Archive for the ‘Folate and B12’ Category

Metformin and vitamin B12 deficiency?

Larry H. Bernstein, MD, FCAP, Curator



Years of taking popular diabetes drug tied to risk of B12 deficiency


Long-term Metformin Use and Vitamin B12 Deficiency in the Diabetes Prevention Program Outcomes Study


Metformin linked to vitamin B12 deficiency

David Holmes   Nature Reviews Endocrinology(2016)

Secondary analysis of data from the Diabetes Prevention Program Outcomes Study (DPPOS), one of the largest and longest studies of metformin treatment in patients at high risk of developing type 2 diabetes mellitus, shows that long-term use of metformin is associated with vitamin B12deficiency.

Aroda, V. R. et al. Long-term metformin use and vitamin B12 deficiency in the Diabetes Prevention Program Outcomes Study. J. Clin. Endocrinol. Metab. (2016)


Long-term Follow-up of Diabetes Prevention Program Shows Continued Reduction in Diabetes Development

San Francisco, California
June 16, 2014

Treatments used to decrease the development of type 2 diabetes continue to be effective an average of 15 years later, according to the latest findings of the Diabetes Prevention Program Outcomes Study, a landmark study funded by the National Institutes of Health (NIH).

The results, presented at the American Diabetes Association’s 74th Scientific Sessions®, come more than a decade after the Diabetes Prevention Program, or DPP, reported its original findings. In 2001, after an average of three years of study, the DPP announced that the study’s two interventions, a lifestyle program designed to reduce weight and increase activity levels and the diabetes medicinemetformin, decreased the development of type 2 diabetes in a diverse group of people, all of whom were at high risk for the disease, by 58 and 31 percent, respectively, compared with a group taking placebo.

The Diabetes Prevention Program Outcomes Study, or DPPOS, was conducted as an extension of the DPP to determine the longer-term effects of the two interventions, including further reduction in diabetes development and whether delaying diabetes would reduce the development of the diabetes complications that can lead to blindness, kidney failure, amputations and heart disease. Funded largely by the NIH’s National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the new findings show that the lifestyle intervention and metformin treatment have beneficial effects, even years later, but did not reduce microvascular complications.

Delaying Type 2 Diabetes

Participants in the study who were originally assigned to the lifestyle intervention and metformin during DPP continued to have lower rates of type 2 diabetes development than those assigned to placebo, with 27 percent and 17 percent reductions, respectively, after 15 years.

“What we’re finding is that we can prevent or delay the onset of type 2 diabetes, a chronic disease, through lifestyle intervention or with metformin, over a very long period of time,” said David M. Nathan, MD, Chairman of the DPP/DPPOS and Professor of Medicine at Harvard Medical School. “After the initial randomized treatment phase in DPP, all participants were offered lifestyle intervention and the rates of diabetes development fell in the metformin and former placebo groups, leading to a reduction in the treatment group differences over time.  However, the lifestyle intervention and metformin are still quite effective at delaying, if not preventing, type 2 diabetes,” Dr. Nathan said. Currently, an estimated 79 million American adults are at high-risk for developing type 2 diabetes.

Microvascular Complications
The DPPOS investigators followed participants for an additional 12 years after the end of the DPP to determine both the extent of diabetes prevention over time and whether the study treatments would also decrease the small vessel -or microvascular- complications, such as eye, nerve and kidney disease. These long-term results did not demonstrate significant differences among the lifestyle intervention, metformin or placebo groups on the microvascular complications, reported Kieren Mather, MD, Professor of Medicine at Indiana University School of Medicine and a study investigator.

“However, regardless of type of initial treatment, participants who didn’t develop diabetes had a 28 percent lower occurrence of the microvascular complications than those participants who did develop diabetes. These findings show that intervening in the prediabetes phase is important in reducing early stage complications,” Dr. Mather noted. The absence of differences in microvascular complications among the intervention groups may be explained by the small differences in average glucose levels among the groups at this stage of follow-up.

Risk for Cardiovascular Disease

The DPP population was relatively young and healthy at the beginning of the study, and few participants had experienced any severe cardiovascular events, such as heart attack or stroke, 15 years later. The relatively small number of events meant that the DPPOS researchers could not test the effects of interventions on cardiovascular disease. However, the research team did examine whether the study interventions, or a delay in the onset of type 2 diabetes, improved cardiovascular risk factors.

“We found that cardiovascular risk factors, such as hypertension, are generally improved by the lifestyle intervention and somewhat less by metformin,” said Ronald Goldberg, MD, Professor of Medicine at the University of Miami and one of the DPPOS investigators. “We know that people with type 2 diabetes are at much higher risk for heart disease and stroke than those who do not have diabetes, so a delay in risk factor development or improvement in risk factors may prove to be beneficial.”

Long-term Results with Metformin

The DPP/DPPOS is the largest and longest duration study to examine the effects of metformin, an inexpensive, well-known and generally safe diabetes medicine, in people who have not been diagnosed with diabetes. For DPPOS participants, metformin treatment was associated with a modest degree of long-term weight loss. “Other than a small increase in vitamin B-12 deficiency, which is a recognized consequence of metformin therapy, it has been extremely safe and well-tolerated over the 15 years of our study,” said Jill Crandall, MD, Professor of Medicine at Albert Einstein College of Medicine and a DPPOS investigator. “Further study will help show whether metformin has beneficial effects on heart disease and cancer, which are both increased in people with type 2 diabetes.”

Looking to the Future

In addition to the current findings, the DPPOS includes a uniquely valuable population that can help researchers understand the clinical course of type 2 diabetes.  Since the participants did not have diabetes at the beginning of the DPP, for those who have developed diabetes, the data show precisely when they developed the disease, which is rare in previous studies. “The DPP and DPPOS have given us an incredible wealth of information by following a very diverse group of people with regard to race and age as they have progressed from prediabetes to diabetes,” said Judith Fradkin, MD, Director of the NIDDK Division of Diabetes, Endocrinology and Metabolic Diseases. “The study provides us with an opportunity to make crucial discoveries about the clinical course of type 2 diabetes.”

Dr. Fradkin noted that the study population held promise for further analyses because researchers would now be able to examine how developing diabetes at different periods of life may cause the disease to progress differently. “We can look at whether diabetes behaves differently if you develop it before the age of 50 or after the age of 60,” she said. “Thanks to the large and diverse population of DPPOS that has remained very loyal to the study, we will be able to see how and when complications first develop and understand how to intervene most effectively.”

She added that NIDDK had invited the researchers to submit an application for a grant to follow the study population for an additional 10 years.

The Diabetes Prevention Program Outcomes Study was funded under NIH grant U01DK048489 by the NIDDK; National Institute on Aging; National Cancer Institute; National Heart, Lung, and Blood Institute; National Eye Institute; National Center on Minority Health and Health Disparities; and the Office of the NIH Director; Eunice Kennedy Shriver National Institute of Child Health and Human Development; Office of Research on Women’s Health; and Office of Dietary Supplements, all part of the NIH, as well as the Indian Health Service, Centers for Disease Control and Prevention and American Diabetes Association. Funding in the form of supplies was provided by Merck Sante, Merck KGaA and LifeScan.

The American Diabetes Association is leading the fight to Stop Diabetes® and its deadly consequences and fighting for those affected by diabetes. The Association funds research to prevent, cure and manage diabetes; delivers services to hundreds of communities; provides objective and credible information; and gives voice to those denied their rights because of diabetes. Founded in 1940, our mission is to prevent and cure diabetes and to improve the lives of all people affected by diabetes. For more information please call the American Diabetes Association at 1-800-DIABETES (1-800-342-2383) or visit Information from both these sources is available in English and Spanish.

Association of Biochemical B12Deficiency With Metformin Therapy and Vitamin B12Supplements  

The National Health and Nutrition Examination Survey, 1999–2006

Lael ReinstatlerYan Ping QiRebecca S. WilliamsonJoshua V. Garn, and Godfrey P. Oakley Jr.
Diabetes Care February 2012 vol. 35 no. 2 327-333

OBJECTIVE To describe the prevalence of biochemical B12deficiency in adults with type 2 diabetes taking metformin compared with those not taking metformin and those without diabetes, and explore whether this relationship is modified by vitamin B12supplements.

RESEARCH DESIGN AND METHODS Analysis of data on U.S. adults ≥50 years of age with (n = 1,621) or without type 2 diabetes (n = 6,867) from the National Health and Nutrition Examination Survey (NHANES), 1999–2006. Type 2 diabetes was defined as clinical diagnosis after age 30 without initiation of insulin therapy within 1 year. Those with diabetes were classified according to their current metformin use. Biochemical B12 deficiency was defined as serum B12concentrations ≤148 pmol/L and borderline deficiency was defined as >148 to ≤221 pmol/L.

RESULTS Biochemical B12 deficiency was present in 5.8% of those with diabetes using metformin compared with 2.4% of those not using metformin (P = 0.0026) and 3.3% of those without diabetes (P = 0.0002). Among those with diabetes, metformin use was associated with biochemical B12 deficiency (adjusted odds ratio 2.92; 95% CI 1.26–6.78). Consumption of any supplement containing B12 was not associated with a reduction in the prevalence of biochemical B12deficiency among those with diabetes, whereas consumption of any supplement containing B12 was associated with a two-thirds reduction among those without diabetes.

CONCLUSIONS Metformin therapy is associated with a higher prevalence of biochemical B12 deficiency. The amount of B12recommended by the Institute of Medicine (IOM) (2.4 μg/day) and the amount available in general multivitamins (6 μg) may not be enough to correct this deficiency among those with diabetes.

It is well known that the risks of both type 2 diabetes and B12deficiency increase with age (1,2). Recent national data estimate a 21.2% prevalence of diagnosed diabetes among adults ≥65 years of age and a 6 and 20% prevalence of biochemical B12 deficiency (serum B12<148 pmol/L) and borderline deficiency (serum B12 ≥148–221 pmol/L) among adults ≥60 years of age (3,4).

The diabetes drug metformin has been reported to cause a decrease in serum B12 concentrations. In the first efficacy trial, DeFronzo and Goodman (5) demonstrated that although metformin offers superior control of glycosylated hemoglobin levels and fasting plasma glucose levels compared with glyburide, serum B12 concentrations were lowered by 22% compared with placebo, and 29% compared with glyburide therapy after 29 weeks of treatment. A recent, randomized control trial designed to examine the temporal relationship between metformin and serum B12 found a 19% reduction in serum B12 levels compared with placebo after 4 years (6). Several other randomized control trials and cross-sectional surveys reported reductions in B12ranging from 9 to 52% (716). Although classical B12 deficiency presents with clinical symptoms such as anemia, peripheral neuropathy, depression, and cognitive impairment, these symptoms are usually absent in those with biochemical B12 deficiency (17).

Several researchers have made recommendations to screen those with type 2 diabetes on metformin for serum B12 levels (6,7,1416,1821). However, no formal recommendations have been provided by the medical community or the U.S. Prevention Services Task Force. High-dose B12 injection therapy has been successfully used to correct the metformin-induced decline in serum B12 (15,21,22). The use of B12supplements among those with type 2 diabetes on metformin in a nationally representative sample and their potentially protective effect against biochemical B12 deficiency has not been reported. It is therefore the aim of the current study to use the nationally representative National Health and Nutrition Examination Survey (NHANES) population to determine the prevalence of biochemical B12deficiency among those with type 2 diabetes ≥50 years of age taking metformin compared with those with type 2 diabetes not taking metformin and those without diabetes, and to explore how these relationships are modified by B12 supplement consumption.

Design overview

NHANES is a nationally representative sample of the noninstitutionalized U.S. population with targeted oversampling of U.S. adults ≥60 years of age, African Americans, and Hispanics. Details of these surveys have been described elsewhere (23). All participants gave written informed consent, and the survey protocol was approved by a human subjects review board.

Setting and participants

Our study included adults ≥50 years of age from NHANES 1999–2006. Participants with positive HIV antibody test results, high creatinine levels (>1.7 mg/dL for men and >1.5 mg/dL for women), and prescription B12 injections were excluded from the analysis. Participants who reported having prediabetes or borderline diabetes (n = 226) were removed because they could not be definitively grouped as having or not having type 2 diabetes. We also excluded pregnant women, those with type 1 diabetes, and those without diabetes taking metformin. Based on clinical aspects described by the American Diabetes Association and previous work in NHANES, those who were diagnosed before the age of 30 and began insulin therapy within 1 year of diagnosis were classified as having type 1 diabetes (24,25). Type 2 diabetes status in adults was dichotomized as yes/no. Participants who reported receiving a physician’s diagnosis after age 30 (excluding gestational diabetes) and did not initiate insulin therapy within 1 year of diagnosis were classified as having type 2 diabetes.

Outcomes and follow-up

The primary outcome was biochemical B12 deficiency determined by serum B12 concentrations. Serum B12 levels were quantified using the Quantaphase II folate/vitamin B12 radioassay kit from Bio-Rad Laboratories (Hercules, CA). We defined biochemical B12 deficiency as serum levels ≤148 pmol/L, borderline deficiency as serum B12 >148 to ≤221 pmol/L, and normal as >221 pmol/L (26).

The main exposure of interest was metformin use. Using data collected in the prescription medicine questionnaire, those with type 2 diabetes were classified as currently using metformin therapy (alone or in combination therapy) versus those not currently using metformin. Length of metformin therapy was used to assess the relationship between duration of metformin therapy and biochemical B12 deficiency. In the final analysis, two control groups were used to allow the comparison of those with type 2 diabetes taking metformin with those with type 2 diabetes not taking metformin and those without diabetes.

To determine whether the association between metformin and biochemical B12 deficiency is modified by supplemental B12 intake, data from the dietary supplement questionnaire were used. Information regarding the dose and frequency was used to calculate average daily supplemental B12 intake. We categorized supplemental B12 intake as 0 μg (no B12 containing supplement), >0–6 μg, >6–25 μg, and >25 μg. The lower intake group, >0–6 μg, includes 6 μg, the amount of vitamin B12 typically found in over-the-counter multivitamins, and 2.4 μg, the daily amount the IOM recommends for all adults ≥50 years of age to consume through supplements or fortified food (1). The next group, >6–25 μg, includes 25 μg, the amount available in many multivitamins marketed toward senior adults. The highest group contains the amount found in high-dose B-vitamin supplements.


In the final analysis, there were 575 U.S. adults ≥50 years of age with type 2 diabetes using metformin, 1,046 with type 2 diabetes not using metformin, and 6,867 without diabetes. The demographic and biological characteristics of the groups are shown in Table 1. Among metformin users, mean age was 63.4 ± 0.5 years, 50.3% were male, 66.7% were non-Hispanic white, and 40.7% used a supplement containing B12. The median duration of metformin use was 5 years. Compared with those with type 2 diabetes not taking metformin, metformin users were younger (P < 0.0001), reported a lower prevalence of insulin use (P < 0.001), and had a shorter duration of diabetes (P = 0.0207). Compared with those without diabetes, metformin users had a higher proportion of nonwhite racial groups (P< 0.0001), a higher proportion of obesity (P < 0.0001), a lower prevalence of macrocytosis (P = 0.0017), a lower prevalence of supplemental folic acid use (P = 0.0069), a lower prevalence of supplemental vitamin B12 use (P = 0.0180), and a lower prevalence of calcium supplement use (P = 0.0002). There was a twofold difference in the prevalence of anemia among those with type 2 diabetes versus those without, and no difference between the groups with diabetes.    

