitine be suboptimal, fatty acid transport into the mitochondria would slow – forcing unused fatty acids to migrate to other cellular systems for oxidation. One such alternative can be found inside the PEROXISOME. When inside the PEROXISOME, fatty acids undergo  -oxidation, leaving behind products seen in the Organix profile. Elevations seen in Adipate, Suberate, or Ethylmalonate will indicate a functional deficiency of CARNITINE. © 2005 Metametrix Clinical Laboratory, Inc. Effects of Carnitine Insufficiency ß-Oxidation

18. Summary Step 1(a) To summarize Step 1, Part (a): High levels of Adipate, Suberate, and Ethylmalonate suggest CARNITINE deficiency. © 2005 Metametrix Clinical Laboratory, Inc.

19. Lipoic ? Carbohydrate Metabolism markers include Pyruvate and Lactate – both end-products of GLYCOLYSIS. The remaining variable, beta-hydroxybutyrate, is a ketone body that provides additional information on glucose tolerance. When the pyruvate marker is high, it is important to quickly scan further down the first page to see if alpha-ketoglutarate, alpha-ketoisovalerate, alpha-ketoisocaproate, and alpha-keto-beta-methylvalerate are also high. All of these analytes share a common enzyme complex – all relying on the same coenzymes: Vitamin B1, B5, lipoic acid, B2, and B3 (in descending order of significance). When both Pyruvate and Lactate are elevated in the Organix report, suspect Lipoic Acid deficiency – as a lipoic acid deficiency will divert glycolytic end-products away from the Kreb’s Cycle and accelerate anaerobic metabolism (producing high levels of Lactic Acid) © 2005 Metametrix Clinical Laboratory, Inc. B 1 B 5 B 2 ? B 3 ? Lactate high too?

20. Lactate elevations, alone (meaning, not in combination with elevated Pyruvate) suggests CoQ10 deficiency. Further corroborative evidence can be found by quickly comparing values for Succinate, Fumarate, Malate, and Hydroxymethylglutarate. Coexisting elevations (especially Succinate and Malate) confirm suspicions of a functional CoQ10 deficiency. © 2005 Metametrix Clinical Laboratory, Inc. Q10? Lactate high too? Lipoic ?

22. Lipoic Summary Step 1(b) When lactate is high, suspect CoQ10 deficiency. Compare with Succinate, Fumarate, Malate, and Hydroxymethylglutarate levels. This patient has sufficiently high levels of both Pyruvate and Lactate to suggest insulin resistance. Specifically, lipoic acid treatment is ideal for this circumstance. © 2005 Metametrix Clinical Laboratory, Inc. Q10?

25. Energy Energy Mitochondrial energy production suffers. Coenzyme Q10 facilitates the creation of ATP in the mitochondria. It is a pivotal component of the electron transport chain that interacts very closely with Succinate, Fumarate, and Malate. In the event of a CoQ10 deficiency, a “bottleneck” will surface in the electron transport chain – interrupting the smooth flow of electrons, and energy, through the mitochondria. Succinate, Fumarate, and Malate will be unable to transfer electrons. With impaired mitochondrial function, diminished capacity for oxidative phophorylation forces a diversion of the glycolytic pathway to greater anaerobic energy production – and an excess of Lactic Acid production. Lactate levels rise. Healthy levels of CoQ10 permit the fluid transfer of electrons from one successive cytochrome to another along the electron transport chain. At each cytochrome junction, energy is released and trapped in a newly formed molecule of ATP. These Kreb’s Cycle intermediates will build-up, eventually to be removed in the urine. © 2005 Metametrix Clinical Laboratory, Inc. Hydroxymethylglutarate (HMG) is commonly associated with cholesterol production. It is a building block of cholesterol whose activity is targeted by STATIN medications (AKA HMG CoA Reductase inhibitors). Statins slow the progress of cholesterol manufacture by slowing the conversion of HMG into further building blocks for cholesterol. HMG is also a building block for CoQ10, and may be equally effected by STATINS. Elevated HMG should prompt the clinician to ask about STATIN treatment, and suspect slowed production of CoQ10. Energy Energy Elevated? CoQ 10 deficiency

