Presentation on theme: "Using the Organix, Organic Acids Testing, in Clinical Practice"— Presentation transcript:
1Using the Organix, Organic Acids Testing, in Clinical Practice Dr. Rachel MarynowskiUnlike amino acids and fatty acids, organic acids are not essential nutrients, but the products of metabolism. Abnormal concentrations of organic acids in the urine, or organic acidurias provide a functional marker for the metabolic effects of micronutrient inadequacies, genetotrophic variations, impaired enzyme function, toxic exposure, neuroendocrine activity, and intestinal bacterial overgrowth. As such, organic acid testing can indicate the functional need for essential or conditionally essential nutrients, diet modification, antioxidant protection, detoxification support and other therapies.
2Stages of Energy Extraction From Food Carbohydrates, fats, and protein from the food we eat serve as energy resources used by our cells to build ATP. In the event we are not eating high quality, nutrient dense foods, are not absorbing and assimilating these nutrients or there is compromise to cellular function, cellular cycles, such as glycolysis and the krebs cycle, do not occur at an optimal rate.In a sub-optimal state, the body kicks out accumulated metabolites which are then analyzed on the Comprehensive Organix Profile. This serves tremendous value in a clinical setting for many reasons, most importantly as a tool to aid practitioners in practicing comprehensive, individualized medicine wherein every patient’s biochemistry is given the consideration it deserves.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.CO2 + H2O + Urea
3The Organix Profile Looks at: Actual values –Examples include analytes such as lactate, sulfate etc.Functional markers –These include analytes such as FIGLU, 5HIA
5General Breakdown Mitochondrial Function and Assessment B Complex Vitamin MarkersMethylation Pathway MarkersNeurotransmitters Metabolism MarkersOxidative Damage and Antioxidant MarkersDetoxification MarkersIntestinal Dysbiosis MarkersOn the first page of a set of results, there is a general breakdown of the analytes measured according to the affiliated pathway or system in the body.- this is in efforts to break the information down into more clinican friendly sections.They include: (read 1-7)
6Evaluation: Step 1 Mitochondrial Function and Assessment Fatty Acid MetabolismCarbohydrate MetabolismEnergy Production (Citric Acid Cycle)
8Adipate, Suberate, and Ethylmalonate are measured to indentify fatty acid metabolism efficiency. Carnitine resides in the mitochondrial membrane. It serves as an intermembrane transport vehicle, shuttling fatty acids into the mitochondria in the form of fatty-acylcarnitine. Here, abundant enzymes reside, capable of oxidizing the fatty acids into ATP, carbon dioxide, and water and therefore, not causing any accumulation or spill over of the aforementioned analytes.If fatty acids are unable to enter the mitochondria, they enter PEROXISOMES instead, undergo oxidation, and leave behind products seen in the Organix profile.Elevations seen in Adipate, Suberate, or Ethylmalonate will indicate a functional deficiency of CARNITINE. Single elevations such as adipate alone may indicate exogenous ingestion of adipate which is found in gelling and flavoring agents.Fatty acid Metabolism markers in the Organix give us clues about how the body utilizes fatty acids
9If Pyruvate is elevated consider: B-vitamins: B1 and B5 Lipoic Acid Under-eatingIf Lactate is elevated consider:CoQ10BiotinLipoic AcidExcess Alcohol intakeCarb metabolism involves glycolysis otherwise known as the breakdown of glucose into pyruvate. The energy liberated in this conversion process leads to the production of ATP… the oh-so-important energy substrate.I took this diagram fromI made a slight change from theirs.
10Requirements…Pyruvate dehydrogenase requires B-vitamin cofactors, thus an elevated pyruvate may be an indicator of a need for B-vitamins Naito et. Al., 1998 Eur J PediatricsCo-enzyme Q10 supplementation has been found to be effective when lactate is elevated.- Sobreira, et. Al., 1997 Neurology
11Pyruvate Dehydrogenase Complex The enzyme complex has multiple copies of the E1, 2, 3 subunits. Each subunit is bound by a different nutrient. Similar dehydrogenase complexes are used for alpha-ketogluerate and the BCAA’s keto acids.The three forms differ in amino acid sequence for E1 and E3, but are simliar for E2. Thus genetic polymorphisms of E2 will manifest metabolic interferences in all the complexes.
