Ronald William Pero
email: rwpero@attglobal.net

Harald Lund

+ AC-11® leads to induction of micromolar levels of nicotinamide and tryptophan in the urine

Keywords: quinic acid, tryptophan, nicotinamide, urine, gastrointestinal tract, HPLC Quinic acid, tryptophan, nicotinamide, urine levels, HPLC, gastrointestinal microflora

Dietary quinic acid supplied as the nutritional supplement AIO + AC-11® leads to induction of micromolar levels of nicotinamide and tryptophan in the urine

Introduction

Beginning back in the early 1900's it was commonly believed that hippuric acid was mainly metabolized in the kidney or liver where it could be easily synthesized via conjugation between benzoic acid with glycine (Quick 1931). Quinic acid in fruit has been recognized as a dietary sourse for catabolic synthesis of hippuric acid, and exposure to benzene-type compounds was another important source (reviewed in Pero et al 2010b). For example, historically it was known man could produce as much as 10 gm quinic acid after eating 450 gm of prunes, and 4.7 gm after eating 305 gm of cranberries (Blatherwick and Long 1923). Likewise, it was also believed in the early literature that benzoic acid and other benzene-type compounds were also primarily metabolized by the liver [START_REF] Beer | The aromatization of hydrogenated derivatives of benzoic acid in animal tissues[END_REF][START_REF] Borsook | The biological synthesis of hippuric acid in vitro[END_REF], and as cited in Pero 2010b). Having this scientific background there slowly has developed a reluctant acceptance of the importance of the GI tract in the primary metabolism of quinic acid to hippuric acid.

The real pivotal breakthrough in the pathway of hippuric acid synthesis was put forward when it was unequivocally established by a series of pioneer scientific reports that (i) quinic acid must be given orally, and not by subcutaneous (SC) or intraperitoneal (IP) injection, in order to produce hippuric acid [START_REF] Adamson | Species differences in the aromatization of quinic acid in vivo and the role of gut bacteria[END_REF], (ii) Co-administration of antibiotics seriously depleted the production of hippuric acid from quinic acid [START_REF] Adamson | Species differences in the aromatization of quinic acid in vivo and the role of gut bacteria[END_REF], Asatoor 1965, Cotrin et al 1960), and (iii) these effects were totally dependent on metabolically functional GI tract microflora [START_REF] Herrmann | The shikimate pathway. Early steps in the biosynthesis of aromatic compounds[END_REF][START_REF] Herrmann | The shikimate pathway[END_REF].

Because hippuric acid synthesis was a well known catabolic metabolism with no known health function except to excrete potential toxic substances like benzene-type substances. For over 70 years it was scientifically ignored, that the shikimate pathway was also a plant metabolic route that could also synthesize other important ingredients such as essential amino acids and nicotinamide.

Connecting hippuric acid levels not only to quinic acid synthesis, but also to pararllel inductive synthesis of tryptophan and nicotinamide also metabolized within the same shikimate pathway was finally realized by the study reports of Pero (Pero 2010b[START_REF] Pero | Antioxidant Metabolism Induced by QuinicAcid. Increased Urinary Excretion of Tryptophan and Nicotinamide[END_REF]. These studies suggest that quinic acid is not only a good surrogate indicator of urinary tract hippuric acid concentration, but also it predicts the inducible health benefit level of the key nutirional ingredients, tryptophan and nicotinamide. Hence, it establishes quinic acid as having profound health benefits and opens up a novel new approach to dietary supplementation benefits. The work presented here verifies our earlier studies and extends the analytical methods that can be used successful to quantify these metabolites.