Association of Biochemical B12Deficiency With Metformin Therapy and Vitamin B12Supplements

Demographic and biological characteristics of U.S. adults ≥50 years of age: NHANES 1999–2006

Table 1
The geometric mean serum B12 concentration among those with type 2 diabetes taking metformin was 317.5 pmol/L. This was significantly lower than the geometric mean concentration in those with type 2 diabetes not taking metformin (386.7 pmol/L; P = 0.0116) and those without diabetes (350.8 pmol/L; P = 0.0011). As seen in Fig. 1, the weighted prevalence of biochemical B12 deficiency adjusted for age, race, and sex was 5.8% for those with type 2 diabetes taking metformin, 2.2% for those with type 2 diabetes not taking metformin (P = 0.0002), and 3.3% for those without diabetes (P = 0.0026). Among the three aforementioned groups, borderline deficiency was present in 16.2, 5.5, and 8.8%, respectively (P < 0.0001). Applying the Fleiss formula for calculating attributable risk from cross-sectional data (27), among all of the cases of biochemical B12 deficiency, 3.5% of the cases were attributable to metformin use; and among those with diabetes, 41% of the deficient cases were attributable to metformin use. When the prevalence of biochemical B12 deficiency among those with diabetes taking metformin was analyzed by duration of metformin therapy, there was no notable increase in the prevalence of biochemical B12 deficiency as the duration of metformin use increased. The prevalence of biochemical B12 deficiency was 4.1% among those taking metformin <1 year, 6.3% among those taking metformin ≥1–3 years, 4.1% among those taking metformin >3–10 years, and 8.1% among those taking metformin >10 years (P = 0.3219 for <1 year vs. >10 years). Similarly, there was no clear increase in the prevalence of borderline deficiency as the duration of metformin use increased (15.9% among those taking metformin >10 years vs. 11.4% among those taking metformin <1 year; P = 0.4365).
Figure 1
Weighted prevalence of biochemical B12 deficiency and borderline deficiency adjusted for age, race, and sex in U.S. adults ≥50 years of age: NHANES 1999–2006. Black bars are those with type 2 diabetes on metformin, gray bars are those with type 2 diabetes not on metformin, and the white bars are those without diabetes. *P = 0.0002 vs. type 2 diabetes on metformin. †P < 0.0001 vs. type 2 diabetes on metformin. ‡P = 0.0026 vs. type 2 diabetes on metformin.
Table 2 presents a stratified analysis of the weighted prevalence of biochemical B12 deficiency and borderline deficiency by B12supplement use. For those without diabetes, B12 supplement use was associated with an ∼66.7% lower prevalence of both biochemical B12deficiency (4.8 vs. 1.6%; P < 0.0001) and borderline deficiency (16.6 vs. 5.5%; P < 0.0001). A decrease in the prevalence of biochemical B12deficiency was seen at all levels of supplemental B12 intake compared with nonusers of supplements. Among those with type 2 diabetes taking metformin, supplement use was not associated with a decrease in the prevalence of either biochemical B12 deficiency (5.6 vs. 5.3%; P= 0.9137) or borderline deficiency (15.5 vs. 8.8%; P = 0.0826). Among the metformin users who also used supplements, those who consumed >0–6 μg of B12 had a prevalence of biochemical B12 deficiency of 14.1%. However, consumption of a supplement containing >6 μg of B12 was associated with a prevalence of biochemical B12 deficiency of 1.8% (P = 0.0273 for linear trend). Similar trends were seen in the association of supplemental B12 intake and the prevalence of borderline deficiency. For those with type 2 diabetes not taking metformin, supplement use was also not associated with a decrease in the prevalence of biochemical B12 deficiency (2.1 vs. 2.0%; P = 0.9568) but was associated with a 54% reduction in the prevalence of borderline deficiency (7.8 vs. 3.4%; P = 0.0057 for linear trend).
Table 2
Comparison of average daily B12 supplement intake by weighted prevalence of biochemical B12 deficiency (serum B12 ≤148 pmol/L) and borderline deficiency (serum B12 >148 to ≤221 pmol/L) among U.S. adults ≥50 years of age: NHANES 1999–2006.
Table 3 demonstrates the association of various risk factors with biochemical B12 deficiency. Metformin therapy was associated with biochemical B12 deficiency (odds ratio [OR] 2.89; 95% CI 1.33–6.28) and borderline deficiency (OR 2.32; 95% CI 1.31–4.12) in a crude model (results not shown). After adjusting for age, BMI, and insulin and supplement use, metformin maintained a significant association with biochemical B12 deficiency (OR 2.92; 95% CI 1.28–6.66) and borderline deficiency (OR 2.16; 95% CI 1.22–3.85). Similar to Table 2, B12 supplements were protective against borderline (OR 0.43; 95% CI 0.23–0.81), but not biochemical, B12 deficiency (OR 0.76; 95% CI 0.34–1.70) among those with type 2 diabetes. Among those without diabetes, B12 supplement use was ∼70% protective against biochemical B12 deficiency (OR 0.26; 95% CI 0.17–0.38) and borderline deficiency (OR 0.27; 95% CI 0.21–0.35).
Table 3
Polytomous logistic regression for potential risk factors of biochemical B12 deficiency and borderline deficiency among U.S. adults ≥50 years of age: NHANES 1999–2006, OR (95% CI)

The IOM has highlighted the detection and diagnosis of B12 deficiency as a high-priority topic for research (1). Our results suggest several findings that add to the complexity and importance of B12 research and its relation to diabetes, and offer new insight into the benefits of B12 supplements. Our data confirm the relationship between metformin and reduced serum B12 levels beyond the background prevalence of biochemical B12 deficiency. Our data demonstrate that an intake of >0–6 μg of B12, which includes the dose most commonly found in over-the-counter multivitamins, was associated with a two-thirds reduction of biochemical B12 deficiency and borderline deficiency among adults without diabetes. This relationship has been previously reported with NHANES and Framingham population data (4,29). In contrast, we did not find that >0–6 μg of B12 was associated with a decrease in the prevalence of biochemical B12 deficiency or borderline deficiency among adults with type 2 diabetes taking metformin. This observation suggests that metformin reduces serum B12 by a mechanism that is additive to or different from the mechanism in older adults. It is also possible that metformin may exacerbate the deficiency among older adults with low serum B12. Our sample size was too small to determine which amount >6 μg was associated with maximum protection, but we did find a dose-response trend.

We were surprised to find that those with type 2 diabetes not using metformin had the lowest prevalence of biochemical B12 deficiency. It is possible that these individuals may seek medical care more frequently than the general population and therefore are being treated for their biochemical B12 deficiency. Or perhaps, because this population had a longer duration of diabetes and a higher proportion of insulin users compared with metformin users, they have been switched from metformin to other diabetic treatments due to low serum B12 concentrations or uncontrolled glucose levels and these new treatments may increase serum B12 concentrations. Despite the observed effects of metformin on serum B12 levels, it remains unclear whether or not this reduction is a public health concern. With lifetime risks of diabetes estimated to be one in three and with metformin being a first-line intervention, it is important to increase our understanding of the effects of oral vitamin B12 on metformin-associated biochemical deficiency (20,21).

The strengths of this study include its nationally representative, population-based sample, its detailed information on supplement usage, and its relevant biochemical markers. This is the first study to use a nationally representative sample to examine the association between serum B12 concentration, diabetes status, and metformin use as well as examine how this relationship may be modified by vitamin B12 supplementation. The data available regarding supplement usage provided specific information regarding dose and frequency. This aspect of NHANES allowed us to observe the dose-response relationship in Table 2 and to compare it within our three study groups.

This study is also subject to limitations. First, NHANES is a cross-sectional survey and it cannot assess time as a factor, and therefore the results are associations and not causal relationships. A second limitation arises in our definition of biochemical B12 deficiency. There is no general consensus on how to define normal versus low serum B12levels. Some researchers include the functional biomarker methylmalonic acid (MMA) in the definition, but this has yet to be agreed upon (3034). Recently, an NHANES roundtable discussion suggested that definitions of biochemical B12 deficiency should incorporate one biomarker (serum B12 or holotranscobalamin) and one functional biomarker (MMA or total homocysteine) to address problems with sensitivity and specificity of the individual biomarkers. However, they also cited a need for more research on how the biomarkers are related in the general population to prevent misclassification (34). MMA was only measured for six of our survey years; one-third of participants in our final analysis were missing serum MMA levels. Moreover, it has recently been reported that MMA values are significantly greater among the elderly with diabetes as compared with the elderly without diabetes even when controlling for serum B12 concentrations and age, suggesting that having diabetes may independently increase the levels of MMA (35). This unique property of MMA in elderly adults with diabetes makes it unsuitable as part of a definition of biochemical B12 deficiency in our specific population groups. Our study may also be subject to misclassification bias. NHANES does not differentiate between diabetes types 1 and 2 in the surveys; our definition may not capture adults with type 2 diabetes exclusively. Additionally, we used responses to the question “Have you received a physician’s diagnosis of diabetes” to categorize participants as having or not having diabetes. Therefore, we failed to capture undiagnosed diabetes. Finally, we could only assess current metformin use. We cannot determine if nonmetformin users have ever used metformin or if they were not using it at the time of the survey.

Our data demonstrate several important conclusions. First, there is a clear association between metformin and biochemical B12 deficiency among adults with type 2 diabetes. This analysis shows that 6 μg of B12 offered in most multivitamins is associated with two-thirds reduction in biochemical B12 deficiency in the general population, and that this same dose is not associated with protection against biochemical B12 deficiency among those with type 2 diabetes taking metformin. Our results have public health and clinical implications by suggesting that neither 2.4 μg, the current IOM recommendation for daily B12 intake, nor 6 μg, the amount found in most multivitamins, is sufficient for those with type 2 diabetes taking metformin.

This analysis suggests a need for further research. One research design would be to identify those with biochemical B12 deficiency and randomize them to receive various doses of supplemental B12chronically and then evaluate any improvement in serum B12concentrations and/or clinical outcomes. Another design would use existing cohorts to determine clinical outcomes associated with biochemical B12 deficiency and how they are affected by B12supplements at various doses. Given that a significant proportion of the population ≥50 years of age have biochemical B12 deficiency and that those with diabetes taking metformin have an even higher proportion of biochemical B12 deficiency, we suggest that support for further research is a reasonable priority.


One research design would be to identify those with biochemical B12 deficiency and randomize them to receive various doses of supplemental B12chronically and then evaluate any improvement in serum B12concentrations and/or clinical outcomes. Another design would use existing cohorts to determine clinical outcomes associated with biochemical B12 deficiency and how they are affected by B12supplements at various doses.
This is of considerable interest.  As far as I can see, there is insufficient data presented to discern all of the variables entangled.  In a study of 8000 hemograms several years ago, it was of some interest that there were a large percentage of patients who were over age 75 years having a MCV of 94 – 100, not considered indicative of macrocytic anemia.  It would have been interesting to explore that set of the data further.

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Protein Energy Malnutrition and Early Child Development

Curator: Larry H. Bernstein, MD, FCAP



In the preceding articles we have seen that poverty and low social class combined with cultural strictures or dependence on a sulfur-poor diet results in childhood stunting and impaired brain development. This is a global health issue.

Protein-Energy Malnutrition

  • Author: Noah S Scheinfeld, JD, MD, FAAD; Chief Editor: Romesh Khardori, MD, PhD, FACP

The World Health Organization (WHO)[1] defines malnutrition as “the cellular imbalance between the supply of nutrients and energy and the body’s demand for them to ensure growth, maintenance, and specific functions.” The term protein-energy malnutrition (PEM) applies to a group of related disorders that includemarasmus, kwashiorkor (see the images below), and intermediate states of marasmus-kwashiorkor. The term marasmus is derived from the Greek wordmarasmos, which means withering or wasting. Marasmus involves inadequate intake of protein and calories and is characterized by emaciation. The term kwashiorkor is taken from the Ga language of Ghana and means “the sickness of the weaning.” Williams first used the term in 1933, and it refers to an inadequate protein intake with reasonable caloric (energy) intake. Edema is characteristic of kwashiorkor but is absent in marasmus.

Studies suggest that marasmus represents an adaptive response to starvation, whereas kwashiorkor represents a maladaptive response to starvation. Children may present with a mixed picture of marasmus and kwashiorkor, and children may present with milder forms of malnutrition. For this reason, Jelliffe suggested the term protein-calorie (energy) malnutrition to include both entities.
Although protein-energy malnutrition affects virtually every organ system, this article primarily focuses on its cutaneous manifestations. Patients with protein-energy malnutrition may also have deficiencies of vitamins, essential fatty acids, and trace elements, all of which may contribute to their dermatosis.

In general, marasmus is an insufficient energy intake to match the body’s requirements. As a result, the body draws on its own stores, resulting in emaciation. In kwashiorkor, adequate carbohydrate consumption and decreased protein intake lead to decreased synthesis of visceral proteins. The resulting hypoalbuminemia contributes to extravascular fluid accumulation. Impaired synthesis of B-lipoprotein produces a fatty liver.

Protein-energy malnutrition also involves an inadequate intake of many essential nutrients. Low serum levels of zinc have been implicated as the cause of skin ulceration in many patients. In a 1979 study of 42 children with marasmus, investigators found that only those children with low serum levels of zinc developed skin ulceration. Serum levels of zinc correlated closely with the presence of edema, stunting of growth, and severe wasting. The classic “mosaic skin” and “flaky paint” dermatosis of kwashiorkor bears considerable resemblance to the skin changes of acrodermatitis enteropathica, the dermatosis of zinc deficiency.

In 2007, Lin et al[2] stated that “a prospective assessment of food and nutrient intake in a population of Malawian children at risk for kwashiorkor” found “no association between the development of kwashiorkor and the consumption of any food or nutrient.”

Marasmus and kwashiorkor can both be associated with impaired glucose clearance that relates to dysfunction of pancreatic beta-cells.[3] In utero, plastic mechanisms appear to operate, adjusting metabolic physiology and adapting postnatal undernutrition and malnutrition to define whether marasmus and kwashiorkor will develop.[4]

In 2012, a report from Texas noted an 18-month-old infant with type 1 glutaric acidemia who had extensive desquamative plaques, generalized nonpitting edema, and red-tinged sparse hair, with low levels of zinc, alkaline phosphatase, albumin, and iron. This patient has a variation on kwashiorkor, and the authors suggest that it be termed acrodermatitis dysmetabolica.[5] On the same note, a boy aged 18 months with type 1 glutaric acidemia suffered from zinc deficiency and acquired protein energy malnutrition.[6]

For complex reasons, sickle cell anemia can predispose suffers to protein malnutrition.[7]

Protein energy malnutrition ramps up arginase activity in macrophages and monocytes.[8]

Protein energy malnutrition (PEM), brain and various facets of child development.

Protein energy malnutrition (PEM) is a global problem. Nearly 150 million children under 5 years in the world and 70-80 million in India suffer from PEM, nearly 20 million in the world and 4 million in India suffer from severe forms of PEM, viz., marasmus, kwashiorkor and marasmic kwashiorkor. The studies in experimental animals in the west and children in developing countries have revealed the adverse effects of PEM on the biochemistry of developing brain which leads to tissue damage and tissue contents, growth arrest, developmental differentiation, myelination, reduction of synapses, synaptic transmitters and overall development of dendritic activity. Many of these adverse effects have been described in children in clinical data, biochemical studies, reduction in brain size, histology of the spinal cord, quantitative studies and electron microscopy of sural nerve, neuro -CT scan, magnetic resonance imaging (MRI) and morphological changes in the cerebellar cells. Longer the PEM, younger the child, poorer the maternal health and literacy, more adverse are the effects of PEM on the nervous system. Just like the importance of nutrients on the developing brain, so are the adverse effects on the child development of lack of environmental stimulation, emotional support and love and affection to the child. When both the adverse factors are combined, the impact is severe. Hence prevention of PEM in pregnant and lactating mothers, breast feeding, adequate home based supplements, family support and love will improve the physical growth, mental development, social competence and academic performance of the child. Hence nutritional rehabilitation, psychosocial and psychomotor development of the child should begin in infancy and continue throughout. It should be at all levels, most important being in family, school, community and various intervention programmes, local, regional and national. Moreover medical students, health personnel, all medical disciplines concerned with total health care and school teachers should learn and concentrate on the developmental stimulation and enrichment of the child.

