Oxygen and nitric oxide readings for Hepatitis C evaluation





University of Medicine and Dentistry of New Jersey performed an independent clinical study on oxygen and nitric oxide readings for Hepatitis C evaluation and it's relation to ROx and BOx.





Technical Information

Abstract

When blood is subjected to a protocol of increasing stress, hidden reservoirs of oxygen (O2) and nitric oxide (NO) are revealed. Blood exposed to a series of stresses (stress profile) releases a unique pattern of O2 and NO concentrations. At first O2 vanishes suddenly and later re-appears, also suddenly. NO appears the same way, indicating a reservoir mechanism, into and from which these molecules are rapidly transported.

O2 and NO are key molecules involved in immune system function, and their transport characteristics in blood are key to the investigation and monitoring of any disease. The purpose of this paper is to present an immunography technique, which exploits blood stress profiles and is useful for evaluating disease level and progress in any patient.

Immunography is the use of an Immunogram Analyzer (IA) to generate blood stress profiles, called immunograms, to measure O2 and NO profiles.

In this paper we demonstrate how to use immunography to evaluate Hepatitis C (Hep C) patients. On the basis of four known Hep C patient samples and 19 controls, we have found higher NO levels, along with higher oxidative activity, in the Hep C samples. Information about blood reservoirs of oxygen (ROX) and cellular boosting of oxygen (BOX) is also presented and discussed.

More information describing The ROX concept and rationale for our hypothesis may be found at: www.bioox.us


Behavior of Blood under stress

Hinshaw, Sofer, and coworkers (1) found when studying an extracorporeal blood recirculation system in canine endotoxic shock models, that the shear stress generated by the extracorporeal pump circulation system led to autoanticoagulation. That is, physical, shear-stressed blood would not clot, in the absence of external anticoagulants such as heparin, even in a vigorously agitated blood circulation system. They further isolated from stressed blood, HLF, a heparin-like factor, which was shown to prevent clotting. Hinshaw an coworkers (2) also noted that such stressed blood in canines led to a 'cure' in the dogs---the dogs were resistant to shock when injected with bacterial endotoxin.

Sofer pursued this problem as a NewJersey State Sponsored Research Professor of Biotechnology, and with his group discovered MOPs, or molecular oxygen peaks (not radical oxygen species) that were generated from stressed blood. Thousands of runs by Sofer's NJIT Biotechnology group (3-6) reinforce the presence of oxygen peaks generated by many other types of stress: chemical, thermal, pH, etc. The questions that arise here are: Where does the oxygen from the MOPs come from, in view of the fact that the MOPs are generated from blood at zero oxygen concentration, where the hemoglobin equilibrium oxygen content is zero? Why does stress release this oxygen? Why does stress strengthen the immune system against bacterial endotoxin attack?


BOX and ROX

Oxygen is a significant and potent tool in the body’s defense against invading foreign chemicals and organisms. The storage, release, and transport of oxygen forms an integral part of the immune system. ROX™ is a marker for oxygen reserves in blood, and is hypothesized to be a measure of the innate ability of the patient to provide oxygen for attack or use. BOX™ is a marker for blood cellular oxidative capability and is hypothesized to be a measure of the adaptive capability of the immune system. ROX™ and BOX™ are numbers that are of potential clinical and diagnostic value for diseases in which immune system operation is of concern. Further work with human blood has led to development of ImmunoGraph™ test, which measures both the amount of Reservoir of Oxygen (ROX ™), and the rate at which it is built (BOX™).

ROX™ and BOX™ exist at high baseline levels in homeostasis. Unlike PSA and c-reactive protein, the baseline is reads '100' with respect to the standard. Therefore both up- and down-fluctuations are readable and of potential value.

Immunotherapy such as with the addition of immunomodulators such as interferons or activated T-cells seeks to trigger the immune system to fight a given disease.Our hypothesis is that the lack of adequate oxygen availability for use, by for example CD8+ cytotoxic T-cells, could be the reason that for many populations, interferons and activated T-cells do not work. For true immunomodulation, interferons and activated T-cells are a necessary, but not sufficient condition to activate the immune system.

The essential concept behind the ROX hypothesis is that oxygen delivery to the site of action is critical.


ROX hypothesis and rationale

The basic tenet of the ROX hypothesis is that post-hemoglobin, O2 transport to and focus at its target site, depends on an elegant mechanism involving the shuttling of O2 in and out of reservoirs in blood and other fluids.

Oxygen drives the body’s life process by combining with food to generate energy for muscular activity, nervous system conduction, immune attack against cancer and viruses and bacteria, detoxification of ingested pollutants and drugs and energy for signaling immune- and nervous-system functions.