Materials and Methods

Dietary Supplement Product being evaluated. The ingredients for AIO + AC-11® nutritional dietary supplement are listed below and quantified based on a dosage of 4 fluid ounces per day as an overall enhancer of cellular health: Volunteers used in the study. Volunteers for this study were recruited from the general population living in the Southern Vermont near Arlington, Vermont 05250. There were 4 females and 1 male. Subject EP was 16 years old, 140 lbs and 5 feet 6 inches, MC was 64 years,192 lbs and 5 feet and 9 inches, MO was 69 years, 198 lbs and 6 feet, LP was 61 years, 170 lbs and 5 feet 7 inches, and BW was 69 years135 lbs and 5 feet 7 inches (range = 16-69 years, mean = 55.8 years). The volunteers were instructed not to change any of their lifestyle factors including concomitant dietary supplements and medications normally being taken during the baseline urine sampling collection period (i.e one week prior to the start of treatment), and again 1 wk after the intervention period (i.e. defined as a week of follow-up with no treatment for urine sample collection only). Besides agreeing not to alter any lifestyle factors during the clinical trial, none were obese, vegetarians, smokers, drug addicts or having untreated disease symptoms. All participants filled out a before and after self-report clinical evaluation. An example is presented below. Trial Design: The AIO + AC-11® trial was carried out in the Arlington,Vermont area (Principle Investigator Ronald W. Pero, 1651 Rupert Road, Sandgate, Vermont 05250, USA) whereas the biochemistry and statistical analysis were undertaken at the University of Lund, Institute of Clinical Medical Sciences, Section of Immunology, BMC D:14, 221 84 Lund, Sweden. The composition of urine is well known to be strongly influenced by dietary factors, so that random A.M. sample collection of urine could vary considerably from day to day and from individual to individual. Hence, we have reasoned that when quantifying ingredients of urine there is likely going to be less variation in the urine sample analyses, if we collected individual before (baseline, no treatment) and after 21 days AIO + AC-11® post intervention urine sample collections for comparison and statistical evaluation.

F

The primary specific aim of this study was to design a clinical evaluation where a small sample size could be utilized to produce significant data to verify previously shown clinical facts:

1. Quinic acid oral administration increases hippuric acid in urine [START_REF] Adamson | Species differences in the aromatization of quinic acid in vivo and the role of gut bacteria[END_REF], Asatoor 1965, Cotrin et al 1960[START_REF] Indahl | Quinic acid aromatization in therat. Urinary hippuric acid and catechol excretion following the singular or repeated administration of quinic acid[END_REF], Pero 2010b).

2.

Quinic acid subcutaneous or intraperitoneal injections have no effect on urinary production of hippuric acid [START_REF] Adamson | Species differences in the aromatization of quinic acid in vivo and the role of gut bacteria[END_REF].

On the other hand, oral administration of quinic acid yields high amounts of hippuric acid which in turn was suppressed by coadministration of antibiotics clearly implicating GI tract microflora [START_REF] Adamson | Species differences in the aromatization of quinic acid in vivo and the role of gut bacteria[END_REF], Asatoor 1965, Cotrin et al 1960). Hence, the GI tract microflora are the main metabolizers of quinic acid to biosynthetic products not synthesized inside animal bodies [START_REF] Adamson | Species differences in the aromatization of quinic acid in vivo and the role of gut bacteria[END_REF], Asatoor 1965, Cotrin 1960, Pero 2010a) 4.

Since quinic acid is a substrate for GI tract microflora, then the metabolic conversion of it to hippuric acid, nicotinamide and tryptophan must be primarily via the shikimate pathway found only in the GI tract of animals, and this pathway is a well known metabolic source for microorganisms or plants [START_REF] Herrmann | The shikimate pathway. Early steps in the biosynthesis of aromatic compounds[END_REF][START_REF] Herrmann | The shikimate pathway[END_REF].

So, the hypothesis being tested by this trial design is elevation of quinic acid concentrations in urine supplied by AIO +AC-11® (4 fluid ounces daily) that contain significant levels of quinic acid, in turn, can also metabolically induce high concentrations of nicotinamide, tryptophan, and hippuric acid in urine.