Cognitive development in children with chronic protein energy malnutrition

Behav Brain Funct. 2008; 4: 31. 
Background: Malnutrition is associated with both structural and functional pathology of the brain. A wide range of cognitive deficits has been reported in malnourished children. Effect of chronic protein energy malnutrition (PEM) causing stunting and wasting in children could also affect the ongoing development of higher cognitive processes during childhood (>5 years of age). The present study examined the effect of stunted growth on the rate of development of cognitive processes using neuropsychological measures.
Methods: Twenty children identified as malnourished and twenty as adequately nourished in the age groups of 5–7 years and 8–10 years were examined. NIMHANS neuropsychological battery for children sensitive to the effects of brain dysfunction and age related improvement was employed. The battery consisted of tests of motor speed, attention, visuospatial ability, executive functions, comprehension and learning and memory
Results: Development of cognitive processes appeared to be governed by both age and nutritional status. Malnourished children performed poor on tests of attention, working memory, learning and memory and visuospatial ability except on the test of motor speed and coordination. Age related improvement was not observed on tests of design fluency, working memory, visual construction, learning and memory in malnourished children. However, age related improvement was observed on tests of attention, visual perception, and verbal comprehension in malnourished children even though the performance was deficient as compared to the performance level of adequately nourished children.
Conclusion: Chronic protein energy malnutrition (stunting) affects the ongoing development of higher cognitive processes during childhood years rather than merely showing a generalized cognitive impairment. Stunting could result in slowing in the age related improvement in certain and not all higher order cognitive processes and may also result in long lasting cognitive impairments.
Malnutrition is the consequence of a combination of inadequate intake of protein, carbohydrates, micronutrients and frequent infections [1]. In India malnutrition is rampant. WHO report states that for the years 1990–1997 52% of Indian children less than 5 years of age suffer from severe to moderate under nutrition [2]. About 35% of preschool children in sub-Saharan Africa are reported to be stunted [3]. Malnutrition is associated with both structural and functional pathology of the brain. Structurally malnutrition results in tissue damage, growth retardation, disorderly differentiation, reduction in synapses and synaptic neurotransmitters, delayed myelination and reduced overall development of dendritic arborization of the developing brain. There are deviations in the temporal sequences of brain maturation, which in turn disturb the formation of neuronal circuits [1]. Long term alterations in brain function have been reported which could be related to long lasting cognitive impairments associated with malnutrition [4]. A wide range of cognitive deficits has been observed in malnourished children in India. In a study, malnourished children were assessed on the Gessell’s developmental schedule from 4 to 52 weeks of age. Children with grades II and III malnutrition had poor development in all areas of behaviour i.e., motor, adaptive, language and personal social [5]. Rural children studying in primary school between the ages of 6–8 years were assessed on measures of social maturity (Vineland social maturity scale), visuomotor co-ordination (Bender gestalt test), and memory (free recall of words, pictures and objects). Malnutrition was associated with deficits of social competence, visuomotor coordination and memory. Malnutrition had a greater effect on the immediate memory of boys as compared with those of girls. Malnourished boys had greater impairment of immediate memory for words, pictures and objects, while malnourished girls had greater impairment of immediate memory for only pictures. Delayed recall of words and pictures of malnourished boys was impaired. Malnourished girls had an impairment of delayed recall of only words. The same authors measured the intelligence of malnourished children using Malin’s Indian adaptation of the Wechsler’s intelligence scale for children. IQ scores decreased with the severity of malnutrition. Significant decreases were observed in performance IQ, as well as on the subtests of information and digit span among the verbal subtests [6]. The above study has shown that though there is decrease in full scale IQ, yet performance on all the subtests was not affected. This suggests that malnutrition may affect different neuropsychological functions to different degrees. Studies done in Africa and South America have focused on the effect of stunted growth on cognitive abilities using verbal intelligence tests based on assessment of reasoning [7]. Such an assessment does not provide a comprehensive and specific assessment of cognitive processes like attention, memory, executive functions, visuo-spatial functions, comprehension as conducted in the present study. Information about the functional status of specific cognitive processes has implications for developing a cognitive rehabilitation program for malnourished children. A neuropsychological assessment would throw light on functional status of brain behaviour relationships affected by malnutrition. Deficits of cognitive, emotional and behavioural functioning are linked to structural abnormalities of different regions of the brain. Brain structures and brain circuits compute different components of cognitive processes [8]. Malnutrition has long lasting effects in the realm of cognition and behaviour, although the cognitive processes like executive functions have not been fully assessed [9]. The differential nature of cognitive deficits associated with malnutrition suggests that different areas of the brain are compromised to different degrees. A neuropsychological assessment would be able to delineate the pattern of brain dysfunction. Malnutrition is a grave problem in our country as 52% of our children are malnourished. Effects of protein-calorie malnutrition are inextricably blended with the effects of social cultural disadvantage; even within the disadvantaged class, literacy environment at home and parental expectation regarding children’s education are powerful variables. Perhaps membership in a higher caste confers some advantage in regard to home literacy, and parental expectation. Short and tall children do differ in some cognitive tests, but not in all as demonstrated in a study done in Orissa, India [10]. But whether or not stunted growth alone is the causative variable for cognitive weakness is not determined as yet. Moreover, the functional integrity of specific cognitive processes is less clear. Chronic PEM resulting in stunting and wasting could result in delay in the development of cognitive processes or in permanent cognitive impairments. Neuropsychological measures can demonstrate delay in normally developing cognitive processes as well as permanent cognitive deficits.
Children in the age range of 5–10 years attending a corporation school in the city of Bangalore participated in the study. Corporation schools in India are government schools with minimal fee attended by children from lowmiddle class. There were 20 children in adequately nourished group and 20 in the malnourished group. The gender distribution was equal. Children in both the groups were from the same ethnic/language background. They were natives of Karnataka living in Bangalore.
After identifying the malnourished and adequately nourished children the coloured progressive matrices test [12] was administered to rule out mental retardation. Children falling at or below the fifth percentile were excluded from the sample, as the 5th percentile is suggestive of intellectually defective range. The percentile points were calculated from the raw scores using Indian norms [13]. Mental retardation was ruled out as otherwise scores on neuropsychological tests would be uniformly depressed and a differentiation of deficits might not occur. Intelligence was not treated as a covariate in the study. The groups did not differ significantly in their scores on CPM (a screening instrument to rule out intellectual impairment in both the groups).
Table 1: Demographic details of the participants
                            Adequately nourished N = 20                  Malnourished N = 20
Mean age              5–7 years        8–10 years                     5–7 years      8–10 years
                               5.8 years        8.8 years                          6.3 years      9.3 years
Gender                   Girls:10           Boys: 10                          Girls:10         Boys: 10
Stunted %
(height for age -2 SD from the median) —-                                  70%
Stunted and wasted %
(height for age and
weight for height: -2 SD from the median) —-                               30%
Exclusion of behaviour problems and history of neurological disorders The children’s behaviour questionnaire form B [14] was administered to the class teachers of the identified children. Children who scored above the cut off score of 9 were not included in the sample. The personal data sheet was filled in consultation with the parents and teachers to rule out any history of any neurological/psychiatric disorders including head injury and epilepsy and one child with epilepsy was excluded. This was one of the exclusion criteria.
Exclusion of behaviour problems and history of neurological disorders The children’s behaviour questionnaire form B [14] was administered to the class teachers of the identified children. Children who scored above the cut off score of 9 were not included in the sample. The personal data sheet was filled in consultation with the parents and teachers to rule out any history of any neurological/psychiatric disorders including head injury and epilepsy and one child with epilepsy was excluded. This was one of the exclusion criteria.
The tests have been grouped under specific cognitive domains on the basis of theoretical rationale and factor analysis. Factor analysis has been done for the battery and the grouping of tests under cognitive functions like executive functions, visuospatial functions, comprehension and learning and memory was done on the basis of the clustering observed in factor analysis as well as on theoretical grounds
The neuropsychological battery consisted of the following tests:
1. Motor speed  Finger tapping test [15]
2. Expressive speech  Expressive speech test was administered to rule out speech related deficits
3. Attention  Color trails test [18] is a measure of focused attention and conceptual tracking.
4. Color cancellation test [21] is a measure of visual scanning/selective attention
5. Executive functions FAS phonemic fluency test is a measure of verbal fluency.
6. Design fluency test [24] is a measure of design fluency, cognitive flexibility and imaginative capacity.
7. Visuo-spatial working memory span task [23]: This test is a measure of visuo-spatial working memory (VSWM) span.
8. Visuospatial functions Motor-free visual perception test [29] is a measure of visuoperceptual ability, having 36 items for visual discrimination, visual closure, figure-ground, perceptual matching and visual memory. Since this test has been originally developed for children between 5–8 years of age, it was modified and items in increasing difficulty level were added by the authors to make it applicable for the children above 8 years. Number of correct responses comprises the score.
9. Picture completion test [30] is a measure of visuoconceptual ability, visual organization and visuo-conceptual reasoning.
10. Block design test [30] is a measure of visuoconstructive ability.
11. Comprehension, learning and memory Token test [31] is a measure of verbal comprehension of commands of increasing complexity.
12. Rey’s auditory verbal learning test (RAVLT) [32] is a measure of verbal learning and memory.
13. Memory for designs test [34] is a measure of visual learning and memory.
Comparison between the performance of adequately nourished children and malnourished children Table 2.0 shows that malnourished group differed significantly from the adequately nourished group on tests of phonemic fluency, design fluency, selective attention, visuospatial working memory, visuospatial functions, verbal comprehension and verbal learning and memory showing poor performance. The two groups did not differ on the test of finger tapping. Since expressive speech was a question answer type assessment looking at repetitive speech, nominative speech and narrative speech, which is like an initial screening for aphasia, like symptoms. Since it did not give a quantitative score, hence was not taken for analysis. As a descriptive account of expressive speech it was observed that malnourished children did not have any difficulty with respect to expressive speech.
Comparison of age related differences in cognitive functions between adequately nourished and malnourished children Data was further subjected to post hoc analysis to compare the two groups across the two age groups to study the rate of improvement with age (Table 2). In both the age groups of 5–7 years and 8–10 years the adequately nourished children performed better than the malnourished children. Figures 1, 2, 3, 4, 5, 6 indicate age related improvement in performance across different cognitive functions in adequately nourished children as compared to malnourished children. Motor speed and coordination was not significantly affected in malnourished children as compared to the adequately nourished children (figure 1). The rate of age related improvement across the two age groups was found rapid on certain functions like selective attention (figure 2) and verbal fluency (figure 3) in malnourished children. However, working memory, design fluency, visuospatial functions, comprehension, learning, and memory showed slowing in terms of age related improvement in malnourished children. Most of the cognitive functions like design fluency (figure 3), working memory (figure 3), Visual perception (figure 4), visuoconceptual reasoning (figure 4), visual construction (figure 4), verbal comprehension (figure 5), verbal and visual memory (figures 6) have shown a very slow rate of improvement with respect to the difference in performance between the two age groups of 5–7 and 8–10 years. On the contrary functions like verbal fluency (figure 3), motor speed (figures 1), and selective attention (figure 2) showed similar rates of improvement in adequately nourished children and malnourished children while comparing the two age groups.
Table 2: Mean comparisons for the cognitive functions across the two age groups of adequately nourished and malnourished children (not shown)
Table 3: Post-hoc comparisons between adequately nourished and malnourished groups across the two age groups (not shown)
Figure 1 Age related comparisons between adequately nourished and malnourished children on motor speed (right and left hand) Age related comparisons between adequately nourished and malnourished children on motor speed (right and left hand). (not shown)
Figure 2 Age related comparisons between adequately nourished and malnourished children on selective attention (color cancellation test). (not shown)
Post-hoc comparisons were computed with Tukey’s posthoc tests to compare the means across age groups between malnourished and adequately nourished children for those test scores that showed significant effects. Hence, post hoc tests were not computed for the finger tapping test scores assessing motor speed. Table 3 presents the post-hoc results with the significance (probability level) levels of the differences across age groups and between adequately nourished and malnourished children. Post hoc results have been done to support our theoretical claims about the lack of age related improvement in certain cognitive functions on one hand and the nature of cognitive impairments on the other in malnourished children. Four comparisons were interpreted i.e., comparing performance between the two age groups of adequately nourished and malnourished children separately. The other comparison was between the adequately nourished and malnourished children for the age group of 5–7 years and similarly for the age group of 8–10 years. Results indicate age related differences within each group as well as between the two groups. Age related differences were found significant for some of the test scores between 5–7 and 8–10 year old children in the adequately nourished group but not for most of the test scores for malnourished group indicative of a delay in development of certain cognitive functions. Differences were found significant between the adequately nourished and malnourished children for the same age group for most of the test scores indicative of a deficit in a particular cognitive function. In few of the tests, performance was not found to be significantly different between the two age groups for both adequately nourished and malnourished children.
Discussion The findings of the present study could be discussed in terms of the effect of chronic malnutrition on neuropsychological performance and with respect to the rate of development of cognitive processes.
Effect of malnutrition on neuropsychological performance Our study indicates that malnourished children perform poor on most of the neuropsychological tests except that of motor speed as compared to adequately nourished children. Malnourished children showed poor performance on tests of higher cognitive functions like cognitive flexibility, attention, working memory, visual perception, verbal comprehension, and memory. These findings are supported by another study on Indian malnourished children, which reported memory impairments in undernourished children and spared fine motor coordination [36]. Malnourished children showed poor performance on novel tasks like tests of executive functions i.e., working memory spatial locations. Poor performance on the tests of fluency and working memory also coincides with very slow rate of improvement between the age groups of 5–7 years and 8–10 years. Poor performance on most of the neuropsychological tests indicated a diffuse impairment including attention, executive functions, visuospatial functions, comprehension and memory.
Effect of malnutrition on cognitive development Both the groups were tested on a neuropsychological battery, which has been found to be sensitive to age related differences in cognitive functions in children (5–15 years). The age trends reported in the present study are based on the assessment that employed the NIMHANS neuropsychological battery for children [13]. The test battery has been standardized based on the growth curve modeling approach for empirical validation of age-related differences in performance on neuropsychological tests. The tests in the battery were found sensitive to show age related differences.
Malnourished children showed poor performance with respect to age as compared to adequately nourished children. The performance of malnourished children in the 5–7 years age group was poor and much lower than the adequately nourished children and did not seem to show much improvement in the 8–10 years age group. The rate of cognitive development was found to be different for different cognitive functions. The rate of development was affected for some of the cognitive functions showing minimal age related improvement across the age range of 5–7 years and 8–10 years such as design fluency, working memory, visual construction, verbal comprehension, learning and memory for verbal and visual material. On the contrary, age related improvement was observed on certain other cognitive functions in malnourished children, where the level of performance was low for both the age groups but the rate of improvement between the two age groups was similar to adequately nourished children.
Not shown
Figure 3 Age related comparisons between adequately nourished and malnourished children on executive functions.
Note: VF: verbal fluency; DF: design fluency; WM: working memory; AN: adequately nourished; MN: malnourished.

MN 5–7 vs 8–10 p > .05 5–7 years AN vs MN p > .05 8–10 years AN vs MN p < .05 Visual memory (memory for designs test) AN 5–7 vs 8–10 p > .05 MN 5–7 vs 8–10 p > .05 5–7 years AN vs MN p < .05 8–10 years AN vs MN p < .05

Figure 4 Age related comparisons between adequately nourished and malnourished children on visuospatial functions.
Figure 5 Age related comparisons between adequately nourished and malnourished children on verbal comprehension and verbal learning.
Motor speed (right and left hand) was not found impaired in malnourished children and the rate of development was also found similar to adequately nourished children.
Executive functions such as design fluency, selective attention and working memory were found deficient in malnourished children also showing poor rate of improvement between the two age groups. All the three tests of executive functions like fluency, selective attention and working memory for spatial locations involved novel stimuli and performance required cognitive flexibility as well as faster information processing which was affected in malnourished children. Results also indicate that malnourished children showed a very slow rate of improvement on these functions.
Visuo-spatial functions like visual perception, visual construction and visuo-conceptual reasoning showed significantly poor performance when compared to the adequately nourished children but showed a steep age related improvement in performance. Performance on functions like visual perception (visual discrimination, perceptual matching, visual closure and visuospatial relationships) and visual construction was severely affected in malnourished children and also showed poor rate of improvement with age.
Verbal comprehension, learning and memory for verbal and visual material was found poor as compared to adequately nourished children but the rate of improvement between 5–7 years age group and 8–10 years age group was similar to that of adequately nourished children. These results suggest that development of comprehension with age might not be affected in malnourished children. However, other than the poor performance on the AVLT test of verbal learning, malnourished children also showed minimal improvement between the two age groups as compared to the greater magnitude of difference between the two age groups in adequately nourished children. Visual memory was most severely affected in malnourished children in terms of the poor performance on delayed recall on design learning test as well as in terms of the difference between the two age groups.
Malnutrition affects brain growth and development and hence future behavioral outcomes [37]. School-age children who suffered from early childhood malnutrition have generally been found to have poorer IQ levels, cognitive function, school achievement and greater behavioral problems than matched controls and, to a lesser extent, siblings. The disadvantages last at least until adolescence. There is no consistent evidence of a specific cognitive deficit [38]. The functional integrity of specific cognitive processes is less clear. Stunting in early childhood is common in developing countries and is associated with poorer cognition and school achievement in later childhood [39]. Deficits in children’s scores have been reported to be smaller at age 11 years than at age 8 years in a longitudinal study on malnourished children stunted children suggesting that adverse effects may decline over time [7]. In our study also all the children in malnourished group were stunted and the cross sectional assessment of age related improvement has shown similar rate of improvement across 5–7 years to 8–10 years age groups as observed in adequately nourished children though the baseline performance was low in malnourished children. These results indicate that the adverse effects of malnutrition (stunting in particular) may decline with age only for certain cognitive functions but the rate of cognitive development for most of the cognitive processes particularly higher cognitive processes including executive processes and visuospatial perception could be severely affected during the childhood years. Decline in the effects of malnutrition overtime has been reported to be independent of differences in educational, socioeconomic and psychosocial resources [7]. Hence, malnutrition (particularly stunting) may result in delayed development of cognitive processes during childhood years rather than a permanent generalized cognitive impairment.
The neuropsychological interpretation of the cognitive processes more severely affected in malnourished children suggests a diffuse cortical involvement. This is with reference to deficits pertaining to functions mediated by dorsolateral prefrontal cortex (poor performance on tests of attention, fluency and working memory), right parietal (poor performance on tests of visuospatial functions) and bilateral temporal cortex (poor performance on tests of comprehension, verbal learning, and memory for verbal and visual material). The prefrontal cortex may be particularly vulnerable to malnutrition [4]. The adverse effects of malnutrition (PEM-stunting) on cognitive development could be related to the delay in certain processes of structural and functional maturation like delayed myelination and reduced overall development of dendritic arborization of the developing brain [1].
The present study highlights two ways in which malnutrition particularly stunting could affect cognitive functions. On one hand age related improvement in cognitive performance is compromised and on the other hand there could be long lasting cognitive impairments as well. However, the effect is nor specific to a particular cognitive domain and is rather more diffuse. Results of the study also indicate that: certain cognitive functions could be vulnerable to the effect of malnutrition in terms of showing impairment but the rate of development of these functions may not be affected. On the other hand, rate of development of certain cognitive functions may be affected and may also show impairment when compared with adequately nourished children.
Conclusion Chronic protein energy malnutrition (stunting) results in cognitive impairments as well as slowing in the rate of the development of cognitive processes. Rate of development of cognitive functions may follow different patterns in children with malnutrition. Chronic protein energy malnutrition affects the development of cognitive processes differently during childhood years rather than merely showing an overall cognitive dysfunction as compared to adequately nourished children. Stunting could result in delay in the development of cognitive functions as well as in permanent cognitive impairments which show minimal improvement with increase in age. Rate of development of attention, executive functions like cognitive flexibility, working memory, visuospatial functions like visual construction is more severely affected by protein energy malnutrition in childhood years, a period that is marked by rapid ongoing development of cognitive functions.
The effects of protein energy malnutrition in early childhood on intellectual and motor abilities in later childhood and adolescence.
Dev Med Child Neurol. 1976 Jun;18(3):330-50.

Three groups of Ugandan children (20 in each group) and one comparison group of 20 children were examined between 11 and 17 years of age. The first three groups had been admitted to hospital for treatment of protein energy malnutrition between the ages of eight to 15, 16 to 21 and 22 to 27 months, respectively. The comparison group had not been clinically malnourished throughout the whole period up to 27 months of age. All the children came from one tribe and were individually matched for sex, age, education and home environment. It was found that the three malnourished groups fell significantly below the comparison group in anthropometric measurements and in tests of intellectual and motor abilities. No evidence was found for a relationship between the deficit and age at admission. Further analysis among the 60 malnourished children revealed that anthropometry and intellectual and motor abilities are the more affected the greater the degree of ‘chronic undernutrition’ at admission, but no correlation was found with the severity of the ‘acute malnutrition’. The results show a general impairment of intellectual abilities, with reasoning and spatial abilities most affected, memory and rote learning intermediately and language ability least, if at all, affected. These findings are discussed in the context of a comprehensive and critical appraisal of the existing literature.

Quake-Hit Nepal Gears up to Tackle Stunting in Children

By Gopal Sharma  July 08, 2015

HECHO, Nepal (Thomson Reuters Foundation) – Shanti Maharjan, who gave birth to a baby girl 10 days ago, has spent the last two months living under corrugated iron sheets with her husband and five others after two major earthquakes reduced her mud-and-brick home to rubble.

Adequate food, drinking water and aid such as tents and blankets have been hard to come by, she says, though scores of aid agencies rushed to the Himalayan nation to help survivors.
What worries the 26-year-old mother most is her inability to produce breastmilk for her new-born daughter, who she fears is at serious risk of malnutrition in the aftermath of the 7.8 and 7.3 magnitude quakes in April and May.