An essential and convenient way to study the availability of energy for the body is to track the delivery of oxygen to tissues. Fuels such as glucose and fats must be oxidized in order to release their energy. Each molecule of glucose requires six molecules of oxygen in order to release its energy. A single molecule of fat can require dozens of molecules of oxygen to unleash its energy stores. In terms of numbers of molecules required for energy, the supply of oxygen is far more critical than the supply of sugars and fats.

Oxygen is the rate-limiting substance for energy generation. Therefore, the simplest way to observe the body’s availability of energy, is to measure the blood’s ability to deliver the oxygen and to focus it at the point of need. This is done by using the Immunogram as a tool.

Our bodies are mainly made of water. But, while sugar and other energy molecules are soluble in water, oxygen is not. For example, the normal blood sugar level is about 100 mg/dl or about 1,000 parts per million (ppm), but oxygen can only dissolve in tissues and plasma to about the 6 ppm level. This is less than 1% of the oxygen needed for glucose oxidation, whereas greater than 1,000 parts per million are needed.

So how can sufficient amounts of oxygen be delivered to, for example, the mitochondrion, an organelle inside the cell, where it is needed for processing sugar into energy?

When a sample of blood is subjected to increasing amounts of shear- and chemical- stress in an IA cell, an Immunogram is generated, measuring the amounts of oxygen and nitric oxide (NO) that are released as the ROX breaks.

Oxygen is paramagnetic and is stored in very concentrated form inside tiny ROX capsules which are also paramagnetic. A magnetic signal such as superoxide attracts paramagnetic compounds such as oxygen and NO, causing the ROX to swarm to the point of the signal. The swarm then releases all the oxygen or NO in concentrated form at one location.


Hepatitis C and ROX

Interferon and Ribavirin (i/r) administration is a standard treatment for Hep C. This treatment is expensive, time consuming, and causes much patient suffering. For a large number of patients, the treatment simply doesn’t work.

The existence of non-responders to interferon treatment of Hepatitis C patients is well known. An excellent review is provided by Dr. William M. Lee at the University of Texas Southwestern Medical School (sciencedaily article). Dr. Lee and colleagues at nine other institutions worked on the HALT-C study from 2002-2007. This study points out that there are 50-60% of non-responders to interferon plus ribavirin treatment. These non-responders are furthermore non-responsive to long-term interferon maintenance strategies in the sense that there is no significant difference in the rate of progression of liver disease between non-responders on interferon and non-responders not on interferon maintenance. The question that arises form this work is: Why is there a difference between responders and non-responders?

Our hypothesis, for patients not responding to i/r, is that insufficient O2 and NO are delivered to the virus for its destruction. Boosting the immune system, without delivering sufficient oxygen for energy creation and viral destruction, is not possible. Immunograms could therefore be of use in bringing more information that may help the patient.


Methods and Results

Four Hep C and 19 control blood samples averaging 3 ml were collected by venipuncture in heparin-anticoagulated Vacutainer vials and frozen at - 15 C for later analysis.

The samples were thawed at room temperature (25 C) for one hour prior to analysis on the IA. The IA was programmed with the following steps:

  1. The IA was calibrated and zeroed with helium before every run. The difference between zero and air saturation represents a concentration of about 258 nmol/ml, or 100 % saturation.

  2. Minor stress was applied (shear stress plus strong light) to yield a sudden drop in oxygen. BOX is the difference of this low level from the baseline saturated with air.

  3. After a pause of 30 seconds, chemical stress (0.45 ml of 5% phenol solution) applied in three successive aliquots, five seconds apart. The sudden rise of oxygen is reported as ROX.

  4. After a pause of 90 seconds, NO was measured and the run ended.



Each run is completed within about 3 minutes.

Step 1: Baseline controls

Upon thawing at room temperature, samples were allowed to stand for approximately 90 minutes. The 16 control samples were divided into 2 groups, depending on the degree of coagulation, of 8 each and analyzed statistically in Table I. Eight had observable coagulation meaning that BOX effects would be reduced (low BOX) in an immunogram. The other eight were standard immunograms. Three more controls were run separately for the data in Table II.

Table I is an analysis of 16 control runs. BOX, ROX, and NO readings from immunograms are tabulated, along with corresponding standard deviations. The controls are statistically significant. Wide variations in ROX, BOX, and NO are expected, due to wide variations in each individual’s oxygen loading at the time of sampling, depending on metabolism and other factors.

Table I: Summary of combined control runs, actual readings in % saturation

Description BOX ROX NO
Control Standard Immunograms n = 8 mean value 67.8 67.8 -10.4
std. deviation 17.7 14.7 11.8
Control low BOX Immunograms n = 8 mean value 5.4 46.4 15.4
std. deviation 5.7 14.6 8.6

Step 2: Normalization

The next step is to normalize BOX, ROX, and NO numbers, and setting baseline controls to equal 100. This is done by dividing each reading by the mean value.