Urine sample collection and transport. Each individual participating in the study donated seven 15 ml Purple-cap VWR test tubes nearly full of urine (13-15 ml), and always collected on separate days during the study. More specifically 3 test tubes were collected 1 week before initiation of the study, or during the baseline period. Another 4 test tubes were collected 21 days after the start of intervention with AIO + AC-11® or 1 week after the intervention period was stopped, and the post intervention urine sampling period began. In such a manner, each individual provided 3 urine specimens for before (baseline) evaluation to be directly compared with 4 urine specimens after the intervention. All urine specimens (n = 34) were stored at + 4 C until shipped to Lund, Sweden on May 27, 2010, also as refrigerated samples (+ 4 C), and arriving within 12 hours of the time shipped. After arrival in Lund the samples were centrifuged at 18,000 x G for 5 minutes and then further stored at -20 C until used experimentally. The average induction ratios for nicotinamide and tryptophan were used for direct comparison to quinic acid in urine. Informed consent was obtained from all participants that included individual permission to obtain urine samples only for use in this study, and with institutional review approval.

HPLC analytical methods. . The urine samples were routinely injected onto the HPLC system without any further clean-up steps. Analytical separations were done on a Genesis® AQ Reversed Phase column. This is a column that is especially suited for 100 % aqueous mobile phases, and maintains stable retention times in them. Particle size: 4µm. Length: 50 mm. Internal Diameter: 4 mm. The mobile phase was delivered with a HP 1050 Series Pumping System with an online ERC-3415 Degasser. The flow rate was 1.0 ml/min. Samples were injected with a HP 1100 Series Thermostatted Autosampler.

Depending on the compounds being analyzed, the mobile phase composition, injection volume and detection method differed. Simultaneous detection of quinic acid and nicotinamide: Mobile phase: 100 % 25 mM ammonium acetate (pH 7). Injection volume: Standard curves were produced for quinic acid and nicotinamide and were linear in the ranges examined. Standards of quinic acid (5-260 mM) were produced in water whereas nicotinamide (0-410 µM) was produced from spiked urine samples. The nicotinamide peak suffered from bad resolution and high background, therefore, spiked urine samples gave a more reliable standard curve, produced in the matrix from which it was analyzed.

Detection of tryptophan was: Mobile phase: 15 mM acetic acid (pH 4) + 1 % acetonitril. Injection volume: 2.0 µl. Tryptophan was detected with an Agilent 1100 Series Fluorescence detector. Excitation wavelength: 286 nm. Emission wavelength: 366 nm. Tryptophan eluted at 9.30-9.70 minutes. Standard curve of tryptophan was linear in the examined range: 1-50 µM.

Statistical analysis procedures. Three statistical approaches were utilized and compared for utility to determine whether quinic acid could significantly induce the levels of nicotinamide and tryptophan in urine. They were unpaired t-test applied to the total sample (n = 34), paired t-test applied to the means of before and after samples of each individual (n = 10) and for each individual group of before and after samples by unpaired t-test (n = 7). In addition, the induction ratios of urinary metabolites for comparing after/before concentrations were calculated by dividing the concentrations of quinic acid, nicotinamide and trypyophan after/before intervention, and then comparing them by linear regression analysis.