12High levels may suggest : Low carbohydrate diet Diabetic ketoacidosis Reduced caloric consumption or Wasting diseases.Insulin resistance (also look at high lactate/pyruvate)beta-hydroxybutyrate is a ketone body formed when fatty acids are broken down to meet energy demands.High levels may suggest :Low carbohydrate dietDiabetic ketoacidosisReduced caloric consumption or Wasting diseases.Insulin resistance (also indicated with high lactate/pyruvate) as seen in metabolic syndrome, for example
13When Citrate, Cis-Aconitase and Isocitrate are elevated it may indicate increased ammonia load in the body.Ammonia toxicity can come from two probable sources:A slow urea cycle (removes ammonia safely)Dysbiosis (introduces ammonia produced by microbes in the gut)
14Citrate, Isocitrate, Cis-Aconitase (Excreted in the urine)BufferCitrate3-(acid)NH4CitrateIsocitrateCis-aconitaseWhen the Urea Cycle no longer has sufficient resources to fully remove ammonia, whatever remains will be eliminated through the kidney…Eliminating “positively” charged AMMONIA is a process facilitated by co-transport with “negatively” charged Citrate, Cis-aconitate, or Isocitrate.Generally you also find Oratate also elevated…If all 3 HIGH, check OROTATE for further assessment of proper ammonia disposal via the liverIf OROTATE ok, look to the organic acid markers of upper bowel bacterial overgrowth…. This helps the overseeing clinician to navigate treatment by determining root causeNH4(Ammonia)Proximal Tubular Cell
15Alpha-ketoglutarate also has a dehydrogenase enzyme and can become elevated when there is a: need for B vitaminswhen there is a mitochondrial need for CoQ10if patient is undereatingalso become elevated with a need for CoQ10.With low levels on many of the Krebs Cycle markers, there is most likely insufficient AA status and/or a failure to refill the krebs cycle intermediates
16Healthy 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.In the event of a CoQ10 deficiency, a “bottleneck” will surface in the electron transport chain and energy, through the mitochondria. Succinate, Fumarate, and Malate will be unable to transfer electrons and then begin to accumulate in the system… hence spilling over in the urine, leading to detection through this profile.With impaired mitochondrial function, diminished capacity for oxidative phosphorylation forces a diversion of the glycolytic pathway to greater anaerobic energy production – and an excess of Lactic Acid production occurs. Make sure to take a peek at Lactate levels which will typically rise with this pattern.These Kreb’s Cycle intermediates will build-up, eventually to be removed in the urine.Elevated?Need for CoQ10
17Statins Hydroxymethylglutarate (HMG) Mevalonic Acid Cholesterol CoQ10 Hydroxymethylglutarate (HMG) is associated with the cholesterol synthesis pathway. It is a building block of cholesterol. It is also the key target for STATIN medications (which are HMG CoA Reductase inhibitors). Statins slow the progress of cholesterol synthesis 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….and the reason why patients on statins should also be on CoQ10 support.