Table II: Typical normalized Immunogram readings

Run Description BOX ROX NO
1 Control 155 108.3 162
2 Control 101 101.1 93
3 Control 44 91.1 45
4 Hep C 187 110.6 237
5 Hep C 173 113.9 205
6 MedX stress standard 200 82.7 206

Table II demonstrates normalization of standard immunograms from a small sample of controls (3) and Hep C (2). An internal, high-stress standard for the IA, MedX, is included as a reference.

From Table II it can be seen that ROX > 110, BOX > 160, and NO > 200 are indicators of Hep C. The purpose of this table is to demonstrate the normalization procedure, and to show individual variations. While it appears that ROX, BOX, and NO are higher for Hep C patients, the sample size is too small and conclusions at this point are not warranted.

However, with a larger sample size, this method would yield acceptable lows and highs for BOX, ROX, and NO.

The three controls in this table are in addition to the 16 controls of Table 1.

Step 3: Examination of NO production

Focussing on NO, since it appears to stand out as a target monitoring marker for Hep C, we next combine the standard controls of Table I (8) with the standard controls of Table II (3), and normalize the data.

Combined normalized data are presented in Table III.

Table III: Summary of normalized NO readings

Sample size NO Standard deviation
Control n = 11 100.0 54.0
Hep C n = 4 203.5 20.8

From Table III, the high standard deviation for the controls indicates a large fluctuation in ‘normal’ NO values. This is also expected, since oxygen consumption on each individual can change drastically within a few minutes, such as brought on by exercise or stress.

On the other hand, for the Hep C samples, the standard deviation is much smaller, indicating more uniform, and higher concentrations, of NO than control values for NO.

A detailed discussion of why NO is high for Hep C is not within the scope of this paper; however, it may be speculated that if NO is indeed high, it may be because the patient is actively fighting the virus. This does not necessarily imply that the same results would hold for other diseases, such as AIDS or cancer. Each immunographic finding for a given disease cannot be extrapolated to another disease. Clinical trials are required for each new finding.

Conclusions

The Immunogram Analyzer (international patents pending) is a new tool which outputs BOX, ROX, and NO values for stressed blood.

We have demonstrated a calculation technique for using the IA to evaluate a disease such as Hep C.

This technique is applicable to diseases other than Hep C.

The sample sizes presented for Hep C are too small from which to draw firm conclusions. A larger study of Hep C should be conducted in order to determine norms for BOX, ROX, and NO.





Contributors

Dharmesh Kaswalla, MD, Michael Demyen, MD, and Zamir Brelvi, MD, PhD
E178 University Hospital
Department of Gastroenterology and Hepatology
University of Medicine and Dentistry of New Jersey
Newark, New Jersey, USA 07103


Sam Sofer, PhD, PE
President, Air & Water Solutions, Inc, Solmedx
ssofer@bioox.us
www.bioox.us
(301) 246-0151





Acknowledgements

Thanks are due for the excellent technical assistance of Ashley Devilliers Cox, and Tom Scharf.





References

  1. Am J Physiol Heart Circ Phys:H742-750 (1980)


  2. Circ. Shock 1979; 6(3)261-9


  3. Acta Toxicol. Ther., Vol. XX, n. 1 (1999) --and-- A Method for Quantitation of Molecular Oxygen Peaks (MOPs) in Blood and Comparison with Hemoglobin-bound Oxygen (1999) S. Sofer, and C. McKenna


  4. A potential rapid in vitro Assay to Quantitate Chemical Stress in Mammalian Blood; S. Sofer, Cristin McKenna, Emerging Therapeutic Targets 3(2) 365-374 (1999)


  5. Molecular Oxygen Peaks (MOPs) in Blood: Their Discovery, Investigation and Implications”; (S. Sofer, C. McKenna), Comparative Haematology International 9:68-71(1999).


  6. The Initial Oxygraphic Response of Bovine Blood as the Basis for a Rapid Assay for Drug Toxicity”; S. Sofer, C. McKenna, Comparative Haematology International 9:72-75 (1999).






Keywords

Interferon treatment; patient blood samples; ROX (reservoir oxygen capacity); Cancer, innate immune viability; AIDs; BOX; blood cell oxygen capacity; adaptive immune system strength; proper medication dosage; clinical studies; Hepatitis C; Diabetes; patient injury and drug toxicity Alzheimer's; ALS; neurodegenerative diseases; Cancer; Diabetes; HIV/AIDS; Huntington's Disease; Parkinson's Disease (PD); Rheumatoid Arthritis (RA) and other auto-immune diseases; and Complex Regional Pain Syndrome (CRPS)]




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