Results

The data supporting the hypothesis that quinic acid in urine is also the main metabolic source for synthesis of nicotinamide and tryptophan in urine are reported in Table 1 and Figure 1. It has been shown from a historical perspective in 1970 [START_REF] Adamson | Species differences in the aromatization of quinic acid in vivo and the role of gut bacteria[END_REF] that quinic acid was metabolized primarily in the GI tract and not in the liver. However, the scientific documentation was based on the catabolic metabolism of quinic acid to hippuric acid, a well known excretory product. More recently [START_REF] Pero | Antioxidant Metabolism Induced by QuinicAcid. Increased Urinary Excretion of Tryptophan and Nicotinamide[END_REF] it was also demonstrated that the synthetic pathway for conversion of quinic acid to other plant metabolites including nicotinamide and tryptophan was also shown in the GI tract via the shikimate pathway [START_REF] Herrmann | The shikimate pathway. Early steps in the biosynthesis of aromatic compounds[END_REF][START_REF] Herrmann | The shikimate pathway[END_REF]. Hence these published data strongly support the logic, that quinic acid is a previously unrecognized major dietary source for essential amino acid synthesis (tryptophan, tyrosine, and phenylalanine) that could directly supply the human body with these nutrients, that could otherwise render the human diet nutritionally deficient. In addition, healthy dietary components such as brightly-colored vegetables and fruits are also the richest sources of dietary quinic acid, and thus supportive of essential amino acid biosynthesis (Engelhardt and Maier 1985; [START_REF] Gorsel | Composition characterization of prune juice[END_REF]Beveridge et al.1999;[START_REF] Jensen | Hydrophilic carboxylic acids and iridoid glycosides in the juice of American and European cranberries (Vaccinium macrocarpon and V. oxycoccos), lingonberries (V. vitis-idaea) and blueberries (V. myrtillus)[END_REF]. Here we show that oral intervention of an anti-aging supplement called AIO + AC-11® (containing 4-6% quinic acid esters as QAE = 16 to 24 mg/day quinic acid) can also elevate urinary nioctinamide and tryptophan, just as dietary sources of quinic acid can do the same thing (Table 1). Quite remarkably, when the inductive ratios of quinic acid were compared with those calculated for nicoinamide/tryptophan there was a strong correlation coefficient (r = 0.96). In other words, when Subject BW had a QA induction ratio < 1 (i.e. 0.99) there were no significant increases in either nicotinamide or tryptophan molar concentrations in urine. However, induction ratios > 1 correlated well with the molar levels of increases between QA and those for nicotinamide and tryptophan (Table 1, Figure 1).

Lifestyle factor analysis before and after intervention with AIO + AC-11® was implemented using a self-report clinical examination form. Summation of the results are recorded in Table 2. Because of the small sample size for this pilot clinical study, no statistical analysis could be employed. Nonetheless, there were some clear trends that should be validated by a larger population study. They are listed below:

1.

The self-report questionnaire comparing each participant before and after intervention response values, yielded some value data as it has done in the past [START_REF] Pero | DNA repair in serum correlates to clinical assessment of antiaging lifestyle criteria in healthy volunteers treated with quinic acid ammonium chelate (Quinmax TM ) in drinking water[END_REF].

2.

When evaluating each subject's overall self report responses, the worse they felt at the start of the study (e.g. Subject EP rated her condition 4 out a possible 7), the greater was the perceived benefit from the AIO + Ac-11® intervention. On the other hand, the four other Subjects in the study were already quite highly self-evaluated for good health at the start, and hence it was impossible to show any improvement on such a small sample size.

3.

Subject EP had the greatest increases in induced urinary levels of quinic acid, nicotinamide and tryptophan (Tabel 1, Figure 1), which also in turn had the greatest health benefit from the AIO + AC1-11 intervention (Table 2). This was taken as an encouraging association to further validate: i.e. if less healthier subjects were evaluated within a larger cohort a more clear clinical improvement in lifestyle responses would be established.

Discussion

One point deserving further detailed discussion is the reliability of the HPLC methods used for quantifying quinic acid, nicotinamide and tryptophan in urine. First of all there are only 2 studies that ever reported on determination of quinic acid in urine. They were carried out and developed by the same laboratory. Hence, they deserve in turn to be summarized and further discussed below. Although both studies had similar trial designs, and the chemical endpoint analyses for quinic acid, nicotinamide, and tryptophan using before/after interventions with AIO + AC-11® or Quinmax TM (i.e.quinic acid ammonium 11 chelate), they also differed greatly in regard to collection time of the urine samples during follow-up being 9 mo for Pero et al 2009 and 1 wk for the post intervention collection period. In addition, the daily doses of quinic acid were also quite different being ≥ 1500 mg/day [START_REF] Pero | Antioxidant Metabolism Induced by QuinicAcid. Increased Urinary Excretion of Tryptophan and Nicotinamide[END_REF] and ≥ 20 mg/day (i.e. estimated from 400 mg dose of AC-11®, this study), respectively. Furthermore, the HPLC analytical procedures varied greatly between the 2 studies in regard to the columns and mobile phases being used (e.g. (i) Pero et al 2009 = C18 150 x 4.6 mm, urine clean-up 1 ml urine: 2 ml ethanol: 4 ethyl acetate (1:2:4 v/v) and mobile phase mobile phase was 0.2% trifluoroacetic (TFA): methanol: acetonitrile (70:30): water in a ratio 8:8:84 (v/v/v), (ii) Whereas this study = Genesis® AQ 50 mm x 4 mm, no clean-up urine, and a mobile phase of 15 mM ammonium acetate. Despite the dramatic differences between these analytical procedures, both of them quantified quinic acid, nicotinamide and tryptophan within approximately the same molar ranges as presented below in the comparative summary studies outline.