“The earthquake destroyed everything, including our food reserves,” said Maharjan, sitting under the iron sheeting on farmland on the outskirts of the capital, Kathmandu.

“There is not enough food. Getting meat, oil and fruits to eat is difficult in this situation. I am worried about my daughter’s nourishment,” she said as the baby, wrapped in a green cloth, lay sleeping on a wooden bed.

The government, aware that disruption caused by the quakes could worsen the country’s already high rate of child malnutrition is sending out teams of community nurses to give advice and food supplements to women and children in the affected areas.

A 2011 government study showed that more than 40% of Napel’s under-five-year-olds were stunted, showing that the country’s child malnutrition rate was one of the world’s highest.
Experts say the two quakes, which killed 8,895 people and destroyed half a million houses, could make things worse as survivors have inadequate food, water, shelter, healthcare and sanitation.

United Nations officials warn that the rate of stunting among children in the South Asian nation could return to the 2001 level of 57%, if authorities and aid agencies do not respond effectively.

“The risk of malnutrition is high and requires the nutrition and other sectors like agriculture, health, water, sanitation, education and social protection to respond adequately,” said Stanley Chitekwe, UNICEF’s nutrition chief in Nepal.


Child malnutrition is an underlying cause of death for 3 million children annually around the world – nearly half of all child deaths – most of whom die from preventable illnesses such as diarrhoea due to weak immune systems.

Those lucky enough to survive grow up without enough energy, protein, vitamins and minerals, causing their brains and bodies to be stunted, and they are often unable to fulfill their potential.

Government officials admit the challenges, citing data showing that almost 70% of Nepali children under the age of two suffer from anaemia caused by iron deficiency.

“This shows that (poor) nutrition is a very big problem. The earthquake will further worsen the situation because people simply don’t have enough to eat, let alone have a nutritious diet,” said Health Ministry official Krishna Prasad Paudel.

Supported by UNICEF, authorities have now launched a drive to reach out to more than 500,000 women and children who need supplementary food and medicines.

More than 10,000 female community volunteers will be fanning out across 14 districts affected by the earthquakes, visiting devastated towns and villages and speaking to new and expectant mothers about breast-feeding their infants.

The volunteers will also advise families on eating locally available nutritious foods such as green vegetables and meat and will distribute vitamin A, iron and folic acid, and other micronutrient supplements to pregnant and breastfeeding women.

In Imadole, a prosperous district on the outskirts of the ancient town of Patan, health volunteer Urmila Sharma Dahal found an extremely thin two-year-old boy weighing 7.5 kg (16.5 pounds) last week, suffering from severe acute malnutrition.

Dahal said she provided his family with sachets of ready-to-use therapeutic food – a paste of peanut, sugar, milk powder, vitamin and oil – and the child gained nearly a kilo (2.2 pounds) in weight in just seven days.

“It does not take much. It can be done with small but right interventions,” said Dahal as she sat next to the child in the family’s brick-and-cement home.

Protein-energy malnutrition occurs due to inadequate intake of food and is a major cause of morbidity and mortality in children in developing countries (Grover and Ee 2009).

Protein energy malnutrition (PEM) has significant negative impacts on children’s growth and development (Grover and Ee 2009). Chronic PEM causes children to have stunted growth (low height for age) and to be underweight (low weight for age); it is estimated that among children under age five, one in every four is stunted and one in every six is underweight. PEM also causes two specific conditions in children: marasmus, which is characterized by an emaciated appearance, and kwashiorkor, in which children develop swollen bellies due to edema (abnormal accumulation of fluid) and discoloration of the hair because of pigment loss among other symptoms (UNWFP 2013b, Ahmed et al. 2012). Countries in sub-Saharan Africa and south Asia have the highest proportions of children suffering from PEM (UNWFP 2013a).

PEM causes direct mortality in children and also increases vulnerability to other serious diseases including diarrhea, pneumonia, and malaria. Children suffering from PEM have compromised immune systems, making them particularly susceptible to infectious diseases.  Furthermore, PEM has negative impacts on children’s brain development, resulting in issues with memory and delayed motor function; these children have decreased ability to learn and have lower productivity as adults. PEM also has serious and potentially long-term impacts on other organ systems including the cardiovascular, respiratory, and gastrointestinal systems (Grover and Ee 2009).

Many adults in developing countries also suffer from PEM, with women disproportionately impacted compared with men, particularly in south Asian countries (UNWFP 2013a). Pregnant women who are undernourished can fall even further behind in their nutritional status due to the increased demand for nutrients by the developing fetus. Women who don’t gain sufficient weight during pregnancy are at increased risk for complications including maternal morbidity and mortality, low birth weight, and neonatal mortality. These women can also have difficulty providing sufficient quantities of breast milk, leading to malnutrition among neonates (Ahmed et al. 2012).

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Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD


Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

SACHS FLYER 2014 Metabolomics SeriesDindividualred-page2

which is now available on Amazon Kindle at

This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:

Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 






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Introduction to Metabolomics

Introduction to Metabolomics

Author: Larry H. Bernstein, MD, FCAP


This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time bachalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career.

In the Preface, I failed to disclose that the term Metabolomics applies to plants, animals, bacteria, and both prokaryotes and eukaryotes.  The metabolome for each organism is unique, but from an evolutionary perspective has metabolic pathways in common, and expressed in concert with the environment that these living creatures exist. The metabolome of each has adaptive accommodation with suppression and activation of pathways that are functional and necessary in balance, for its existence.  Was it William Faulkner who said in his Nobel Prize acceptance that mankind shall not merely exist, but survive? That seems to be the overlying theme for all of life. If life cannot persist, a surviving “remnant” might continue. The history of life may well be etched into the genetic code, some of which is not expressed.

This work is apportioned into chapters in a sequence that is first directed at the major sources for the energy and the structure of life, in the carbohydrates, lipids, and fats, which are sourced from both plants and animals, and depending on their balance, results in an equilibrium, and a disequilibrium we refer to as disease.  There is also a need to consider the nonorganic essentials which are derived from the soil, from water, and from the energy of the sun and the air we breathe, or in the case of water-bound metabolomes, dissolved gases.

In addition to the basic essential nutrients and their metabolic utilization, they are under cellular metabolic regulation that is tied to signaling pathways.  In addition, the genetic expression of the organism is under regulatory control by the interaction of RNAs that interact with the chromatin genetic framework, with exosomes, and with protein modulators.This is referred to as epigenetics, but there are also drivers of metabolism that are shaped by the interactions between enzymes and substartes, and are related to the tertiary structure of a protein.  The framework for diseases in a separate chapter.  Pharmaceutical interventions that are designed to modulate specific metabolic targets are addressed as the pathways are unfolded. Neutraceuticals and plant based nutrition are covered in Chapter 8.

Chapter 1: Metabolic Pathways

Chapter 2. Lipid Metabolism

Chapter 3. Cell Signaling

Chapter 4. Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8. Impairments in Pathological States: Endocrine Disorders; Stress Hypermetabolism and Cancer

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Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics

Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics

Reviewer and Curator: Larry H. Bernstein, MD, FCAP 


The human genome is estimated to encode over 30,000 genes, and to be responsible for generating more than 100,000 functionally distinct proteins. Understanding the interrelationships among

  1. genes,
  2. gene products, and
  3. dietary habits

is fundamental to identifying those who will benefit most from or be placed at risk by intervention strategies.

Unraveling the multitude of

  • nutrigenomic,
  • proteomic, and
  • metabolomic patterns

that arise from the ingestion of foods or their

  • bioactive food components

will not be simple but is likely to provide insights into a tailored approach to diet and health. The use of new and innovative technologies, such as

  • microarrays,
  • RNA interference, and
  • nanotechnologies,

will provide needed insights into molecular targets for specific bioactive food components and

  • how they harmonize to influence individual phenotypes(1).

Nutrigenetics asks the question how individual genetic disposition, manifesting as

  • single nucleotide polymorphisms,
  • copy-number polymorphisms and
  • epigenetic phenomena,

affects susceptibility to diet.

Nutrigenomics addresses the inverse relationship, that is how diet influences

  • gene transcription,
  • protein expression and
  • metabolism.

A major methodological challenge and first pre-requisite of nutrigenomics is integrating

  • genomics (gene analysis),
  • transcriptomics (gene expression analysis),
  • proteomics (protein expression analysis) and
  • metabonomics (metabolite profiling)

to define a “healthy” phenotype. The long-term deliverable of nutrigenomics is personalised nutrition (2).

Science is beginning to understand how genetic variation and epigenetic events

  • alter requirements for, and responses to, nutrients (nutrigenomics).

At the same time, methods for profiling almost all of the products of metabolism in a single sample of blood or urine are being developed (metabolomics). Relations between

  • diet and nutrigenomic and metabolomic profiles and
  • between those profiles and health

have become important components of research that could change clinical practice in nutrition.

Most nutrition studies assume that all persons have average dietary requirements, and the studies often

  • do not plan for a large subset of subjects who differ in requirements for a nutrient.

Large variances in responses that occur when such a population exists

  • can result in statistical analyses that argue for a null effect.

If nutrition studies could better identify responders and differentiate them from nonresponders on the basis of nutrigenomic or metabolomic profiles,

  • the sensitivity to detect differences between groups could be greatly increased, and
  • the resulting dietary recommendations could be appropriately targeted (3).

In recent years, nutrition research has moved from classical epidemiology and physiology to molecular biology and genetics. Following this trend,

  • Nutrigenomics has emerged as a novel and multidisciplinary research field in nutritional science that
  • aims to elucidate how diet can influence human health.

It is already well known that bioactive food compounds can interact with genes affecting

  • transcription factors,
  • protein expression and
  • metabolite production.

The study of these complex interactions requires the development of

  • advanced analytical approaches combined with bioinformatics.

Thus, to carry out these studies

  • Transcriptomics,
  • Proteomics and
  • Metabolomics

approaches are employed together with an adequate integration of the information that they provide(4).

Metabonomics is a diagnostic tool for metabolic classification of individuals with the asset of quantitative, non-invasive analysis of easily accessible human body fluids such as urine, blood and saliva. This feature also applies to some extent to Proteomics, with the constraint that

  • the latter discipline is more complex in terms of composition and dynamic range of the sample.

Apart from addressing the most complex “Ome”, Proteomics represents

  • the only platform that delivers not only markers for disposition and efficacy
  • but also targets of intervention.

Application of integrated Omic technologies will drive the understanding of

  • interrelated pathways in healthy and pathological conditions and
  • will help to define molecular ‘switchboards’,
  • necessary to develop disease related biomarkers.

This will contribute to the development of new preventive and therapeutic strategies for both pharmacological and nutritional interventions (5).

Human health is affected by many factors. Diet and inherited genes play an important role. Food constituents,

  • including secondary metabolites of fruits and vegetables, may
  • interact directly with DNA via methylation and changes in expression profiles (mRNA, proteins)
  • which results in metabolite content changes.

Many studies have shown that

  • food constituents may affect human health and
  • the exact knowledge of genotypes and food constituent interactions with
  • both genes and proteins may delay or prevent the onset of diseases.

Many high throughput methods have been employed to get some insight into the whole process and several examples of successful research, namely in the field of genomics and transcriptomics, exist. Studies on epigenetics and RNome significance have been launched. Proteomics and metabolomics need to encompass large numbers of experiments and linked data. Due to the nature of the proteins, as well as due to the properties of various metabolites, experimental approaches require the use of

  • comprehensive high throughput methods and a sufficiency of analysed tissue or body fluids (6).

New experimental tools that investigate gene function at the subcellular, cellular, organ, organismal, and ecosystem level need to be developed. New bioinformatics tools to analyze and extract meaning

  • from increasingly systems-based datasets will need to be developed.

These will require, in part, creation of entirely new tools. An important and revolutionary aspect of “The 2010 Project”  is that it implicitly endorses

  • the allocation of resources to attempts to assign function to genes that have no known function.

This represents a significant departure from the common practice of defining and justifying a scientific goal based on the biological phenomena. The rationale for endorsing this radical change is that

  • for the first time it is feasible to envision a whole-systems approach to gene and protein function.

This whole-systems approach promises to be orders of magnitude more efficient than the conventional approach (7).

The Institute of Medicine recently convened a workshop to review the state of the various domains of nutritional genomics research and policy and to provide guidance for further development and translation of this knowledge into nutrition practice and policy (8). Nutritional genomics holds the promise to revolutionize both clinical and public health nutrition practice and facilitate the establishment of

(a) genome-informed nutrient and food-based dietary guidelines for disease prevention and healthful aging,

(b) individualized medical nutrition therapy for disease management, and

(c) better targeted public health nutrition interventions (including micronutrient fortification and supplementation) that

  • maximize benefit and minimize adverse outcomes within genetically diverse human populations.

As the field of nutritional genomics matures, which will include filling fundamental gaps in

  • knowledge of nutrient-genome interactions in health and disease and
  • demonstrating the potential benefits of customizing nutrition prescriptions based on genetics,
  • registered dietitians will be faced with the opportunity of making genetically driven dietary recommendations aimed at improving human health.

The new era of nutrition research translates empirical knowledge to evidence-based molecular science (9). Modern nutrition research focuses on

  • promoting health,
  • preventing or delaying the onset of disease,
  • optimizing performance, and
  • assessing risk.

Personalized nutrition is a conceptual analogue to personalized medicine and means adapting food to individual needs. Nutrigenomics and nutrigenetics

  • build the science foundation for understanding human variability in
  • preferences, requirements, and responses to diet and
  • may become the future tools for consumer assessment

motivated by personalized nutritional counseling for health maintenance and disease prevention.

The primary aim of ―omic‖ technologies is

  • the non-targeted identification of all gene products (transcripts, proteins, and metabolites) present in a specific biological sample.

By their nature, these technologies reveal unexpected properties of biological systems.

A second and more challenging aspect of ―omic‖ technologies is

  • the refined analysis of quantitative dynamics in biological systems (10).

For metabolomics, gas and liquid chromatography coupled to mass spectrometry are well suited for coping with

  • high sample numbers in reliable measurement times with respect to
  • both technical accuracy and the identification and quantitation of small-molecular-weight metabolites.

This potential is a prerequisite for the analysis of dynamic systems. Thus, metabolomics is a key technology for systems biology.

In modern nutrition research, mass spectrometry has developed into a tool

  • to assess health, sensory as well as quality and safety aspects of food.

In this review, we focus on health-related benefits of food components and, accordingly,

  • on biomarkers of exposure (bioavailability) and bioefficacy.

Current nutrition research focuses on unraveling the link between

  • dietary patterns,
  • individual foods or
  • food constituents and

the physiological effects at cellular, tissue and whole body level

  • after acute and chronic uptake.

The bioavailability of bioactive food constituents as well as dose-effect correlations are key information to understand

  • the impact of food on defined health outcomes.

Both strongly depend on appropriate analytical tools

  • to identify and quantify minute amounts of individual compounds in highly complex matrices–food or biological fluids–and
  • to monitor molecular changes in the body in a highly specific and sensitive manner.

Based on these requirements,

  • mass spectrometry has become the analytical method of choice
  • with broad applications throughout all areas of nutrition research (11).

Recent advances in high data-density analytical techniques offer unrivaled promise for improved medical diagnostics in the coming decade. Genomics, proteomics and metabonomics (as well as a whole slew of less well known ―omics‖ technologies) provide a detailed descriptor of each individual. Relating the large quantity of data on many different individuals to their current (and possibly even future) phenotype is a task not well suited to classical multivariate statistics. The datasets generated by ―omics‖ techniques very often violate the requirements for multiple regression. However, another statistical approach exists, which is already well established in areas such as medicinal chemistry and process control, but which is new to medical diagnostics, that can overcome these problems. This approach, called megavariate analysis (MVA),

  • has the potential to revolutionise medical diagnostics in a broad range of diseases.

It opens up the possibility of expert systems that can diagnose the presence of many different diseases simultaneously, and

  • even make exacting predictions about the future diseases an individual is likely to suffer from (12).

Cardiovascular diseases

Cardiovascular diseases are the leading cause of morbidity and mortality in Western countries. Although coronary thrombosis is the final event in acute coronary syndromes,

  • there is increasing evidence that inflammation also plays a role in development of atherosclerosis and its clinical manifestations, such as
  • myocardial infarction, stroke, and peripheral vascular disease.

The beneficial cardiovascular health effects of

  • diets rich in fruits and vegetables are in part mediated by their flavanol content.

This concept is supported by findings from small-scale intervention studies with surrogate endpoints including

  1. endothelium-dependent vasodilation,
  2. blood pressure,
  3. platelet function, and
  4. glucose tolerance.

Mechanistically, short term effects on endothelium-dependent vasodilation

  • following the consumption of flavanol-rich foods, as well as purified flavanols,
  • have been linked to an increased nitric oxide bioactivity.

The critical biological target(s) for flavanols have yet to be identified (13), but we are beginning to see over the horizon.

Nutritional sciences

Nutrition sciences apply

  1. transcriptomics,
  2. proteomics and
  3. metabolomics

to molecularly assess nutritional adaptations.

Transcriptomics can generate a

  • holistic overview on molecular changes to dietary interventions.

Proteomics is most challenging because of the higher complexity of proteomes as compared to transcriptomes and metabolomes. However, it delivers

  • not only markers but also
  • targets of intervention, such as
  • enzymes or transporters, and
  • it is the platform of choice for discovering bioactive food proteins and peptides.

Metabolomics is a tool for metabolic characterization of individuals and

  • can deliver metabolic endpoints possibly related to health or disease.

Omics in nutrition should be deployed in an integrated fashion to elucidate biomarkers

  • for defining an individual’s susceptibility to diet in nutritional interventions and
  • for assessing food ingredient efficacy (14).