26. Summary Step 1(c) Although only one of the three indicators for ammonia toxicity is high in this patient, it may be worthwhile to look further ahead in the Organix profile to see if there is evidence of either UREA CYCLE impairment or DYSBIOSIS. These topics will be covered in later sections. There is strong evidence of CoQ10 deficiency in this patient – a likely contributor to her complaints of chronic fatigue… … punctuated by the alarming elevation in Lactate. © 2005 Metametrix Clinical Laboratory, Inc. Ammonia Toxicity Elevated? CoQ 10 deficiency

28. 1 of 2 markers for B 6 deficiency All use similar dehydrogenase enzyme complex Branched-Chain Amino Acids (BCAAs) are typically broken down to provide energy or they may assist as building blocks for amino acids needed elsewhere in the body. Human muscle tissue contains a massive reservoire of BCAAs – always available to serve metabolic needs in other areas of the body. Interim products of BCAA breakdown can be measured to reflect the presence (or deficiency) of vitamin coenzymes. With the assistance of Vitamin B1, B5, lipoic acid, B2, and B3, Valine will produce the indermediary, alpha-ketoisovalerate… … Leucine will produce alpha-ketoisocaproate… … and Isoleucine will produce alph-keto-beta-methylbutyrate. The enzyme complex used to break down BCAAs, as you may recall, use the same coenzymes as two previously-discussed analytes: Pyruvate and alpha-ketoglutarate. In fact, the enzymes are all very similar in each case. A build-uup of any of these 5 analytes is evidence of a deficiency of these coenzymes. © 2005 Metametrix Clinical Laboratory, Inc. The first of two Vitamin B6 markers is Xanthurenate. High levels of Xanthurenate provides the strongest evidence of the two markers that a B6 deficiency exists. Already discussed B 1 B 5 B 2 ? B 3 ? Lipoic ? Isoleucine Leucine Valine Branched-chain Amino Acids

30. Biotin? Summary Step 2(a) This patient does not appear to have any B-Complex Vitamin deficiencies. Biotin status, although close to being high, is still within the normal range. © 2005 Metametrix Clinical Laboratory, Inc.  -hydroxyisovalerate

31. B 12 Megaloblastic Marrow Methylmalonate Methylmalonic aciduria Macrocytosis Low RBC B 12 Hypersegmentation Low Serum B 12 Anemia Still another amino acid catabolite, Methylmalonate relies on Vitamin B12 for it to be recycled as succinate. In the absence of B12, Methylmalonate cannot be converted into Succinate, and builds up. The more deficient the B12 status, the greater the elevation of Methylmalonate. Most signs and symptoms of Vitamin B12 deficiency require lengthy periods of deficiency to manifest. Megaloblastic anemia takes over 4 months of B12 deficiency before becoming evident. Methylmalonate is the earliest known factor to show B12 deficiency – appearing only 10 days after deficiency. © 2005 Metametrix Clinical Laboratory, Inc. Valine B 12 deficiency - Before Anemia Succinate 10 22 49 days 123 days 127 days 134 days 137 days

32. Folate Required in FIGLU Metabolism Folate Formiminoglutamate (or FIGLU) is a biochemical intermediate of Histidine metabolism. Degradation of Histidine relies on assistance from FOLATE so that it may be converted to Glutamate © 2005 Metametrix Clinical Laboratory, Inc. In the absence of Folate, FIGLU will accumulate and appear in the urine. The higher the value of FIGLU, the more severe the functional folate deficit.

33. Folate Required in FIGLU Metabolism Summary Step 2(b) To summarize the “Methylation Cofactor Markers” from section 2: The patient has no apparent deficiencies of either Vitamin B12, or Folate. © 2005 Metametrix Clinical Laboratory, Inc.