Taken together it would appear to be obvious the following conclusions can safely be drawn:

1.

The urine analysis of quinic acid, nicotinamide or tryptophan were not influenced by the analytical procedures used, and hence reflect the true molar concentrations in urine.

2.

The after/before urine sample collection design allows for appropriate quantitative estimation of the of induction ratios representing the induced molar concentrations of quinic acid, nicotinamide and tryptophan in urine.

3.

Substantial variation in collection time of the urine samples during follow-up from 9 mo [START_REF] Pero | Antioxidant Metabolism Induced by QuinicAcid. Increased Urinary Excretion of Tryptophan and Nicotinamide[END_REF] to 1 wk (this study) when there was no intervention treatment going on, did not seriously alter the measured the concentrations of these parameters established after 3-4 wk of daily intervention.

4.

The composition of AIO + AC-11® was consistent with providing a natural blend of quinic acid by the fact that comparable stimulation of quinic acid synthesis was observed (see details below). This point was supported by the fact that similar increases in stimulation of quinic acid, tryptophan and nioctinamide levels were observed for ≥ 20 mg AIO + AC-11® dose compared to ≥ 1500 mg pure quinic acid (i.e. quinic acid ammonium chelate). 1. The influence and statistical analysis of the metabolism and uptake of quinic acid, nicotinamide and tryptophan in urine treated with 2 X 2 fluid ounces per day 1 hour before breakfast and dinner for 21 consecutive days of the dietary supplement AIO + AC-11®. The statistical design was that each subject was base lined by providing 3 different daily urine samples 1 week before the AIO + AC-11® intervention was initiated for 21 days, and then another 4 days of different daily urine samples day 22-26 days after the 21 day intervention period. In such a way, each subject could be controlled against his/her own individual variation. Because individuals eat differently on a daily basis, and because diet reflects urinary composition, then this protocol was believed to be appropriate and less variable. Boldface print = presentation of means, or approaching statistical significance whether analyzed by unpaired t-test, paired t-test, or patient by patient group analyses. 2. The analysis of the influence of lifestyle factors before and after supplementation with AIO +AC-11® evaluated from a physician assisted self-reported clinical examination questionnaire, an example of which is presented in the "Volunteers used in this study" section found in Materials and Methods. There were no adverse events recorded, and no parameters being scored by a graded number response decreased when comparing the before and after responses. This was interpreted as either there were no adverse events induced, or there was no effect yet induced by the intervention.

Patient

Positive changes for each subject are presented below. Inductive urinary levels of nicotinamde and tryptophan calculated as their after/before ratio of concentrations in urine after treatment for 21 days with AIO + AC-11®containig about 4-5% quinic acid esters (QAEs). The data points for 5 individuals had a highly significant linear correlation coefficient (r = 0.96, n = 5, p < 0.01), thus providing strong statistical evidence that the levels of quinic acid in urine induced the synthesis of nico and trypto in a positive linear matter from quinic acid. The subjects initials appear next to the individual data points and are also independently presented in Table 1. These data were analyzed by linear regression analysis as the induction ratios calculated as after/before urinary concentrations of QA, nico and trypto.

  µl. Compounds were detected with a HP 1100 Series Diode Array Detector at 215 nm. Quinic acid eluted at 0.75-0.77 minutes and nicotinamide at 6.70-7.00 minutes.

Figure 1 .