The more elaborate tools offered by metabolomics opened the door to exploring an active role played by adipose tissue that is affected by diet, race, sex, and probably age and activity. When the multifactorial is brought into play, and the effect of changes in diet and activities studied we leave the study of metabolomics and enter the world of ―metabonomics‖. Adiponectin and adipokines arrive (15-22). We shall discuss ―adiposity‖ later.

Potential Applications of Metabolomics

Either individually or grouped as a profile, metabolites are detected by either

  • nuclear magnetic resonance spectroscopy or mass spectrometry.

There is potential for a multitude of uses of metabolome research, including

  1. the early detection and diagnosis of cancer and as
  2. both a predictive and pharmacodynamic marker of drug effect.

However, the knowledge regarding metabolomics, its technical challenges, and clinical applications is unappreciated

  • even though when used as a translational research tool,
  • it can provide a link between the laboratory and clinic.

Precise numbers of human metabolites is unknown, with estimates ranging from the thousands to tens of thousands. Metabolomics is a term that encompasses several types of analyses, including

(a) metabolic fingerprinting, which measures a subset of the whole profile with little differentiation or quantitation of metabolites;

(b) metabolic profiling, the quantitative study of a group of metabolites, known or unknown, within or associated with a particular metabolic pathway; and

(c) target isotope-based analysis, which focuses on a particular segment of the metabolome by analyzing

  • only a few selected metabolites that comprise a specific biochemical pathway.


Dynamic Construct of the –Omics

Dynamic Construct of the –Omics


Dynamic Construct of the –Omics



Iron metabolism – Anemia

Hepcidin is a key hormone governing mammalian iron homeostasis and may be directly or indirectly involved in the development of most iron deficiency/overload and inflammation-induced anemia. The anemia of chronic disease (ACD) is characterized by macrophage iron retention induced by cytokines and hepcidin regulation. Hepcidin controls cellular iron efflux on binding to the iron export protein ferroportin. While patients present with both ACD and iron deficiency anemia (ACD/IDA), the latter results from chronic blood loss. Iron retention during inflammation occurs in macrophages and the spleen, but not in the liver. In ACD, serum hepcidin concentrations are elevated, which is related to reduced duodenal and macrophage expression of ferroportin. Individuals with ACD/IDA have significantly lower hepcidin levels than ACD subjects. ACD/IDA patients, in contrast to ACD subjects, were able to absorb dietary iron from the gut and to mobilize iron from macrophages. Hepcidin elevation may affect iron transport in ACD and ACD/IDA and it is more responsive to iron demand with IDA than to inflammation. Hepcidin determination may aid in selecting appropriate therapy for these patients (23).

There is correlation between serum hepcidin, iron and inflammatory indicators associated with anemia of chronic disease (ACD), ACD, ACD concomitant iron-deficiency anemia (ACD/IDA), pure IDA and acute inflammation (AcI) patients. Hepcidin levels in anemia types were statistically different, from high to low: ACD, AcI > ACD/IDA > the control > IDA. Serum ferritin levels were significantly increased in ACD and AcI patients but were decreased significantly in ACD/IDA and IDA. Elevated serum EPO concentrations were found in ACD, ACD/IDA and IDA patients but not in AcI patients and the controls. A positive correlation exists between hepcidin and IL-6 levels only in ACD/IDA, AcI and the control groups. A positive correlation between hepcidin and ferritin was marked in the control group, while a negative correlation between hepcidin and ferritin was noted in IDA. The significant negative correlation between hepcidin expression and reticulocyte count was marked in both ACD/IDA and IDA groups. If the hepcidin role in pathogenesis of ACD, ACD/IDA and IDA, it could be a potential marker for detection and differentiation of these anemias (24).


Because cancer cells are known to possess a highly unique metabolic phenotype, development of specific biomarkers in oncology is possible and might be used in identifying fingerprints, profiles, or signatures to detect the presence of cancer, determine prognosis, and/or assess the pharmacodynamic effects of therapy (25).

HDM2, a negative regulator of the tumor suppressor p53, is over-expressed in many cancers that retain wild-type p53. Consequently, the effectiveness of chemotherapies that induce p53 might be limited, and inhibitors of the HDM2–p53 interaction are being sought as tumor-selective drugs. A binding site within HDM2 has been dentified which can be blocked with peptides inducing p53 transcriptional activity. A recent report demonstrates the principle using drug-like small molecules that target HDM2 (26).

Obesity, CRP, interleukins, and chronic inflammatory disease

Elevated CRP levels and clinically raised CRP levels were present in 27.6% and 6.7% of the population, respectively. Both overweight (body mass index [BMI], 25-29.9 kg/m2) and obese (BMI, 30 kg/m2) persons were more likely to have elevated CRP levels than their normal-weight counterparts (BMI, <25 kg/m2). After adjusting for potential confounders, the odds ratio (OR) for elevated CRP was 2.13 for obese men and 6.21 for obese women. In addition, BMI was associated with clinically raised CRP levels in women, with an OR of 4.76 (95% CI, 3.42-6.61) for obese women. Waist-to-hip ratio was positively associated with both elevated and clinically raised CRP levels, independent of BMI. Restricting the analyses to young adults (aged 17-39 years) and excluding smokers, persons with inflammatory disease, cardiovascular disease, or diabetes mellitus and estrogen users did not change the main findings (27).

A study of C-reactive protein and interleukin-6 with measures of obesity and of chronic infection as their putative determinants related levels of C-reactive protein and interleukin-6 to markers of the insulin resistance syndrome and of endothelial dysfunction. Levels of C-reactive protein were significantly related to those of interleukin-6 (r=0.37, P<0.0005) and tumor necrosis factor-a (r=0.46, P<0.0001), and concentrations of C-reactive protein were related to insulin resistance as calculated from the homoeostasis model and to markers of endothelial dysfunction (plasma levels of von Willebrand factor, tissue plasminogen activator, and cellular fibronectin). A mean standard deviation score of levels of acute phase markers correlated closely with a similar score of insulin resistance syndrome variables (r=0.59, P<0.00005) and the data suggested that adipose tissue is an important determinant of a low level, chronic inflammatory state as reflected by levels of interleukin-6, tumor necrosis factor-a, and C-reactive protein (28).

A number of other studies have indicated the inflammatory ties of visceral obesity to adipose tissue metabolic profiles, suggesting a role in ―metabolic syndrome‖. There is now a concept of altered liver metabolism in ―non-alcoholic‖ fatty liver disease (NAFLD) progressing from steatosis to steatohepatitis (NASH) (31,32).

These unifying concepts were incomprehensible 50 years ago. It was only known that insulin is anabolic and that insulin deficiency (or resistance) would have consequences in the point of entry into the citric acid cycle, which generates 16 ATPs. In fat catabolism, triglycerides are hydrolyzed to break them into fatty acids and glycerol. In the liver the glycerol can be converted into glucose via dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by way of gluconeogenesis. In the case of this cycle there is a tie in with both catabolism and anabolism.





For bypass of the Pyruvate Kinase reaction of Glycolysis, cleavage of 2 ~P bonds is required. The free energy change associated with cleavage of one ~P bond of ATP is insufficient to drive synthesis of phosphoenolpyruvate (PEP), since PEP has a higher negative G of phosphate hydrolysis than ATP.

The two enzymes that catalyze the reactions for bypass of the Pyruvate Kinase reaction are the following:

(a) Pyruvate Carboxylase (Gluconeogenesis) catalyzes:

pyruvate + HCO3 + ATP — oxaloacetate + ADP + Pi

(b) PEP Carboxykinase (Gluconeogenesis) catalyzes:

oxaloacetate + GTP — phosphoenolpyruvate + GDP + CO2

The concept of anomalies in the pathways with respect to diabetes was sketchy then, and there was much to be filled in. This has been substantially done, and is by no means complete. However, one can see how this comes into play with diabetic ketoacidosis accompanied by gluconeogenesis and in severe injury or sepsis with peripheral proteolysis to provide gluconeogenic precursors. The reprioritization of liver synthetic processes is also brought into play with the conundrum of protein-energy malnutrition.

The picture began to be filled in with the improvements in technology that emerged at the end of the 1980s with the ability to profile tissue and body fluids by NMR and by MS. There was already a good inkling of a relationship of type 2 diabetes to major indicators of CVD (29,30). And a long suspected relationship between obesity and type 2 diabetes was evident. But how did it tie together?

End Stage Renal Disease and Cardiovascular Risk

Mortality is markedly elevated in patients with end-stage renal disease. The leading cause of death is cardiovascular disease.

As renal function declines,

  • the prevalence of both malnutrition and cardiovascular disease increase.

Malnutrition and vascular disease correlate with the levels of

  • markers of inflammation in patients treated with dialysis and in those not yet on dialysis.

The causes of inflammation are likely to be multifactorial. CRP levels are associated with cardio-vascular risk in the general population.

The changes in endothelial cell function,

  • in plasma proteins, and
  • in lpiids in inflammation

are likely to be atherogenic.

That cardiovascular risk is inversely correlated with serum cholesterol in dialysis patients, suggests that

  • hyperlipidemia plays a minor role in the incidence of cardiovascular disease.

Hypoalbuminemia, ascribed to malnutrition, has been one of the most powerful risk factors that predict all-cause and cardiovascular mortality in dialysis patients. The presence of inflammation, as evidenced by increased levels of specific cytokines (interleukin-6 and tumor necrosis factor a) or acute-phase proteins (C-reactive protein and serum amyloid A), however, has been found to be associated with vascular disease in the general population as well as in dialysis patients. Patients have

  • loss of muscle mass and changes in plasma composition—decreases in serum albumin, prealbumin, and transferrin levels, also associated with malnutrition.

Inflammation alters

  • lipoprotein structure and function as well as
  • endothelial structure and function

to favor atherogenesis and increases

  • the concentration of atherogenic proteins in serum.

In addition, proinflammatory compounds, such as

  • advanced glycation end products, accumulate in renal failure, and
  • defense mechanisms against oxidative injury are reduced,

contributing to inflammation and to its effect on the vascular endothelium (33,34).

Endogenous copper can play an important role in postischemic reperfusion injury, a condition associated with endothelial cell activation and increased interleukin 8 (IL-8) production. Excessive endothelial IL-8 secreted during trauma, major surgery, and sepsis may contribute to the development of systemic inflammatory response syndrome (SIRS), adult respiratory distress syndrome (ARDS), and multiple organ failure (MOF). No previous reports have indicated that copper has a direct role in stimulating human endothelial IL-8 secretion. Copper did not stimulate secretion of other cytokines. Cu(II) appeared to be the primary copper ion responsible for the observed increase in IL-8 because a specific high-affinity Cu(II)-binding peptide, d-Asp-d-Ala-d-Hisd-Lys (d-DAHK), completely abolished this effect in a dose-dependent manner. These results suggest that Cu(II) may induce endothelial IL-8 by a mechanism independent of known Cu(I) generation of reactive oxygen species (35).

Blood coagulation plays a key role among numerous mediating systems that are activated in inflammation. Receptors of the PAR family serve as sensors of serine proteinases of the blood clotting system in the target cells involved in inflammation. Activation of PAR_1 by thrombin and of PAR_2 by factor Xa leads to a rapid expression and exposure on the membrane of endothelial cells of both adhesive proteins that mediate an acute inflammatory reaction and of the tissue factor that initiates the blood coagulation cascade. Other receptors that can modulate responses of the cells activated by proteinases through PAR receptors are also involved in the association of coagulation and inflammation together with the receptors of the PAR family. The presence of PAR receptors on mast cells is responsible for their reactivity to thrombin and factor Xa , essential to the inflammation and blood clotting processes (36).

The understanding of regulation of the inflammatory process in chronic inflammatory diseases is advancing.

Evidence consistently indicates that T-cells play a key role in initiating and perpetuating inflammation, not only via the production of soluble mediators but also via cell/cell contact interactions with a variety of cell types through membrane receptors and their ligands. Signalling through CD40 and CD40 ligand is a versatile pathway that is potently involved in all these processes. Many inflammatory genes relevant to atherosclerosis are influenced by the transcriptional regulator nuclear factor κ B (NFκB). In these events T-cells become activated by dendritic cells or inflammatory cytokines, and these T-cells activate, in turn, monocytes / macrophages, endothelial cells, smooth muscle cells and fibroblasts to produce pro-inflammatory cytokines, chemokines, the coagulation cascade in vivo, and finally matrix metalloproteinases, responsible for tissue destruction. Moreover, CD40 ligand at inflammatory sites stimulates fibroblasts and tissue monocyte/macrophage production of VEGF, leading to angiogenesis, which promotes and maintains the chronic inflammatory process.

NFκB plays a pivotal role in co-ordinating the expression of genes involved in the immune and inflammatory response, evoking tumor necrosis factor α (TNFα), chemokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-8, matrix metalloproteinase enzymes (MMP), and genes involved in cell survival. A complex array of mechanisms, including T cell activation, leukocyte extravasation, tissue factor expression, MMP expression and activation, as well induction of cytokines and chemokines, implicated in atherosclerosis, are regulated by NFκB.

Expression of NFκB in the atherosclerotic milieu may have a number of potentially harmful consequences. IL-1 activates NFκB upregulating expression of MMP-1, -3, and -9. Oxidized LDL increases macrophage MMP-9, associated with increased nuclear binding of NFκB and AP-1. Expression of tissue factor, initiating the coagulation cascade, is regulated by NFκB. In atherosclerotic plaque cells, tissue factor antigen and activity were inhibited following over-expression of IκBα and dominant-negative IKK-2, but not by dominant negative IKK-1 or NIK. Tis supports the concept that activation of the ―canonical‖ pathway upregulates pro-thrombotic mediators involved in disease. Many of the cytokines and chemokines which have been detected in human atherosclerotic plaques are also regulated by NFκB. Over-expression of IκBα inhibits release of TNFα, IL-1, IL-6, and IL-8 in macrophages stimulated with LPS and CD40 ligand (CD40L). This report describes how NFκB activation upregulates major pro-inflammatory and pro-thrombotic mediators of atherosclerosis (37-41).

This review is both focused and comprehensive. The details of evolving methods are avoided in order to build the argument that a very rapid expansion of discovery has been evolving depicting disease, disease mechanisms, disease associations, metabolic biomarkers, study of effects of diet and diet modification, and opportunities for targeted drug development. The extent of future success will depend on the duration and strength of the developed interventions, and possibly the avoidance of dead end interventions that are unexpectedly bypassed. I anticipate the prospects for the interplay between genomics, metabolomics, metabonomics, and personalized medicine may be realized for several of the most common conditions worldwide within a few decades (42-44).


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  5. Kim K, Valentine RJ, Shin Y, Gong K. Association of visceral adiposity and exercise participation with C- reactive protein, insulin resistance, and endothelial dysfunction in Korean healthy adults. Metabolism 2008;57:1181-9. [(VAT-EC exhibits a marked angiogenic and proinflammatory state];
  6. Villaret A, Galitzky J, Decaunes P, Exteve D, et al. Adipose tisue endothelial cells from obese human subjects: differences among depots in angiogenic, metabolic, and inflammatory gene expression and cellular senescence. Diabetes 2010;59:2755-63;
  7. van Dijk -, Feskens EJ, Bos MB, Hoelen DW, et al. A saturated fatty acid-rich diet induces an obesity-linked proinflammatory gene expression profile in adipose tissue of subjects at risk of metabolic syndrome. Am J Clin Nutr 2009;90:1656-64.[MUFA in LDL lowering].
  8. Theurl I, Aigner E, Theurl M, Nairz M, et al. Regulation of iron homeostasis in anemia of chronic disease and iron deficiency anemia: diagnostic and therapeutic implications. Blood. 2009;113(21):5277-86
  9. Cheng PP, Jiao XY, Wang XH, Lin JH, Cai YM. Hepcidin expression in anemia of chronic disease and concomitant iron-deficiency anemia. Clin Exp Med. 2010 May 25. [Epub ahead of print].
  10. Spratlin JL, Serkova NJ, and Eckhardt SG. Clinical Applications of Metabolomics in Oncology: A Review. Clin Cancer Res. 2009 ;15; 15(2): 431–440.
  11. Fischer PM, Lane DP. Small molecule inhibitors of thep53 suppressor HDM2: have protein-protein interactions come of age as drug targets? Trends in Pharm Sci 2004;25(7):343-346.
  12. Visser M, Bouter LM, McQuillan GM, Wener HM. Elevated C-Reactive Protein Levels in Overweight and Obese Adults. JAMA. 1999;282:2131-2135.
  13. Yudkin JS, Stehouwer CDA, Emeis JJ, Coppack SW. C-Reactive Protein in Healthy Subjects: Associations With Obesity, Insulin Resistance, and Endothelial Dysfunction : A Potential Role for Cytokines Originating From Adipose Tissue? Arterioscler. Thromb. Vasc. Biol. 1999; 19:972-978.
  14. Visvikis-Siest S, Siest G. The STANISLAS cohort: a 10-year followup of supposed healthy families. Gene-environment interactions, reference values and evaluation of biomarkers in prevention of cardiovascular diseases. Clin Chem Lab Med 2008;46:733-47.
  15. Schmidt MI, Duncan BB. Diabesity: an inflammatory metabolic condition. Clin Chem Lab Med 2003;41:1120-1130.
  16. Fenkci S, Rota S, Sabir N, Akdag B. Ultrasonographic and biochemical evaluation of visceral obesity in obese women with non-alcoholic fatty liver disease. Eur J Med Res 2007;12:68-73. (VAT, HOMA)
  17. Lee JW, Lee HR, Shim JY, Im JA, et al. Viscerally obese women with normal body weight have greater brachial-ankle pulse wave velocity than non viscerally obese women with excessive body weight. Clin Endocrinol (Oxf) 2007;66:572-8. [visceral obesity – high trigly, high baPWV, greater SFA and thigh SFA].
  18. Kaysen GE. The Microinflammatory State in Uremia: Causes and Potential Consequences. J Am Soc Nephrol 2001;12:1549–1557.
  19. Kaysen GE. Role of Inflammation and Its Treatment in ESRD Patients. Blood Purif 2002;20:70–80.
  20. Bar-Or D, Thomas GW, Yukl RL, Rael LT, et al. Copper stimulates the synthesis and release of interleukin-8 in human endothelial cells: a possible early role in systemic inflammatory responses. Shock 2003;20(2):154–158.
  21. Dugina TN, Kiseleva EV, Chistov IV, Umarova BA, and Strukova SM. Receptors of the PAR Family as a Link between Blood Coagulation and Inflammation. Biochemistry (Moscow), 2002; 67(1):65-74. [Translated from Biokhimiya 2002;67(1):77-87].
  22. Monaco C, Andreakos E, Kiriakidis S, Feldmann M, and and Ewa Paleolog. T-Cell-Mediated Signalling in Immune, Inflammatory and Angiogenic Processes: The Cascade of Events Leading to Inflammatory Diseases. Current Drug Targets – Inflammation & Allergy, 2004, 3, 35-42.
  23. Monaco C, Grosjean J, and Paleolog E. The role of the NFκB pathway in atherosclerosis. [E-mail:]
  24. Libby P, Ridker PM, and Maseri A. Inflammation and atherosclerosis. Circulation 2002;105:1135-43.
  25. Karin M, Yamamoto Y, Wang QM. The IKK NF-kappa B system: a treasure trove for drug development. Nat Rev Drug Discov 2004;3:17-26.
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  28. Faca V, Krasnoselsky A, and Hanash S. Innovative proteomic approaches for cancer biomarker discove.
  29. Sharp, P, and MIT faculty. ‘Convergence’ offers potential for revolutionary advance in biomedicine. The Third Revolution: Convergence of the Life Sciences, Physical Sciences and Engineering. White paper. Reported in Biotechnology Jan 5, 2011. [Convergence is a new paradigm that can yield critical advances in a broad array of sectors]


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Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Author and Curator: Larry H Bernstein, MD, FCAP

The evolution of progress we have achieved in cancer research, diagnosis, and therapeutics has  originated from an emergence of scientific disciplines and the focus on cancer has been recent. We can imagine this from a historical perspective with respect to two observations. The first is that the oldest concepts of medicine lie with the anatomic dissection of animals and the repeated recurrence of war, pestilence, and plague throughout the middle ages, and including the renaissance.  In the awakening, architecture, arts, music, math, architecture and science that accompanied the invention of printing blossomed, a unique collaboration of individuals working in disparate disciplines occurred, and those who were privileged received an education, which led to exploration, and with it, colonialism.  This also led to the need to increasingly, if not without reprisal, questioning long-held church doctrines.