37. 80% 20% Aldehyde reductase +MAO VMA + COMT Urinary Epi/NE Dopamine Serotonin HVA 5-HIA Neurotransmitters, once manufactured, are bound in vescicles. Upon stimulation of a presynaptic nerve fiber, the vesicles are sent into motion – bound for the synapse. Like so… Upon release, most of the neurotransmitter – rather than signaling the postsynaptic nerve – 80% actually gets reabsorbed (known also as reuptake) into the presynaptic nerve. This is a self-regulating mechanism to ensure the nerve doesn’t “overshoot” its intended stimulus. In effect, reuptake sends a message – turning “off” the nerve quickly after the neurotransmitter is released into the synaptic gap. Just enough to signal the next nerve… Dopamine, when degraded, leaves homovanillate (HVA) to appear in the urine. When Epinephrine or Norepinephrine are catabolized, they appear as Vanilmandelate in the urine (VMA) Serotonin will break down to produce 5-hydroxyidoleacetate. When the signal transfer is complete, a portion of synaptic neurotransmitter and some of the reuptaken neurotransmitter is subject to enzymatic degradation. A series of enzymes will successively convert neurotransmitters into the products we see in the organix profile… © 2005 Metametrix Clinical Laboratory, Inc. Neurotransmitter Function Signal, Re-uptake, and Disposal of Neurotransmitters

38. SSRIs Aldehyde reductase +MAO 5-HIA + COMT Urinary Serotonin 5-HIA Serotonin Patients on antidepressant therapy can significantly alter Organix neurotransmitter marker output. It is important to understand the mechanisms that underlie these changes, because they can also explain non-pharmaceutically induced changes in these Organix analytes. Here is how selective serotonin reuptake inhibitors (SSRIs) work… SSRIs block neurotransmitter reuptake of serotonin… With reuptake impaired, feedback inhibition of neurotransmitter release is no longer an effective means of slowing serotonin output… The synaptic compartment is flooded with excess neurotransmitter, with even more on the way. © 2005 Metametrix Clinical Laboratory, Inc. The inevitable consequence of reuptake inhibition is a higher rate of neurotransmitter wasting. Neurotransmitter cannot be recycled, and is alternatively subject to degradation – appearing on the Organix test as elevated 5-HIA. The inevitable consequence of reuptake inhibition is a higher rate of neurotransmitter wasting. Neurotransmitter cannot be recycled, and is alternatively subject to degradation – appearing on the Organix test as elevated 5-HIA. Antidepressant Effects: Selective Serotonin Re-uptake Inhibitors

39. Aldehyde reductase +MAO + COMT Urinary? HVA + VMA + 5-HIA MAOIs Epi/NE Dopamine Serotonin Monoamine Oxidase Inhibitors (MAOIs) work quite differently than SSRIs, yet continue to influence Organix results. MAOIs slow the degradation of neurotransmitters. In effect, keeping neurotransmitters around the synapse for longer periods of time. Here is how MAOIs work… Monoamine Oxidase (MAO) activity is inhibited by MAOIs, slowing the breakdown of the neurotransmitter. Monoamine Oxidase (MAO), as an enzyme works on both adrenergic and serotonergic neurotransmitters. Any changes in MAO activity, therefore, may effect one or all of the neurotransmitters covered on the Organix test. Monoamine Oxidase (MAO), as an enzyme works on both adrenergic and serotonergic neurotransmitters. Any changes in MAO activity, therefore, may effect one or all of the neurotransmitters covered on the Organix test. As MAOIs take effect, urinary excretion of VMA, HVA, and 5-HIA diminish. The life of the neurotransmitter is extended, sustaining a larger, more prolonged stimulus on the postsynaptic nerve. © 2005 Metametrix Clinical Laboratory, Inc. Antidepressant Effects: Monoamine Oxidase Inhibitors (MAOIs)

40. Urinary? Now that you have a basic understanding of nerve function and neurotransmitter regulation, perhaps the neurotransmitter analytes on the Organix provide greater insight into the neurological health of your patient. © 2005 Metametrix Clinical Laboratory, Inc.