 1 Figure1. Interdependence of the µM urinary levels of nicotinamide (nico) and tryptophan (trypto) on the mM amounts of quinic acid (QA) in urine. Inductive urinary levels of nicotinamde and tryptophan calculated as their after/before ratio of concentrations in urine after treatment for 21 days with AIO + AC-11®containig about 4-5% quinic acid esters (QAEs). The data points for 5 individuals had a highly significant linear correlation coefficient (r = 0.96, n = 5, p < 0.01), thus providing strong statistical evidence that the levels of quinic acid in urine induced the synthesis of nico and trypto in a positive linear matter from quinic acid. The subjects initials appear next to the individual data points and are also independently presented in Table1. These data were analyzed by linear regression analysis as the induction ratios calculated as after/before urinary concentrations of QA, nico and trypto.

facts % daily value a Serving facts: (4 fluid ounces daily)

	Amto puree
	Pomegranate puree
	Blueberry puree
	Mango puree
	Camu Camu puree
	Acerola puree
	Calories Acai puree		76
	Saturated fat Dark cherry concentrate	0
	Trans fat		0
	Total dietary fiber Other ingredients:	.64 gm
	Total carbohydrates USP 32 purified water, Nisin	16 gm
	F o r Proteins _________________________________________________ -.1.80 gm Sugars b a percentage of daily value are based on a 2,000 calorie diet 2.20 gm AIO + AC-11® daily health: Vitamin A 9000 IU Vitamin B1 Vitamin B2 204 mcg r 1800 mcg o F b daily value not established
	Vitamin B6 Vitamin B12 Vitamin C Vitamin D3 Vitamin E Biotin	P e e r 24 mg P 7.2 mcg 496 mg 3000 IU 36 IU 360 mcg e e r
	Folic acid Pantothenic acid Potassium Selenium Inositol Lycopene Lutein Manganese Zinc Calcium	R e v i e w 480 mcg 16 mg 400 mg 120 mcg 12 mg 20 mg 20 mg 8 mg 12 mg 41.2 mg R e v i e w
	Chromium		560 mcg
	AIO + AC-11® premium cellular and DNA health:
	Reservatrol		300 mg
	AC-11®		400 mg
	Carnosine		200 mg
	Niacin		120 mg
	Green tea		80 mg
	AIO + AC-11® premium antioxidant blend:
	53,000mg mg whole fruit per daily serving:
			5

4 Supplement

6 Example Physician-Assisted Self Report Clinical Examination Summery data sheet for all physician-assisted and self-reported clinical examinations used for final analysis.

	Concentration
	previous month
	(poor(1), mod.(2)
	good(3)
	Self perceived
	health: poor
	(1,2,3,4) and
	good (5,6,7)
	Patient Name:		Birth date:	Treatment:
	Energy previous
	Observations month poor (1),		Baseline	+21 days treated
	mod.(2) good (3)	Before Trial Initiation	After Trial Initiation	Comments
	Body Weight (kg) Height BMI Doctor Signed	o F o
	Work Attendance Previous month	r r
	(%) Flu incidences previous month # sore throats previous month		P e e r P e e r
	# headaches previous month # diarrhea /constipation previous month? appetite previous week bad (1), normal (2), (good (3)	R e v i e w R e v i e w
	Rashs previous
	month (Yes, No)
	Pain previous
	month Yes(1),
	some(2), No(3)
	Fatigue previous
	month: Yes(1),
	some(2), No(3)
					7

Table

	Pero et al 2009 Quinmax (QA-NH 4	+ ) 4 wk treat + 9 mo
	n = 36, urine	= ≥ 1500 mg/day	follow-up	58-184 21-453 13-30
	samples analyzed
	This study	AIO + AC-11®	3 wk treat + 1 wk
	n = 34, urine	(AC-11® 400 mg	follow-up	38-83 16-60 28-163
	samples analyzed = > 20 mg QA)
	F o F o F o
		r r r
		P P P
		e e r e e r e e r
			R e v i e w R e v i e w R e v i e w
	Comparative summary of studies comparing urinary quinic acid (QA) to urinary
	nicotinamide (nico) and tryptophan (trypto)
	_______________________________________________________________________
	QA -containing		Samples analyzed during
	agent being tested	Evaluation	follow-up (value ranges)
	Trial Data	in clinic	period	QA mM Nico µM Trypt µM
	_______________________________________________________________________
				12 14