It was in Vienna that Rokitansky developed the discipline of pathology, and his student Semelweis identified an association between then unknown infection and childbirth fever. The extraordinary accomplishments of John Hunter in anatomy and surgery came during the twelve years war, and his student, Edward Jenner, observed the association between cowpox and smallpox resistance. The development of a nursing profession is associated with the work of Florence Nightengale during the Crimean War (at the same time as Leo Tolstoy). These events preceded the work of Pasteur, Metchnikoff, and Koch in developing a germ theory, although Semelweis had committed suicide by infecting himself with syphilis. The first decade of the Nobel Prize was dominated by discoveries in infectious disease and public health (Ronald Ross, Walter Reed) and we know that the Civil War in America saw an epidemic of Yellow Fever, and the Armed Services Medical Museum was endowed with a large repository of osteomyelitis specimens. We also recall that the Russian physician and playwriter, Anton Checkov, wrote about the conditions in prison camps.

But the pharmacopeia was about to open with the discoveries of insulin, antibiotics, vitamins, thyroid action (Mayo brothers pioneered thyroid surgery in the thyroid iodine-deficient midwest), and pitutitary and sex hormones (isolatation, crystal structure, and synthesis years later), and Karl Landsteiner’s discovery of red cell antigenic groups (but he also pioneered in discoveries in meningitis and poliomyelitis, and conceived of the term hapten) with the introduction of transfusion therapy that would lead to transplantation medicine.  The next phase would be heralded by the discovery of cancer, which was highlighted by the identification of leukemia by Rudolph Virchow, who cautioned about the limitations of microscopy. This period is highlighted by the classic work – “Microbe Hunters”.

John Hunter

John Hunter

Walter Reed

Walter Reed

Robert Koch

Robert Koch

goldberger 1916 Pellagra

goldberger 1916 Pellagra

Louis Pasteur

Louis Pasteur

A multidisciplinary approach has led us to a unique multidisciplinary or systems view of cancer, with different fields of study offering their unique expertise, contributions, and viewpoints on the etiology of cancer.  Diverse fields in immunology, biology, biochemistry, toxicology, molecular biology, virology, mathematics, social activism and policy, and engineering have made such important contributions to our understanding of cancer, that without cooperation among these diverse fields our knowledge of cancer would never had evolved as it has. In a series of posts “Heroes in Medical Research:” the work of researchers are highlighted as examples of how disparate scientific disciplines converged to produce seminal discoveries which propelled the cancer field, although, at the time, they seemed like serendipitous findings.  In the post Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin (Translating Basic Research to the Clinic) discusses the seminal yet serendipitous discoveries by bacteriologist Dr. Barnett Rosenberg, which eventually led to the development of cisplatin, a staple of many chemotherapeutic regimens. Molecular biologist Dr. Robert Ting, working with soon-to-be Nobel Laureate virologist Dr. James Gallo on AIDS research and the associated Karposi’s sarcoma identified one of the first retroviral oncogenes, revolutionizing previous held misconceptions of the origins of cancer (described in Heroes in Medical Research: Dr. Robert Ting, Ph.D. and Retrovirus in AIDS and Cancer).   Located here will be a MONTAGE of PHOTOS of PEOPLE who made seminal discoveries and contributions in every field to cancer   Each of these paths of discovery in cancer research have led to the unique strategies of cancer therapeutics and detection for the purpose of reducing the burden of human cancer.  However, we must recall that this work has come at great cost, while it is indeed cause for celebration. The current failure rate of clinical trials at over 70 percent, has been a cause for disappointment, and has led to serious reconsideration of how we can proceed with greater success. The result of the evolution of the cancer field is evident in the many parts and chapters of this ebook.  Volume 4 contains chapters that are in a predetermined order:

  1. The concepts of neoplasm, malignancy, carcinogenesis,  and metastatic potential, which encompass:

(a)     How cancer cells bathed in an oxygen rich environment rely on anaerobic glycolysis for energy, and the secondary consequences of cachexia and sarcopenia associated with progression



ARTS protein and cancer

ARTS protein and cancer



Krebs cycle

Krebs cycle

Metabolic control analysis of respiration in human cancer tissue

Metabolic control analysis of respiration in human cancer tissue



(b)     How advances in genetic analysis, molecular and cellular biology, metabolomics have expanded our basic knowledge of the mechanisms which are involved in cellular transformation to the cancerous state.



Methylation of adenine

Methylation of adenine





(c)  How molecular techniques continue to advance our understanding  of how genetics, epigenetics, and alterations in cellular metabolism contribute to cancer and afford new pathways for therapeutic intervention.

 genomic effects

genomic effects

LKB1AMPK pathway

LKB1AMPK pathway



AMPK-activating drugs metformin or phenformin might provide protection against cancer

AMPK-activating drugs metformin or phenformin might provide protection against cancer





2. The distinct features of cancers of specific tissue sites of origin

3.  The diagnosis of cancer by

(a)     Clinical presentation

(b)     Age of onset and stage of life

(c)     Biomarker features

hairy cell leukemia

hairy cell leukemia

lymphoma leukemia

lymphoma leukemia

(d)     Radiological and ultrasound imaging

  1. Treatments
  2. Prognostic differences within and between cancer types

We have introduced the emergence of a disease of great complexity that has been clouded in more questions than answers until the emergence of molecular biology in the mid 20th century, and then had to await further discoveries going into the 21st century.  What gave the research impetus was the revelation of

1     the mechanism of transcription of the DNA into amino acid sequences

Proteins in Disease

Proteins in Disease

2     the identification of stresses imposed on cellular function

NO beneficial effects

NO beneficial effects

3     the elucidation of the substructure of the cell – cell membrane, mitochondria, ribosomes, lysosomes – and their functions, respectively

pone.0080815.g006  AKIP1 Expression Modulates Mitochondrial Function

AKIP1 Expression Modulates Mitochondrial Function

4     the elucidation of oligonucleotide sequences

















5     the further elucidation of functionally relevant noncoding lncDNA

lncRNA-s   A summary of the various functions described for lncRNA

6     the technology to synthesis mRNA and siRNA sequences

RNAi_Q4 Primary research objectives

Figure. RNAi and gene silencing

7     the repeated discovery of isoforms of critical enzymes and their pleiotropic properties

8.     the regulatory pathways involved in signaling

signaling pathjways map

Figure. Signaling Pathways Map

This is a brief outline of the modern progression of advances in our understanding of cancer.  Let us go back to the beginning and check out a sequence of  Nobel Prizes awarded and related discoveries that have a historical relationship to what we know.  The first discovery was the finding by Louis Pasteur that fungi that grew in an oxygen poor environment did not put down filaments.  They did not utilize oxygen and they produced used energy by fermentation.  This was the basis for Otto Warburg sixty years later to make the comparison to cancer cells that grew in the presence of oxygen, but relied on anaerobic glycolysis. He used a manometer to measure respiration in tissue one cell layer thick to measure CO2 production in an adiabatic system.

video width=”1280″ height=”720″ caption=”1741-7007-11-65-s1 Macromolecular juggling by ubiquitylation enzymes.” mp4=”“][/video]

An Introduction to the Warburg Apparatus

Lavoisier Antoine-Laurent and Laplace Pierre-Simon (1783) Memoir on heat. Mémoirs de l’Académie des sciences. Translated by Guerlac H, Neale Watson Academic Publications, New York, 1982.

Instrumental background 200 years later:   Gnaiger E (1983) The twin-flow microrespirometer and simultaneous calorimetry. In Gnaiger E, Forstner H, eds. Polarographic Oxygen Sensors. Springer, Heidelberg, Berlin, New York: 134-166.



Warburg apparatus

The Warburg apparatus is a manometric respirometer which was used for decades in biochemistry for measuring oxygen consumption of tissue homogenates or tissue slices.

The Warburg apparatus has its name from the German biochemist Otto Heinrich Warburg (1883-1970) who was awarded the Nobel Prize in physiology or medicine in 1931 for his “discovery of the nature and mode of action of the respiratory enzyme” [1].

The aqueous phase is vigorously shaken to equilibrate with a gas phase, from which oxygen is consumed while the evolved carbon dioxide is trapped, such that the pressure in the constant-volume gas phase drops proportional to oxygen consumption. The Warburg apparatus was introduced to study cell respiration, i.e. the uptake of molecular oxygen and the production of carbon dioxide by cells or tissues. Its applications were extended to the study of fermentation, when gas exchange takes place in the absence of oxygen. Thus the Warburg apparatus became established as an instrument for both aerobic and anaerobic biochemical studies [2, 3].

The respiration chamber was a detachable glass flask (F) equipped with one or more sidearms (S) for additions of chemicals and an open connection to a manometer (M; pressure gauge). A constant temperature was provided by immersion of the Warburg chamber in a constant temperature water bath. At thermal mass transfer equilibrium, an initial reading is obtained on the manometer, and the volume of gas produced or absorbed is determined at specific time intervals. A limited number of ‘titrations’ can be performed by adding the liquid contained in a side arm into the main reaction chamber. A Warburg apparatus may be equipped with more than 10 respiration chambers shaking in a common water bath.   Since temperature has to be controlled very precisely in a manometric approach, the early studies on mammalian tissue respiration were generally carried out at a physiological temperature of 37 °C.

The Warburg apparatus has been replaced by polarographic instruments introduced by Britton Chance in the 1950s. Since Chance and Williams (1955) measured respiration of isolated mitochondria simultaneously with the spectrophotometric determination of cytochrome redox states, a water chacket could not be used, and measurements were carried out at room temperature (or 25 °C). Thus most later studies on isolated mitochondria were shifted to the artifical temperature of 25 °C.

Today, the importance of investigating mitochondrial performance at in vivo temperatures is recognized again in mitochondrial physiology.  Incubation times of 1 hour were typical in experiments with the Warburg apparatus, but were reduced to a few or up to 20 min, following Chance and Williams, due to rapid oxygen depletion in closed, aqueous phase oxygraphs with high sample concentrations.  Today, incubation times of 1 hour are typical again in high-resolution respirometry, with low sample concentrations and the option of reoxygenations.

“The Nobel Prize in Physiology or Medicine 1931”. 27 Dec 2011

  1. Oesper P (1964) The history of the Warburg apparatus: Some reminiscences on its use. J Chem Educ 41: 294.
  2. Koppenol WH, Bounds PL, Dang CV (2011) Otto Warburg’s contributions to current concepts of cancer metabolism. Nature Reviews Cancer 11: 325-337.
  3. Gnaiger E, Kemp RB (1990) Anaerobic metabolism in aerobic mammalian cells: information from the ratio of calorimetric heat flux and respirometric oxygen flux. Biochim Biophys Acta 1016: 328-332. – “At high fructose concen­trations, respiration is inhibited while glycolytic end products accumulate, a phenomenon known as the Crabtree effect. It is commonly believed that this effect is restric­ted to microbial and tumour cells with uniquely high glycolytic capaci­ties (Sussman et al, 1980). How­ever, inhibition of respiration and increase of lactate production are observed under aerobic condi­tions in beating rat heart cell cultures (Frelin et al, 1974) and in isolated rat lung cells (Ayuso-Parrilla et al, 1978). Thus, the same general mechanisms respon­sible for the integra­tion of respiration and glycolysis in tumour cells (Sussman et al, 1980) appear to be operating to some extent in several isolated mammalian cells.”

Mitochondria are sometimes described as “cellular power plants” because they generate most of the cell’s supply of adenosine triphosphate (ATP), used as a source of chemical energy.[2] In addition to supplying cellular energy, mitochondria are involved in other tasks such as signalingcellular differentiationcell death, as well as the control of the cell cycle and cell growth.[3]   The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. In humans, 615 distinct types of proteins have been identified from cardiac mitochondria,[9   Leonor Michaelis discovered that Janus green can be used as a supravital stain for mitochondria in 1900.  Benjamin F. Kingsbury, in 1912, first related them with cell respiration, but almost exclusively based on morphological observations.[13] In 1913 particles from extracts of guinea-pig liver were linked to respiration by Otto Heinrich Warburg, which he called “grana”. Warburg and Heinrich Otto Wieland, who had also postulated a similar particle mechanism, disagreed on the chemical nature of the respiration. It was not until 1925 when David Keilin discovered cytochromes that the respiratory chain was described.[13]    

The Clark Oxygen Sensor

Dr. Leland Clark – inventor of the “Clark Oxygen Sensor” (1954); the Clark type polarographic oxygen sensor remains the gold standard for measuring dissolved oxygen in biomedical, environmental and industrial applications .   ‘The convenience and simplicity of the polarographic ‘oxygen electrode’ technique for measuring rapid changes in the rate of oxygen utilization by cellular and subcellular systems is now leading to its more general application in many laboratories. The types and design of oxygen electrodes vary, depending on the investigator’s ingenuity and specific requirements of the system under investigation.’Estabrook R (1967) Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol. 10: 41-47.   “one approach that is underutilized in whole-cell bioenergetics, and that is accessible as long as cells can be obtained in suspension, is the oxygen electrode, which can obtain more precise information on the bioenergetic status of the in situ mitochondria than more ‘high-tech’ approaches such as fluorescent monitoring of Δψm.” Nicholls DG, Ferguson S (2002) Bioenergetics 3. Academic Press, London.

Great Figures in Cancer

Dr. Elizabeth Blackburn,

Dr. Elizabeth Blackburn,

j_michael_bishop onogene

j_michael_bishop onogene

Harold Varmus

Harold Varmus

Potts and Habener (PTH mRNA, Harvard MIT)  JCI

Potts and Habener (PTH mRNA, Harvard MIT) JCI

JCI Fuller Albright and hPTH AA sequence

JCI Fuller Albright and hPTH AA sequence

Dr. E. Donnall Thomas  Bone Marrow Transplants

Dr. E. Donnall Thomas Bone Marrow Transplants

Dr Haraldzur Hausen  EBV HPV

Dr Haraldzur Hausen EBV HPV

Dr. Craig Mello

Dr. Craig Mello

Dorothy Hodgkin  protein crystallography

Lee Hartwell - Hutchinson Cancer Res Center

Lee Hartwell – Hutchinson Cancer Res Center

Judah Folkman, MD

Judah Folkman, MD

Gertrude B. Elien (1918-1999)

Gertrude B. Elien (1918-1999)

The Nobel Prize in Physiology or Medicine 1922   

Archibald V. Hill, Otto Meyerhof

AV Hill –

“the production of heat in the muscle” Hill started his research work in 1909. It was due to J.N. Langley, Head of the Department of Physiology at that time that Hill took up the study on the nature of muscular contraction. Langley drew his attention to the important (later to become classic) work carried out by Fletcher and Hopkins on the problem of lactic acid in muscle, particularly in relation to the effect of oxygen upon its removal in recovery. In 1919 he took up again his study of the physiology of muscle, and came into close contact with Meyerhof of Kiel who, approaching the problem differently, arrived at results closely analogous to his study. In 1919 Hill’s friend W. Hartree, mathematician and engineer, joined in the myothermic investigations – a cooperation which had rewarding results.