46. NIACIN (Only in the liver) Tissue-Specific Kynurenine Pathway Effects In the liver, the Kynurenine pathway has one additional step. An added enzyme is available in hepatocytes that allows quinolinate to be converted to NIACIN (B3)! © 2005 Metametrix Clinical Laboratory, Inc. L-Tryptophan Kynurenine 3-Hydroxykynurenine Quinolinate B 6

47. NIACIN (Only in the liver) Tissue-Specific Kynurenine Pathway Effects L-Tryptophan Kynurenine 3-Hydroxykynurenine Quinolinate B 6 Kynurenate Xanthurenate Quinolinate B 6 Urinary Kynurenate Xanthurenate In the liver, the Kynurenine pathway has one additional step. An added enzyme is available in hepatocytes that allows quinolinate to be converted to NIACIN (B3)! The forward progress of this reaction requires Vitamin B6. In the event of a B6 deficiency, a buildup of Kynurenine and 3-Hydroxykynurenine intermediates will result. The Kynurenine pathway gets diverted. Xanthurenate and Kynurenate are products of this diverted pathway – and they are detected on the Organix test when excreted. © 2005 Metametrix Clinical Laboratory, Inc. deficiency B 6

48. Summary Step 3 Elevated Vanilmandelate suggests the patient may be stressed. Sympathetic stimulation would causes a higher use and turnover of epinephrine and norepinephrine. High 5-HIA indicates the patient may have already been prescribed an anti-depressant for her mental state. These results imply that the clinician inquire as to whether she is on anti-depressants, or is supplementing with 5-HTP. © 2005 Metametrix Clinical Laboratory, Inc. We know patient is depressed. Is she on anti-depressants? 5-HTP supps? STRESSED?

51. Summary Step 4 The patient does not show any particular deficits in terms of her antioxidant status. With her 8-OHDG in the upper 4 th quintile, it may be beneficial to suggest a moderate-dose multi antioxidant formula – just to be on the safe side. © 2005 Metametrix Clinical Laboratory, Inc. Multi Antioxidant formula – just to be safe?

55. PI PII Conjugation & Elimination Glucose Glucarate is a remnant of Glucuronic Acid production. This analyte tells two stories. It gives us details about Phase I Liver detoxification and about Phase II Liver Glucuronic Acid conjugation. Glucarate represents both accelerated Phase I activity… When the demand for Glucuronidation is high, the body constructs Glucuronic acid from Glucose. … and slow Phase II Glucuronidation status. This process requires intervention from Phase I, leaving Glucarate behind as a remnant. When glucarate is high, consider taking measures to slow Phase I Detoxification. Keep in mind: the purpose of Phase I is to activate toxins for entry into Phase II detoxification. Elevated Glucarate is telling us that Phase I is highly active, and that Phase II Glucuronidation is likely slow. An overactive Phase I produces highly reactive, carcinogenic and oxidative intermediates that can potentially cause damage to surrounding tissues if not immediately admitted into Phase II detoxification. © 2005 Metametrix Clinical Laboratory, Inc. Glucarate (remnant) Glucuronic Acid