Table

		induction ratio (after/before) induction ratio (after/before)			1.9 2.0		1.2 1.6		1.2 1.2
		unpaired t-test unpaired t-test
		(before vs after) (before vs after)		0.099 0.047		0.369 0.114		0.295 0.077
		n = 7 n=7
	MC	before			mean		mean		mean
	Total sample	#1 before (n=14)		14.6 36.5		29.2 21.1		22.8 66.7
	mean (n=34)	#2* after (n=20)	#NAME? 54.1		105,1 34.8		77,5 84.4
		#3 Unpaired t-test		13.6	14.1	24.6	26.9	13.2	18.0
		after (before vs after)		0.044		0.017		0.146
		#4 n=34		38.4		87.8		19.7
	F o #5 #6 Paired t-test #7 induction ratio (after/before) F o (before vs after of means) n=10 * = unanalyzable by current available techniques for this method 33.5 83.1 41.0 35.9 T-test 40.2 38.3 32.6 2.7 0.142 F o	59.9 2.2 0.050	17.3 41.3 34.6	28.2 1.6 0.039
		r unpaired t-test r r
		P (before vs after) n = 6 P P	0.0002		0.056		0.128
	EP BW	QA mM mean 12.0 9.6 e e r mean 90.5 70.6 93.5 84.9 e e r e e r 12.1 11.3 31.5 74.9 86.4 43.1 59.0 induction ratio before #1 #2 before #1 #2 #3 #3 after #4 #5 #6 #7 (after/before) 5.2 unpaired t-test (before vs after) 0.017 R e v i 12.4 Nico µM mean 9.5 mean 29.3 14.3 13.8 19.1 4.8 8.9 8.9 26.7 38.9 21.7 24.1 2.7 0.044 e w after #4 42.9 19.7 #5 48.8 14.8 #6 35.0 19.5 #7 55.7 45.6 10.6 16.2 induction ratio (after/before) 0.5 0.8 unpaired t-test (before vs after) n = 7 0.007 0.315 R e v i e w R e v i e w	Trypto µM after 30.5 mean 61.7 77.2 77.8 72.2 51.9 55.3 45.9 74.7 104.1 127.5 96.0 100.6 2.2 0.005 79.7 85.5 86.5 76.3 82.0 1.1 0.098
	MO	n = 7 before			mean		mean		mean
	LP	before #1 #1 #2 #2 #3 #3 after #4 after #4 #5 #5 #6 #6 #7		34.4 20.9 70.3 10.2 19.4 39.5 81.7 18.5 52.6 48.6 113.1 72.3 85.8	mean 41.4 23.5 83.3	23.5 28.9 45.5 14.2 25.3 19.8 23.2 7.8 42.8 26.5 75.0 47.3 56.8	mean 31.4 21.0 49.5	130.9 153.2 45.4 139.3 25.6 49.7 152.3 161.2 16.0 195.5 54.0 67.6 144.2	mean 141.1 40.2 163.3
		#7		38.5	44.5	15.9	24.4	54.0	47.9
								15 16 17

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Acknowledgements

We are indebted to Promethean Corp (Matthew T. Hennineger, CEO) for supporting this study, and to Professors Tomas Leanderson and Fredrik Ivars for providing research accommodations at Lund University, Lund, Sweden. Harald Lund received a research stipend for his work from Promethean Corp.

Acknowledgements

The authors would like to thank Daniel Zwiren CEO Optigenex, Matthew T. Henninger CEO Promethean Corp, Professor Fredrick Ivars Immunology Lund University and Professor Tomas Leanderson Immunology Lund University for providing financial and research support to this project. Harald Lund received a research stipend for his work from Promethean Corp. Moreover, we are grateful to the study participants for supplying numerous urine samples as well as cooperation to this study as well.