Otto Meyerhof



lactic acid production in muscle contraction Under the influence of Otto Warburg, then at Heidelberg, Meyerhof became more and more interested in cell physiology.  In 1923 he was offered a Professorship of Biochemistry in the United States, but Germany was unwilling to lose him.  In 1929 he was he was placed in charge of the newly founded Kaiser Wilhelm Institute for Medical Research at Heidelberg.  From 1938 to 1940 he was Director of Research at the Institut de Biologie physico-chimique at Paris, but in 1940 he moved to the United States, where the post of Research Professor of Physiological Chemistry had been created for him by the University of Pennsylvania and the Rockefeller Foundation.  Meyerhof’s own account states that he was occupied chiefly with oxidation mechanisms in cells and with extending methods of gas analysis through the calorimetric measurement of heat production, and especially the respiratory processes of nitrifying bacteria. The physico-chemical analogy between oxygen respiration and alcoholic fermentation caused him to study both these processes in the same subject, namely, yeast extract. By this work he discovered a co-enzyme of respiration, which could be found in all the cells and tissues up till then investigated. At the same time he also found a co-enzyme of alcoholic fermentation. He also discovered the capacity of the SH-group to transfer oxygen; after Hopkins had isolated from cells the SH bodies concerned, Meyerhof showed that the unsaturated fatty acids in the cell are oxidized with the help of the sulfhydryl group. After studying closer the respiration of muscle, Meyerhof investigated the energy changes in muscle. Considerable progress had been achieved by the English scientists Fletcher and Hopkins by their recognition of the fact that lactic acid formation in the muscle is closely connected with the contraction process. These investigations were the first to throw light upon the highly paradoxical fact, already established by the physiologist Hermann, that the muscle can perform a considerable part of its external function in the complete absence of oxygen.

But it was indisputable that in the last resort the energy for muscle activity comes from oxidation, so the connection between activity and combustion must be an indirect one, and observed that in the absence of oxygen in the muscle, lactic acid appears, slowly in the relaxed state and rapidly in the active state, disappearing in the presence of oxygen. Obviously, then, oxygen is involved when muscle is in the relaxed state.

The Nobel Prize committee had been receiving nominations intermittently for the previous 14 years (for Eijkman, Funk, Goldberger, Grijns, Hopkins and Suzuki but, strangely, not for McCollum in this period). Tthe Committee for the 1929 awards apparently agreed that it was high time to honor the discoverer(s) of vitamins; but who were they? There was a clear case for Grijns, but he had not been re-nominated for that particular year, and it could be said that he was just taking the relatively obvious next steps along the new trail that had been laid down by Eijkman, who was also now an old man in poor health, but there was no doubt that he had taken the first steps in the use of an animal model to investigate the nutritional basis of a clinical disorder affecting millions. Goldberger had been another important contributor, but his recent death put him out of consideration. The clearest evidence for lack of an unknown “something” in a mammalian diet was presented by Gowland Hopkins in 1912. This Cambridge biochemist was already well known for having isolated the amino acid tryptophan from a protein and demonstrated its essential nature. He fed young rats on an experimental diet, half of them receiving a daily milk supplement, and only those receiving milk grew well, Hopkins suggested that this was analogous to human diseases related to diet, as he had suggested already in a lecture published in 1906. Hopkins, the leader of the “dynamic biochemistry” school in Britain and an influential advocate for the importance of vitamins, was awarded the prize jointly with Eijkman. A door was opened. Recognition of work on the fat-soluble vitamins begun by McCollum. The next award related to vitamins was given in 1934 to George WhippleGeorge Minot and William Murphy “for their discoveries concerning liver therapy in cases of [then incurable pernicious] anemia,” The essential liver factor (cobalamin, or vitamin B12) was isolated in 1948, and Vitamin B12  was absent from plant foods. But William Castle in 1928 had demonstrated that the stomachs of pernicious anemia patients were abnormal in failing to secrete an “intrinsic factor”.

1937   Albert von Szent-Györgyi Nagyrápolt

” the biological combustion processes, with special reference to vitamin C and the catalysis of fumaric acid”

structure of fumarate

Szent-Györgyi was a Hungarian biochemist who had worked with Otto Warburg and had a special interest in oxidation-reduction mechanisms. He was invited to Cambridge in England in 1927 after detecting an antioxidant compound in the adrenal cortex, and there, he isolated a compound that he named hexuronic acid. Charles Glen King of the University of Pittsburgh reported success In isolating the anti-scorbutic factor in 1932, and added that his crystals had all the properties reported by Szent-Györgyi for hexuronic acid. But his work on oxidation reactions was also important. Fumarate is an intermediate in the citric acid cycle used by cells to produce energy in the form of adenosine triphosphate (ATP) from food. It is formed by the oxidation of succinate by the enzyme succinate dehydrogenase. Fumarate is then converted by the enzyme fumarase to malate. An enzyme adds water to the fumarate molecule to form malate. The malate is created by adding one hydrogen atom to a carbon atom and then adding a hydroxyl group to a carbon next to a terminal carbonyl group.

In the same year, Norman Haworth from the University of Birmingham in England received a Nobel prize from the Chemistry Committee for having advanced carbohydrate chemistry and, specifically, for having worked out the structure of Szent-Györgyi’s crystals, and then been able to synthesize the vitamin. This was a considerable achievement. The Nobel Prize in Chemistry was shared with the Swiss organic chemist Paul Karrer, cited for his work on the structures of riboflavin and vitamins A and E as well as other biologically interesting compounds. This was followed in 1938 by a further Chemistry award to the German biochemist Richard Kuhn, who had also worked on carotenoids and B-vitamins, including riboflavin and pyridoxine. But Karrer was not permitted to leave Germany at that time by the Nazi regime. However, the American work with radioisotopes at Lawrence Livermore Laboratory, UC Berkeley, was already ushering in a new era of biochemistry that would enrich our studies of metabolic pathways. The importance of work involving vitamins was acknowledged in at least ten awards in the 20th century.

1.   Carpenter, K.J., Beriberi, White Rice and Vitamin B, University of California Press, Berkeley (2000).

2.  Weatherall, M.W. and Kamminga, H., The making of a biochemist: the construction of Frederick Gowland Hopkins’ reputation. Medical History vol.40, pp. 415-436 (1996).

3.  Becker, S.L., Will milk make them grow? An episode in the discovery of the vitamins. In Chemistry and Modern Society (J. Parascandela, editor) pp. 61-83, American Chemical Society,

Washington, D.C. (1983).

4.  Carpenter, K.J., The History of Scurvy and Vitamin C, Cambridge University Press, New York (1986).

Transport and metabolism of exogenous fumarate and 3-phosphoglycerate in vascular smooth muscle.

D R FinderC D Hardin

Molecular and Cellular Biochemistry (Impact Factor: 2.33). 05/1999; 195(1-2):113-21.

The keto (linear) form of exogenous fructose 1,6-bisphosphate, a highly charged glycolytic intermediate, may utilize a dicarboxylate transporter to cross the cell membrane, support glycolysis, and produce ATP anaerobically. We tested the hypothesis that fumarate, a dicarboxylate, and 3-phosphoglycerate (3-PG), an intermediate structurally similar to a dicarboxylate, can support contraction in vascular smooth muscle during hypoxia. 3-PG improved maintenance of force (p < 0.05) during the 30-80 min period of hypoxia. Fumarate decreased peak isometric force development by 9.5% (p = 0.008) but modestly improved maintenance of force (p < 0.05) throughout the first 80 min of hypoxia. 13C-NMR on tissue extracts and superfusates revealed 1,2,3,4-(13)C-fumarate (5 mM) metabolism to 1,2,3,4-(13)C-malate under oxygenated and hypoxic conditions suggesting uptake and metabolism of fumarate. In conclusion, exogenous fumarate and 3-PG readily enter vascular smooth muscle cells, presumably by a dicarboxylate transporter, and support energetically important pathways.

Comparison of endogenous and exogenous sources of ATP in fueling Ca2+ uptake in smooth muscle plasma membrane vesicles.

C D HardinL RaeymaekersR J Paul

The Journal of General Physiology (Impact Factor: 4.73). 12/1991; 99(1):21-40.

A smooth muscle plasma membrane vesicular fraction (PMV) purified for the (Ca2+/Mg2+)-ATPase has endogenous glycolytic enzyme activity. In the presence of glycolytic substrate (fructose 1,6-diphosphate) and cofactors, PMV produced ATP and lactate and supported calcium uptake. The endogenous glycolytic cascade supports calcium uptake independent of bath [ATP]. A 10-fold dilution of PMV, with the resultant 10-fold dilution of glycolytically produced bath [ATP] did not change glycolytically fueled calcium uptake (nanomoles per milligram protein). Furthermore, the calcium uptake fueled by the endogenous glycolytic cascade persisted in the presence of a hexokinase-based ATP trap which eliminated calcium uptake fueled by exogenously added ATP. Thus, it appears that the endogenous glycolytic cascade fuels calcium uptake in PMV via a membrane-associated pool of ATP and not via an exchange of ATP with the bulk solution. To determine whether ATP produced endogenously was utilized preferentially by the calcium pump, the ATP production rates of the endogenous creatine kinase and pyruvate kinase were matched to that of glycolysis and the calcium uptake fueled by the endogenous sources was compared with that fueled by exogenous ATP added at the same rate. The rate of calcium uptake fueled by endogenous sources of ATP was approximately twice that supported by exogenously added ATP, indicating that the calcium pump preferentially utilizes ATP produced by membrane-bound enzymes.

Evidence for succinate production by reduction of fumarate during hypoxia in isolated adult rat heart cells.

C HohlR OestreichP RösenR WiesnerM Grieshaber

Archives of Biochemistry and Biophysics (Impact Factor: 3.37). 01/1988; 259(2):527-35.   It has been demonstrated that perfusion of myocardium with glutamic acid or tricarboxylic acid cycle intermediates during hypoxia or ischemia, improves cardiac function, increases ATP levels, and stimulates succinate production. In this study isolated adult rat heart cells were used to investigate the mechanism of anaerobic succinate formation and examine beneficial effects attributed to ATP generated by this pathway. Myocytes incubated for 60 min under hypoxic conditions showed a slight loss of ATP from an initial value of 21 +/- 1 nmol/mg protein, a decline of CP from 42 to 17 nmol/mg protein and a fourfold increase in lactic acid production to 1.8 +/- 0.2 mumol/mg protein/h. These metabolite contents were not altered by the addition of malate and 2-oxoglutarate to the incubation medium nor were differences in cell viability observed; however, succinate release was substantially accelerated to 241 +/- 53 nmol/mg protein. Incubation of cells with [U-14C]malate or [2-U-14C]oxoglutarate indicates that succinate is formed directly from malate but not from 2-oxoglutarate. Moreover, anaerobic succinate formation was rotenone sensitive.

We conclude that malate reduction to succinate occurs via the reverse action of succinate dehydrogenase in a coupled reaction where NADH is oxidized (and FAD reduced) and ADP is phosphorylated. Furthermore, by transaminating with aspartate to produce oxaloacetate, 2-oxoglutarate stimulates cytosolic malic dehydrogenase activity, whereby malate is formed and NADH is oxidized.

In the form of malate, reducing equivalents and substrate are transported into the mitochondria where they are utilized for succinate synthesis.

1953 Hans Adolf Krebs –

 ” discovery of the citric acid cycle” and In the course of the 1920’s and 1930’s great progress was made in the study of the intermediary reactions by which sugar is anaerobically fermented to lactic acid or to ethanol and carbon dioxide. The success was mainly due to the joint efforts of the schools of Meyerhof, Embden, Parnas, von Euler, Warburg and the Coris, who built on the pioneer work of Harden and of Neuberg. This work brought to light the main intermediary steps of anaerobic fermentations.

In contrast, very little was known in the earlier 1930’s about the intermediary stages through which sugar is oxidized in living cells. When, in 1930, I left the laboratory of Otto Warburg (under whose guidance I had worked since 1926 and from whom I have learnt more than from any other single teacher), I was confronted with the question of selecting a major field of study and I felt greatly attracted by the problem of the intermediary pathway of oxidations.

These reactions represent the main energy source in higher organisms, and in view of the importance of energy production to living organisms (whose activities all depend on a continuous supply of energy) the problem seemed well worthwhile studying.

Interactive Krebs cycle

There are different points where metabolites enter the Krebs’ cycle. Most of the products of protein, carbohydrates and fat metabolism are reduced to the molecule acetyl coenzyme A that enters the Krebs’ cycle. Glucose, the primary fuel in the body, is first metabolized into pyruvic acid and then into acetyl coenzyme A. The breakdown of the glucose molecule forms two molecules of ATP for energy in the Embden Meyerhof pathway process of glycolysis.

On the other hand, amino acids and some chained fatty acids can be metabolized into Krebs intermediates and enter the cycle at several points. When oxygen is unavailable or the Krebs’ cycle is inhibited, the body shifts its energy production from the Krebs’ cycle to the Embden Meyerhof pathway of glycolysis, a very inefficient way of making energy.  

Fritz Albert Lipmann –

 “discovery of co-enzyme A and its importance for intermediary metabolism”.

In my development, the recognition of facts and the rationalization of these facts into a unified picture, have interplayed continuously. After my apprenticeship with Otto Meyerhof, a first interest on my own became the phenomenon we call the Pasteur effect, this peculiar depression of the wasteful fermentation in the respiring cell. By looking for a chemical explanation of this economy measure on the cellular level, I was prompted into a study of the mechanism of pyruvic acid oxidation, since it is at the pyruvic stage where respiration branches off from fermentation.

For this study I chose as a promising system a relatively simple looking pyruvic acid oxidation enzyme in a certain strain of Lactobacillus delbrueckii1.   In 1939, experiments using minced muscle cells demonstrated that one oxygen atom can form two adenosine triphosphate molecules, and, in 1941, the concept of phosphate bonds being a form of energy in cellular metabolism was developed by Fritz Albert Lipmann.

In the following years, the mechanism behind cellular respiration was further elaborated, although its link to the mitochondria was not known.[13]The introduction of tissue fractionation by Albert Claude allowed mitochondria to be isolated from other cell fractions and biochemical analysis to be conducted on them alone. In 1946, he concluded that cytochrome oxidase and other enzymes responsible for the respiratory chain were isolated to the mitchondria. Over time, the fractionation method was tweaked, improving the quality of the mitochondria isolated, and other elements of cell respiration were determined to occur in the mitochondria.[13]

The most important event during this whole period, I now feel, was the accidental observation that in the L. delbrueckii system, pyruvic acid oxidation was completely dependent on the presence of inorganic phosphate. This observation was made in the course of attempts to replace oxygen by methylene blue. To measure the methylene blue reduction manometrically, I had to switch to a bicarbonate buffer instead of the otherwise routinely used phosphate. In bicarbonate, pyruvate oxidation was very slow, but the addition of a little phosphate caused a remarkable increase in rate. The phosphate effect was removed by washing with a phosphate free acetate buffer. Then it appeared that the reaction was really fully dependent on phosphate.

A coupling of this pyruvate oxidation with adenylic acid phosphorylation was attempted. Addition of adenylic acid to the pyruvic oxidation system brought out a net disappearance of inorganic phosphate, accounted for as adenosine triphosphate.   The acetic acid subunit of acetyl CoA is combined with oxaloacetate to form a molecule of citrate. Acetyl coenzyme A acts only as a transporter of acetic acid from one enzyme to another. After Step 1, the coenzyme is released by hydrolysis to combine with another acetic acid molecule and begin the Krebs’ Cycle again.

Hugo Theorell

the nature and effects of oxidation enzymes”

From 1933 until 1935 Theorell held a Rockefeller Fellowship and worked with Otto Warburg at Berlin-Dahlem, and here he became interested in oxidation enzymes. At Berlin-Dahlem he produced, for the first time, the oxidation enzyme called «the yellow ferment» and he succeeded in splitting it reversibly into a coenzyme part, which was found to be flavin mononucleotide, and a colourless protein part. On return to Sweden, he was appointed Head of the newly established Biochemical Department of the Nobel Medical Institute, which was opened in 1937.

Succinate is oxidized by a molecule of FAD (Flavin Adenine Dinucleotide). The FAD removes two hydrogen atoms from the succinate and forms a double bond between the two carbon atoms to create fumarate.






Watson & Crick double helix model 

A landmark in this journey

They followed the path that became clear from intense collaborative work in California by George Beadle, by Avery and McCarthy, Max Delbruck, TH Morgan, Max Delbruck and by Chargaff that indicated that genetics would be important.


François Jacob, André Lwoff and Jacques Monod  –

” genetic control of enzyme and virus synthesis”.

In 1958 the remarkable analogy revealed by genetic analysis of lysogeny and that of the induced biosynthesis of ß-galactosidase led François Jacob, with Jacques Monod, to study the mechanisms responsible for the transfer of genetic information as well as the regulatory pathways which, in the bacterial cell, adjust the activity and synthesis of macromolecules. Following this analysis, Jacob and Monod proposed a series of new concepts, those of messenger RNA, regulator genes, operons and allosteric proteins.

Francois Jacob

Having determined the constants of growth in the presence of different carbohydrates, it occurred to me that it would be interesting to determine the same constants in paired mixtures of carbohydrates. From the first experiment on, I noticed that, whereas the growth was kinetically normal in the presence of certain mixtures (that is, it exhibited a single exponential phase), two complete growth cycles could be observed in other carbohydrate mixtures, these cycles consisting of two exponential phases separated by a-complete cessation of growth.

Lwoff, after considering this strange result for a moment, said to me, “That could have something to do with enzyme adaptation.”

“Enzyme adaptation? Never heard of it!” I said.

Lwoff’s only reply was to give me a copy of the then recent work of Marjorie Stephenson, in which a chapter summarized with great insight the still few studies concerning this phenomenon, which had been discovered by Duclaux at the end of the last century.  Studied by Dienert and by Went as early as 1901 and then by Euler and Josephson, it was more or less rediscovered by Karström, who should be credited with giving it a name and attracting attention to its existence.