18Included in interpretive guide that accompanies test results… also on our website
19Evaluation Step 2 Mitochondrial Function and Assessment B Complex Vitamin MarkersThiamin (B1)Riboflavin (B2)Niacin (B3)Pantothenic acid (B5)Pyridoxine (B6)Lipoic AcidBiotin
20B-Complex (B1, B2, B3, B5)Branched-Chain Amino Acids (BCAAs) are typically broken down to provide energy or are used as building blocks for amino acids needed elsewhere in the body. Human muscle tissue contains a massive reservoir of BCAAs – always available to serve our metabolic needs. Interim products of BCAA breakdown can be measured to reflect the presence (or deficiency) of vitamin coenzymes.The enzyme complex used to break down BCAAs, utilizes the same coenzymes as two previously-discussed analytes: Pyruvate and alpha-ketoglutarate. In fact, the enzymes are all very similar in each case. So an accumulation of any of these 5 analytes under the B complex section, is evidence of a deficiency of these coenzymes.More specfically, if any of the alpha-keto’s are elevated, the coenzymes above are deficient (a-keto’s are supposed to go on to form CO2 & water). This is where supplementation becomes the most appropriate therapy and often times the patient responds very well to a nice B complex product.P. 342 in textbook
21Each of these uses a similar dehydrogenase enzyme to move on… Pyruvatea-ketoglutaratea-ketoisovaleratea-ketoisocaproatea-keto-β-methylvalerateThe dehydrogenase enzyme requires vitamins:B1, B5, B2, B3 and lipoic acid
22Vitamin B6 (Pyridoxine) The 2nd to last analyte within the B vitamin section… XANTHURENATEIt’s a product of tryptophan catabolism as you can see here… in fact an L-tryptophan challenge test is utilized to evaluate xanthurenate or need for pyridoxineHigh levels of Xanthurenate provides solid evidence that a B6 deficiency exists. (Docs use pyridoxine (100mg/d) and or pyridoxine-5-phosphate (P5P: 30mg/d)
23The DRI for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline ( IOM)“One of the earliest markers for B6 deficiency was the urinary excretion of xanthurenic acid, which is normally a minor tryptophan catabolite. The major pathway of tryptophan catabolism proceeds via the PLP-dependent kynureninase reaction.” (Shane and Contractor, 1980)Indicators of vitamin B6 status have traditionally been described as direct (vitamin concentrations in plasma, blood cells, or urine), indirect, or as functional (erythrocyte aminotransferase saturation by pyridoxal 5'-phosphate [PLP] or tryptophan metabolites). In most instances, the concentrations of these indicators change with increases or decreases in vitamin intake. As such, they are useful indicators of relative B6 status, especially in controlled depletion-repletion studies (Leklem, 1990; Sauberlich et al., 1972). However, there is little scientific information concerning which concentration of a particular indicator represents a clinical deficiency or inadequate status of the vitamin. Because of this, B6 requirements have often been evaluated by using a combination of status indicators. However, this does not overcome the problem of establishing absolute values reflecting impaired status. The increase in methionine metabolites after a methionine load has also been used as an indicator of B6 status (Leklem, 1994) but it has not found extensive use in B6 requirement studies.
24The last analyte within the B complex vitamin markers… beta-hydroxyisovalerate. Several factors are responsible for biotin deficiency:The absence of biotin and biotin precursors in the diet.Antibiotic use will destroy bacteria that creates biotin.Pregnancy significantly increases biotin need.beta-hydroxyisovalarate is a product of incomplete catabolism of Leucine. Leucine generally goes to alpha-ketoisocaproate. In the absence of biotin, Leucine will be diverted to beta-hydroxyisovalerate – and it will appear elevated in the urine.Many biotin food sources, eggs, milk, whole grains, legumes, cereals, cruciferous veggies, beef…
25Severe biotin deficiency is rare in the industrialized countries; (IOM, 1998). Marginal biotin deficiency may be fairly common; (Mock, 1990).”Leucine goes on to a-ketoisocaproate and then eventually to CO2 & water, the other direction of the reaction after transamination, and AA catabolism, it moves on to B-hydroxyisovaerate and with biotin as its cofactor, should also move on to CO2 & water…. Without biotin, b-hydroxyisovalerate accumulates.Biotin
26Excretion of β-hydroxyisovalerate (3-HIA) and biotin are early and sensitive indicators of biotin deficiency.(Mock et al., 2002)The indicators of biotin status that have been validated to the greatest extent are an abnormally decreased urinary excretion of biotin and an abnormally increased urinary excretion of 3-hydroxyisovaleric acid.(DRI Institute of Medecine 1998)