61. © 2005 Metametrix Clinical Laboratory, Inc. Dysbiosis Markers help the clinician to understand whether the gut may factor into the condition of the patient. The markers identify dysbiosis from bacterial and fungal sources. The markers, by and large, do not represent individual microorganisms, but chemicals created by organisms in the small bowel. These chemicals are absorbed and subsequently discovered in the urine. Benzoate, Hippurate, Phenylacetate, and Phenylpropionate are all derived from gut transformation of the amino acid, Phenylalanine. Benzoate may appear elevated when foods preserved with benzoate are consumed in quantity. For example, cranberries, pickles, and lunch meats all contain benzoate. Bacterial action on Tyrosine will cause para-Hydroxybenzoate and para-Hydroxyphenylacetate to appear elevated in the urine. Tryptophan is converted to Indole in the gut – and subsequently excreted as Indican. Carbohydrates can be converted by bacteria to for Tricarballylate. Candida in the small intestine will consume simple sugars, yielding D-Arabinitol as a byproduct. Giardia has been implicated among a number of potential species of bacteria the produce p-Hydroxyphenylacetate. Generally, if most other bacterial dysbiosis markers are low and p-Hydroxyphenylacetate alone is high, consider treating for a Giardia infection. Studies have shown that the probiotic species, Saccromyces boulardii can effectively treat Clostridial infections. High readings for Dihydroxyphenylproprionate calls for S. boulardii. Candida yeast species overgrowth is indicated when the analyte, D-Arabinitol has high readings. Dysbiosis Markers Tyr Phe Trp CHO Giardia? Cranberries? Pickles? Luncheon meats? S. boulardii Candida sp.

63. One final facet of the dysbiosis panel, in fact, has to do with probiotics. Few realize that much Lactobacillus supplementation can cause D-Lactate to accumulate – demonstrating an overgrowth of these beneficial bacteria. Jejunoileostomy patients have the highest risk of developing D-lactic acidosis and accompanying encephalopathy. Elevated D-lactate may serve as a warning to patients to limit intake of Lactobacillus supplements – and also to limit carbohydrate consumption – a food source for Lactobacillus. © 2005 Metametrix Clinical Laboratory, Inc. Too much Lactobacillus? CHO Probiotics

64. Summary Step 6(b) There is no apparent concern about overpopulated L. acidophillus with this patient. © 2005 Metametrix Clinical Laboratory, Inc. CHO Probiotics

65. © 2005 Metametrix Clinical Laboratory, Inc. 6 Step Organix Test A quick overview of the Organix Test Result and our case of a woman with depression, chronic fatigue, insomnia, digestive bloating, and tiredness around 3pm. Carnitine and CoQ10 markers suggest a Q10 deficiency – which might explain her fatigue. Some evidence also exists for ammonia toxicity. Although, with only one ammonia toxicity marker high, that is uncertain. B-Complex markers did not show any B-vitamin deficiencies. There were some obvious concerns posed by elevations in 5-HIA. Ask the patient if she is currently taking antidepressants or supplemental Tryptophan or 5-HTP. Any of these will cause 5-HIA to rise above normal. High Vanilmandelate may be the result of stress. There is very little evidence to suggest any problem with oxidative stress. Regardless, a reading for 8-OHDG in the upper 4 th quintile might suggest some prophylactic antioxidant supplementation. The Dysbiosis profile suggest the patient take supplemental Glycine to reduce Benzoate toxicity. Elevated Dihyroxyphenylpropionate indicate a small intestinal Clostridial infection, which can be treated using S. boulardii probiotics. Detox markers indicate a surge of Glutathione synthesis. Low sulfate suggests that there may have been a recent glut of available sulfur compounds (like cysteine and methionine) to build Glutathione, causing urinary sulfur to plummet. Dietary sulfate may have solved the problem for this patient… for now. NAC or Vitamin C treatment is advised. 1(a) Carnitine (?) 1(b) Lipoic, Q10 (?) 1(c) NH3 (?) Q10 2(a) Biotin (?) 2(b) Folate (?) 3 Stress (?) SSRI (?) 5-HTTP (?) 4 Multi A/Ox 5 GSH, NAC. 6 Glycine, S. boulardii

66. Guide to Supplementation from Organix™ Profile At the end of every Organix report, a comprehensive list of supplement suggestions is provided for the doctor to pass on to the patient, or to submit to a compounding pharmacist for formulation. These recommendations are tailored to the specific needs of the patient. © 2005 Metametrix Clinical Laboratory, Inc.

67. Guide to Supplementation from Organix™ Profile © 2005 Metametrix Clinical Laboratory, Inc.