27Evaluation Step 3 Mitochondrial Function and Assessment B Complex Vitamin MarkersMethylation Pathway MarkersCyanocobalamin (B12)Folic Acid
28B12 Urinary Excretion Isoleucine Valine O3, O6, O9 Methylmalonate Methylmalonate should feed into the krebs cycle but requires B12… if accumulation, indicative of B12 deficiency or insufficiencyMethylmalonateB12Urinary Excretion
29Methylmalonate was shown to be superior to serum B12 Urinary MMA levels are a good indication of cobalamin distribution and function since they are directly related to a cobalamin-dependent metabolic pathway.-EJ Norman, 1982; 59(6):Methylmalonic acid (MMA) is increased in blood and urine of patients with vitamin B12 deficiency.-Clinical Chemistry. 2001;47:
30Degradation of histidine relies on assistance from folate (FIGLU)Formiminoglutamate (FIGLU) is a biochemical intermediate of Histidine metabolism. Labs began looking into FIGLU to determine folate sufficiency as early as 1965.Degradation of Histidine relies on assistance from FOLATE (as tetrahydrate folate = THF)so that it may be converted to Glutamatic acidIn the absence of Folate or inadequate supplies of folate cause FIGLU to accumulate and appear in the urine. The higher the value of FIGLU, the more severe the functional folate deficit. High urinary FIGLU appears after about 90 days of insufficient folate availability.Role of Folate: AA metabolism, cell division + replication, nucleic acid synthesis (deficiency = inhibition of DNA synthesis, impaired cell division, and alterations in protein synthesis)•
31Evaluation Step 4 Mitochondrial Function and Assessment B Complex Vitamin MarkersMethylation Pathway MarkersNeurotransmitters Metabolism Markers
32Iron and BH4 Iron and BH4 Vitamin B6 Copper Magnesium Magnesium The adrenergic neurotransmitters are all derived from dietary Phenylalanine and/or Tyrosine. The absence of adequate precursor may lead to deficiencies in neurological activity, showing as an accumulation of these organic acids on test results.We have established that Vanilmandelate is a degradation product of both epinephrine and norepinephrine… … and Homovanillate is created from the breakdown of dopamine.Notice the importance of both vitamin coenzymes and mineral cofactors necessary for the transition of Phenylalanine and Tyrosine to their ultimate end products:IronVitamin B6Copper and Vitamin CS-AdenosylmethionineMagnesiumMagnesium
33Low levels of HVA and VMA are associated with depression, fatigue, sleep disturbance. Clinicians should consider stress and cortisol levels, and may consider adding amino acid supplements.B6Vit C, CuFeSAMeL-dopaDopamineNorepinephrineEpinephrineTyrosineLow levels of VMA and HVA signify heightened sympathetic activity indicative of chronic stress and/or adrenal insufficiency,If markers are HIGH, it may also be indicative of a chronic stress picture because the body is pumping out neurotransmitters in full-force and we see breakdown products spilling over into the urine. It may also be indicative of nutrient/coenzyme deficiencies (such as copper and amino acids)If drastically elevated, the possibility of a neuroblastic tumor exists, however this would be a rare finding and potentially not something to convey to the patient right away to avoid unnecessary worry.Again this is where labs are our tool but ultimately we as docs have to pair results with that of the patients specific presentation, past medical history and symptom picture to determine how to navigate diagnosis and treatment.Homovanillate (HVA)Vanilmandelate(VMA)
34The next marker…5-Hydroxyindoleacetate is a marker of serotonin turnover in the gut & brain Tryptophan is absorbed into the nerve cell from the bloodstream. There, it is converted into Serotonin.
355-Hydroxyindoleacetate (5-HIA) is a degradation product of Serotonin. _________________________________________________________________5-HIA will likely be elevated whenL-Tryptophan, SSRIs or 5-HTP supplements are taken.B6FeTryptophan5-HTPSerotonin5-HIAA low reading for 5-HIA can be caused by low levels of serotonin production, cofactor deficiency (iron & B6) and tryptophan deficiency. On a clinical interpretation note, it may imply untreated depression. Tryptophan deficiency will also lower 5-HIA. Low serotonin production (indicated by low 5-HIA may also limit production of Melatonin – leading to insomnia)_____Treatment of depression will dramatically elevate 5-HIA levels. So always consider medication (SSRI’s) and supplements when analyzing a patient’s abnormally high 5-HIA level.Other factors to consider w/ significantly high levels include increased release of serotonin from 1 of 3 sites- CNS, gut, platelets; high intake of tryptophan or 5-HTP including in food (turkey, bananas, milk, eggs, lentils) and lastly, a carcinoid tumor, one of serotonin-producing enterochromaffin tissue.
36NMDA agonist Interferon-gamma Inflammation Viral or bacterial source? Increased nociceptionIncreased irritationPossible sleep disturbanceThe last 2 markers in this section: Kynurenate & Quinolinate.Interferon release at the site of infection or injury ignites macrophage activity.Elevations in Kynurenate and Quinolinate have been linked to induced macrophage activity. Injury, inflammation, viral and bacterial infection will all induce elevated macrophage responses – mediated by release of interferon-g.Quinolinate will amplify NMDA neuronal transmission, facilitating nociception and irritation – and may, unabated, induce neurodegeneration.Macrophage metabolism induces the production of quinolinic acid. In appropriate quantity, quinolinate will bind to NMDA receptors in much the same way that glutamate does.Interferon-gammaInflammation
38Evaluation Step 5 Mitochondrial Function and Assessment B Complex Vitamin MarkersMethylation Pathway MarkersNeurotransmitters Metabolism MarkersOxidative Damage and Antioxidant Markers
39P-Hydroxyphenyllactate is a marker for cellular mitosis P-Hydroxyphenyllactate is a marker for cellular mitosis. While a small amount of production is normal, indicating metabolic cell turnover, excessive production can indicate injury or possibly tumorogenesis.8-OHdG has been used as an assessment of oxidative damage in; toxic exposure, hematological diseases, cancer,, liver disease, ALS, Parkinson disease, sleep apnea, environmental exposure, smoking2, and diabetes. It has also been used as an assessment when evaluating the possible beneficial treatment effects of; calcium channel blockers, treatments for hypertension, cancer treatment,and dietary interventions. Numerous studies have indicated that urinary 8-OHdG is not only a biomarker of generalized, cellular oxidative stress but might also be a risk factor for disease states, expanding its importance in research and clinical guidance.8-Hydroxydeoxyguanosine (8-OHDG) is a residue resulting from free radical damage to guanosine in DNA. Levels correlate with the rate of DNA damage and repair.
40Evaluation Step 6 Mitochondrial Function and Assessment B Complex Vitamin MarkersMethylation Pathway MarkersNeurotransmitters Metabolism MarkersOxidative Damage and Antioxidant MarkersDetoxification Markers
41Xylene exposure sources: Xylene is converted to 2-Methylhippurate so by analyzing this marker, we’re looking at the liver’s ability to breakdown xylene via phase I & II hepatic detoxification. Elevated 2-Methylhippurate suggest hepatic burden & Glycine deficiency.Xylene exposure sources:Cigarette smokeNew paint, spray paint, paint thinnersNew carpetNew carsDry cleaning productsFuel & Exhaust fumes
42Excessive ammonia production: From gut-derived bacteria Orotate levels may confirm earlier suspicions of ammonia toxicity. An earlier clue would be elevated Citrate, cis-Aconitate, and Isocitrate in the Krebs cycle markers. Orotate confirms that the urea cycle is saturated and unable to detoxify ammonia efficiently. This can occur for one of two reasons:When the capacity of the ammonia clearance system (the hepatic and renal urea cycle) is exceeded, the conversion of Carbamoyl phosphate to urea becomes stalled. Carbamoyl converts to orotate to help increase ammonia clearance. Orotate will then spill over into the urine.Many things can increase ammonia loading including: heavy protein intake, dysbiosis (intestinal bacterial ammonia production), arginine deficiency, magnesium deficiency, and long term oral glutamine supplementation.Excessive ammonia production:From gut-derived bacteriaElevated Amino Acid catabolism
43Glucarate Conjugation & Elimination 2. …and slow Phase II glucuronidation statusPhase IIGlucarate(remnant)Glucuronic Acid1. Glucarate represents both accelerated Phase I activity…(This process requires intervention from Phase I, leaving Glucarate behind as a remnant.)Phase IGlucarate is a remnant of Glucuronic Acid production. It gives us details about Liver detoxification and about Glucuronic Acid conjugation.When glucarate is high, consider taking measures to slow Phase I 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 if not immediately admitted into Phase II detoxification.Causes of high levels: toxins (pesticides, fungicides, herbicides, petrochem, alcohol) intestinal dysbiosis, food components, nitrosamines (rubber products, cured meat), medicationsThe excretion of D-glucaric acid in the urine (uGA) correlates with the total liver content of hepatic cytochrome P-450.Glucose
44Alpha-hydroxybutyrate, Pyroglutamate and Sulfate… 3 clues of glutathione status! The last 3 markers in this section represent glutathione status in the body…
45Cysteine lost in the urine will appear as urinary sulfate With every molecule of cystathionine committed to making Glutathione, one molecule of α-Hydroxybutyrate is made.GlutamateGlycinePathway used for:1.Phase II sulfation-Glutathione serves many important purposes – mainly to detoxify free radicals and chemicals using Hepatic Mercapten and Sulfate conjugation pathways. These detox pathways rely very heavily on the cysteine component of Glutathione to create sulfur bridges with toxic compounds – binding them (preparing them) for renal excretion…2.Antioxidant;3. Amino Acid absorption;Cysteine joins with two other amino acids, Glycine and Glutamate, to form the tripeptide, Glutathione.The Methionine/homocysteine cycle is the ultimate source of the most important building block for Glutathione: cysteine.With every molecule of cystathionine committed to making Glutathione, one molecule of a-Hydroxybutyrate is made.A heavy toxic load puts a strain on the chemical balance of Glutathione. Cysteine gets diverted away from recycling Glutathione and is instead lost in the urine – complexed with toxins. Any cysteine lost in the urine will appear as urinary sulfate – on the Organix report.After extensive toxic exposure, available sources of cysteine run dry. Unable to recycle Glutathione, Pyroglutamate is left without a purpose, and abandons the Glutathione recycling pathway. Pyroglutamate is lost to the urine. When pyroglutamate is being produced (from Glutathione) at a faster rate than it can be recycled, it will appear elevated on the Organix profile.When pyroglutamate is being produced at a faster rate than it can be recycled, it will appear elevated on the Organix profile.Cysteine lost in the urine will appear as urinary sulfate
46Evaluation Step 7 Mitochondrial Function and Assessment B Complex Vitamin MarkersMethylation Pathway MarkersNeurotransmitters Metabolism MarkersOxidative Damage and Antioxidant MarkersDetoxification MarkersMarkers of Intestinal Dysbiosis – Yeast/Fungal
47The markers are compounds created by organisms in the small bowel, that are absorbed and excreted in the urineThe conversion of Benzoate to Hippurate is a glycine conjugation step. Supplemental glycine may help with the conversion.PheTyrTryptophan is converted to Indole in the gut – and subsequently excreted as Indican.TrpDysbiosis 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.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.High readings for Dihydroxyphenylproprionate may be indicative of Clostridial infections. Studies have shown that the probiotic species, Saccromyces boulardii can effectively treat Clostridial infections.Tricarbalylate represents a bacteria that likes to bind Magnesium.Candida yeast species overgrowth is indicated when, D-Arabinitol is high
48Causes of Dysbiosis Use of antibiotics Use of antacids Loss of digestive functionGastric, pancreatic, hepaticDietary intake (sugar, alcohol, etc.)Jejuno-ileal bypass or resectionBlind loop, physiological abnormalityCeliac disease
49Heal the GI Mucosal Lining L-GlutamineN-acetyl GlucosamineBotanicals:LicoriceOkraAloe LeafSlippery ElmMarshmallowMSMMUCINButyric AcidZinc-carnosinePre & ProbioticsHigh Fiber diet
50Guide to Supplementation from Organix™ Profile 1.2.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.