Lwoff’s intuition was correct. The phenomenon of “diauxy” that I had discovered was indeed closely related to enzyme adaptation, as my experiments, included in the second part of my doctoral dissertation, soon convinced me. It was actually a case of the “glucose effect” discovered by Dienert as early as 1900.   That agents that uncouple oxidative phosphorylation, such as 2,4-dinitrophenol, completely inhibit adaptation to lactose or other carbohydrates suggested that “adaptation” implied an expenditure of chemical potential and therefore probably involved the true synthesis of an enzyme.

With Alice Audureau, I sought to discover the still quite obscure relations between this phenomenon and the one Massini, Lewis, and others had discovered: the appearance and selection of “spontaneous” mutants.   We showed that an apparently spontaneous mutation was allowing these originally “lactose-negative” bacteria to become “lactose-positive”. However, we proved that the original strain (Lac-) and the mutant strain (Lac+) did not differ from each other by the presence of a specific enzyme system, but rather by the ability to produce this system in the presence of lactose.  This mutation involved the selective control of an enzyme by a gene, and the conditions necessary for its expression seemed directly linked to the chemical activity of the system.


Albert Claude, Christian de Duve and George E. Palade –

” the structural and functional organization of the cell”.

I returned to Louvain in March 1947 after a period of working with Theorell in Sweden, the Cori’s, and E Southerland in St. Louis, fortunate in the choice of my mentors, all sticklers for technical excellence and intellectual rigor, those prerequisites of good scientific work. Insulin, together with glucagon which I had helped rediscover, was still my main focus of interest, and our first investigations were accordingly directed on certain enzymatic aspects of carbohydrate metabolism in liver, which were expected to throw light on the broader problem of insulin action. But I became distracted by an accidental finding related to acid phosphatase, drawing most of my collaborators along with me. The studies led to the discovery of the lysosome, and later of the peroxisome.

In 1962, I was appointed a professor at the Rockefeller Institute in New York, now the Rockefeller University, the institution where Albert Claude had made his pioneering studies between 1929 and 1949, and where George Palade had been working since 1946.  In New York, I was able to develop a second flourishing group, which follows the same general lines of research as the Belgian group, but with a program of its own.


Robert W. Holley, Har Gobind Khorana and Marshall W. Nirenberg –

“interpretation of the genetic code and its function in protein synthesis”.


Max Delbrück, Alfred D. Hershey and Salvador E. Luria –

” the replication mechanism and the genetic structure of viruses”.

1975 David Baltimore, Renato Dulbecco and Howard Martin Temin –

” the interaction between tumor viruses and the genetic material of the cell”.


Baruch S. Blumberg and D. Carleton Gajdusek –

” new mechanisms for the origin and dissemination of infectious diseases” The editors of the website of the Nobel Foundation have asked me to provide a supplement to the autobiography that I wrote in 1976 and to recount the events that happened after the award. Much of what I will have to say relates to the scientific developments since the last essay. These are described in greater detail in a scientific memoir first published in 2002 (Blumberg, B. S., Hepatitis B. The Hunt for a Killer Virus, Princeton University Press, 2002, 2004).


Baruj Benacerraf, Jean Dausset and George D. Snell 

” genetically determined structures on the cell surface that regulate immunological reactions”.


Edmond H. Fischer and Edwin G. Krebs 

“for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism”


Alfred G. Gilman and Martin Rodbell –

“G-proteins and the role of these proteins in signal transduction in cells”


Bruce A. Beutler and Jules A. Hoffmann –

the activation of innate immunity and the other half to Ralph M. Steinman – “the dendritic cell and its role in adaptive immunity”.

Renato L. Baserga, M.D.

Kimmel Cancer Center and Keck School of Medicine

Dr. Baserga’s research focuses on the multiple roles of the type 1 insulin-like growth factor receptor (IGF-IR) in the proliferation of mammalian cells. The IGF-IR activated by its ligands is mitogenic, is required for the establishment and the maintenance of the transformed phenotype, and protects tumor cells from apoptosis. It, therefore, serves as an excellent target for therapeutic interventions aimed at inhibiting abnormal growth. In basic investigations, this group is presently studying the effects that the number of IGF-IRs and specific mutations in the receptor itself have on its ability to protect cells from apoptosis.

This investigation is strictly correlated with IGF-IR signaling, and part of this work tries to elucidate the pathways originating from the IGF-IR that are important for its functional effects. Baserga’s group has recently discovered a new signaling pathway used by the IGF-IR to protect cells from apoptosis, a unique pathway that is not used by other growth factor receptors. This pathway depends on the integrity of serines 1280-1283 in the C-terminus of the receptor, which bind 14.3.3 and cause the mitochondrial translocation of Raf-1.

Another recent discovery of this group has been the identification of a mechanism by which the IGF-IR can actually induce differentiation in certain types of cells. When cells have IRS-1 (a major substrate of the IGF-IR), the IGF-IR sends a proliferative signal; in the absence of IRS-1, the receptor induces cell differentiation. The extinction of IRS-1 expression is usually achieved by DNA methylation.

Janardan Reddy, MD

Northwestern University

The central effort of our research has been on a detailed analysis at the cellular and molecular levels of the pleiotropic responses in liver induced by structurally diverse classes of chemicals that include fibrate class of hypolipidemic drugs, and phthalate ester plasticizers, which we designated hepatic peroxisome proliferators. Our work has been central to the establishment of several principles, namely that hepatic peroxisome proliferation is associated with increases in fatty acid oxidation systems in liver, and that peroxisome proliferators, as a class, are novel nongenotoxic hepatocarcinogens.

We introduced the concept that sustained generation of reactive oxygen species leads to oxidative stress and serves as the basis for peroxisome proliferator-induced liver cancer development. Furthermore, based on the tissue/cell specificity of pleiotropic responses and the coordinated transcriptional regulation of fatty acid oxidation system genes, we postulated that peroxisome proliferators exert their action by a receptor-mediated mechanism. This receptor concept laid the foundation for the discovery of

  • a three member peroxisome proliferator-activated receptor (PPARalpha-, ß-, and gamma) subfamily of nuclear receptors.
  •  PPARalpha is responsible for peroxisome proliferator-induced pleiotropic responses, including
    • hepatocarcinogenesis and energy combustion as it serves as a fatty acid sensor and regulates fatty acid oxidation.

Our current work focuses on the molecular mechanisms responsible for PPAR action and generation of fatty acid oxidation deficient mouse knockout models. Transcription of specific genes by nuclear receptors is a complex process involving the participation of multiprotein complexes composed of transcription coactivators.  

Jose Delgado de Salles Roselino, Ph.D.

Leloir Institute, Brazil

Warburg effect, in reality “Pasteur-effect” was the first example of metabolic regulation described. A decrease in the carbon flux originated at the sugar molecule towards the end metabolic products, ethanol and carbon dioxide that was observed when yeast cells were transferred from anaerobic environmental condition to an aerobic one. In Pasteur´s works, sugar metabolism was measured mainly by the decrease of sugar concentration in the yeast growth media observed after a measured period of time. The decrease of the sugar concentration in the media occurs at great speed in yeast grown in anaerobiosis condition and its speed was greatly reduced by the transfer of the yeast culture to an aerobic condition. This finding was very important for the wine industry of France in Pasteur time, since most of the undesirable outcomes in the industrial use of yeast were perceived when yeasts cells took very long time to create a rather selective anaerobic condition. This selective culture media was led by the carbon dioxide higher levels produced by fast growing yeast cells and by a great alcohol content in the yeast culture media. This finding was required to understand Lavoisier’s results indicating that chemical and biological oxidation of sugars produced the same calorimetric results. This observation requires a control mechanism (metabolic regulation) to avoid burning living cells by fast heat released by the sugar biological oxidative processes (metabolism). In addition, Lavoisier´s results were the first indications that both processes happened inside similar thermodynamics limits.

In much resumed form, these observations indicates the major reasons that led Warburg to test failure in control mechanisms in cancer cells in comparison with the ones observed in normal cells. Biology inside classical thermo dynamics poses some challenges to scientists. For instance, all classical thermodynamics must be measured in reversible thermodynamic conditions. In an isolated system, increase in P (pressure) leads to decrease in V (volume) all this in a condition in which infinitesimal changes in one affects in the same way the other, a continuum response. Not even a quantic amount of energy will stand beyond those parameters. In a reversible system, a decrease in V, under same condition, will led to an increase in P.

In biochemistry, reversible usually indicates a reaction that easily goes from A to B or B to A. This observation confirms the important contribution of E Schrodinger in his What´s Life: “This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject-matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

Hans Krebs describes the cyclic nature of the citrate metabolism. Two major research lines search to understand the mechanism of energy transfer that explains how ADP is converted into ATP. One followed the organic chemistry line of reasoning and therefore, searched how the breakdown of carbon-carbon link could have its energy transferred to ATP synthesis. A major leader of this research line was B. Chance who tried to account for two carbon atoms of acetyl released as carbon dioxide in the series of Krebs cycle reactions. The intermediary could store in a phosphorylated amino acid the energy of carbon-carbon bond breakdown. This activated amino acid could transfer its phosphate group to ADP producing ATP. Alternatively, under the possible influence of the excellent results of Hodgkin and Huxley a second line of research appears.

The work of Hodgkin & Huxley indicated the storage of electrical potential energy in transmembrane ionic asymmetries and presented the explanation for the change from resting to action potential in excitable cells. This second line of research, under the leadership of P Mitchell postulated a mechanism for the transfer of oxide/reductive power of organic molecules oxidation through electron transfer as the key for energetic transfer mechanism required for ATP synthesis. Paul Boyer could present how the energy was transduced by a molecular machine that changes in conformation in a series of 3 steps while rotating in one direction in order to produce ATP and in opposite direction in order to produce ADP plus Pi from ATP (reversibility). Nonetheless, a victorious Peter Mitchell obtained the correct result in the conceptual dispute, over the B. Chance point of view, after he used E. Coli mutants to show H gradients in membrane and its use as energy source.

However, this should not detract from the important work of Chance. B. Chance got the simple and rapid polarographic assay method of oxidative phosphorylation and the idea of control of energy metabolism that bring us back to Pasteur. This second result seems to have been neglected in searching for a single molecular mechanism required for the understanding of the buildup of chemical reserve in our body. In respiring mitochondria the rate of electron transport, and thus the rate of ATP production, is determined primarily by the relative concentrations of ADP, ATP and phosphate in the external media (cytosol) and not by the concentration of respiratory substrate as pyruvate. Therefore, when the yield of ATP is high as is in aerobiosis and the cellular use of ATP is not changed, the oxidation of pyruvate and therefore of glycolysis is quickly (without change in gene expression), throttled down to the resting state. The dependence of respiratory rate on ADP concentration is also seen in intact cells. A muscle at rest and using no ATP has very low respiratory rate.

I have had an ongoing discussion with Jose Eduardo de Salles Roselino, inBrazil. He has made important points that need to be noted.

  1. The constancy of composition which animals maintain in the environment surrounding their cells is one of the dominant features of their physiology. Although this phenomenon, homeostasis, has held the interest of biologists over a long period of time, the elucidation of the molecular basis for complex processes such as temperature control and the maintenance of various substances at constant levels in the blood has not yet been achieved. By comparison, metabolic regulation in microorganisms is much better understood, in part because the microbial physiologist has focused his attention on enzyme-catalyzed reactions and their control, as these are among the few activities of microorganisms amenable to quantitative study. Furthermore, bacteria are characterized by their ability to make rapid and efficient adjustments to extensive variations in most parameters of their environment; therefore, they exhibit a surprising lack of rigid requirements for their environment, and appears to influence it only as an incidental result of their metabolism. Animal cells on the other hand have only a limited capacity for adjustment and therefore require a constant milieu. Maintenance of such constancy appears to be a major goal in their physiology (Regulation of Biosynthetic Pathways H.S. Moyed and H EUmbarger Phys Rev,42 444 (1962)).
  2. A living cell consists in a large part of a concentrated mixture of hundreds of different enzymes, each a highly effective catalyst for one or more chemical reactions involving other components of the cell. The paradox of intense and highly diverse chemical activity on the one hand and strongly poised chemical stability (biological homeostasis) on the other is one of the most challenging problems of biology (Biological feedback Control at the molecular Level D.E. Atkinson Science vol. 150: 851, 1965). Almost nothing is known concerning the actual molecular basis for modulation of an enzyme`s kinetic behavior by interaction with a small molecule. (Biological feedback Control at the molecular Level D.E. Atkinson Science vol. 150: 851, 1965). In the same article, since the core of Atkinson´s thinking seems to be strongly linked with Adenylates as regulatory effectors, the previous phrases seems to indicate a first step towards the conversion of homeostasis to an intracellular phenomenon and therefore, one that contrary to Umbarger´s consideration could be also studied in microorganisms.
  3.  Most biochemical studies using bacteria, were made before the end of the third upper part of log growth phase. Therefore, they could be considered as time-independent as S Luria presented biochemistry in Life an Unfinished Experiment. The sole ingredient on the missing side of the events that led us into the molecular biology construction was to consider that proteins, a macromolecule, would never be affected by small molecules translational kinetic energy. This, despite the fact that in a catalytic environment and its biological implications S Grisolia incorporated A K Balls observation indicating that the word proteins could be related to Proteus an old sea god that changed its form whenever he was subjected to inquiry (Phys Rev v 4,657 (1964).
  1. In D.E. Atkinson´s work (Science vol 150 p 851, 1965), changes in protein synthesis acting together with factors that interfere with enzyme activity will lead to “fine-tuned” regulation better than enzymatic activity regulation alone. Comparison of glycemic regulation in granivorous and carnivorous birds indicate that when no important nutritional source of glucose is available, glycemic levels can be kept constant in fasted and fed birds. The same was found in rats and cats fed on high protein diets. Gluconeogenesis is controlled by pyruvate kinase inhibition. Therefore, the fact that it can discriminate between fasting alone and fasting plus exercise (carbachol) requirement of gluconeogenic activity (correspondent level of pyruvate kinase inhibition) the control of enzyme activity can be made fast and efficient without need for changes in genetic expression (20 minute after stimulus) ( Migliorini,R.H. et al Am J. Physiol.257 (Endocrinol. Met. 20): E486, 1989). Regrettably, this was not discussed in the quoted work. So, when the control is not affected by the absorption of nutritional glucose it can be very fast, less energy intensive and very sensitive mechanism of control despite its action being made in the extracellular medium (homeostasis).

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Erythropoietin (EPO) and Intravenous Iron (Fe) as Therapeutics for Anemia in Severe and Resistant CHF: The Elevated N-terminal proBNP Biomarker


Co-Author of the FIRST Article: Larry H. Bernstein, MD, FCAP

Reviewer and Curator of the SECOND and of the THIRD Articles: Larry H. Bernstein, MD, FCAP


Article Architecture Curator: Aviva Lev-Ari, PhD, RN

This article presents Advances in the Treatment using Subcutaneous Erythropoietin (EPO) and Intravenous Iron (Fe) for IMPROVEMENT of Severe and Resistant Congestive Heart Failure and its resultant Anemia.  The Leading Biomarker for Congestive Heart Failure is an Independent Predictor identified as an Elevated N-terminal proBNP.

NT-proBNP schematic diagram-Copy.pdf_page_1


Anemia as an Independent Predictor of Elevated N-terminal proBNP

Salman A. Haq, MD1, Mohammad E. Alam2, Larry Bernstein, MD, FCAP3,  LB Banko 1, Leonard Y. Lee, MD, FACS4, Barry I. Saul, MD, FACC5, Terrence J. Sacchi, MD, FACC6,  John F. Heitner, MD, FACC7
1Cardiology Fellow,  2  Clinical Chemistry Laboratories, 3 Program Director, Cardiothoracic Surgery, 4 Division of Cardiology,  Department of Medicine, New York Methodist Hospital-Weill Cornell, Brooklyn, NY

(Unpublished manuscript)  Poster Presentation


The effect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study

Donald S Silverberg, MDa; Dov Wexler, MDa; David Sheps, MDa; Miriam Blum, MDa; Gad Keren, MDa; Ron Baruch, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Shoshana Steinbruch, RNa; Itzhak Shapira, MDa; Shlomo Laniado, MDa; Adrian Iaina, MDa

J Am Coll Cardiol. 2001;37(7):1775-1780. doi:10.1016/S0735-1097(01)01248-7


The use of subcutaneous erythropoietin and intravenous iron for the treatment of the anemia of severe, resistant congestive heart failure improves cardiac and renal function and functional cardiac class, and markedly reduces hospitalizations

Donald S Silverberg, MDa; Dov Wexler, MDa; Miriam Blum, MDa; Gad Keren, MDa; David Sheps, MDa; Eyal Leibovitch, MDa; David Brosh, MDa; Shlomo Laniado, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Itzhak Shapira, MDa; Dov Gavish, MDa; Ron Baruch, MDa; Bella Koifman, MDa; Carl Kaplan, MDa; Shoshana Steinbruch, RNa; Adrian Iaina, MDa

J Am Coll Cardiol. 2000;35(7):1737-1744. doi:10.1016/S0735-1097(00)00613-6


This THREE article sequence is related by investigations occurring by me, a very skilled cardiologist and his resident, and my premedical student at New York Methodist Hospital-Weill Cornell, in Brooklyn, NY, while a study had earlier been done applying the concordant discovery, which the team in Israel had though was knowledge neglected.  There certainly was no interest in the problem of the effect of anemia on the patient with severe congestive heart failure, even though erythropoietin was used widely in patients with end-stage renal disease requiring dialysis, and also for patients with myelofibrosis.  The high cost of EPO was only one factor, the other being a guideline to maintain the Hb concentration at or near 11 g/dl – not higher.  In the first article, the authors sought to determine whether the amino terminal pro– brain type natriuretic peptide (NT-pro BNP) is affected by anemia, and to determine that they excluded all patients who had renal insufficiency and/or CHF, since these were associated with elevated NT-proBNP.  It was already well established that this pro-peptide is secreted by the heart with the action as a urinary sodium retention hormone on the kidney nephron, the result being an increase in blood volume.  Perhaps the adaptation would lead to increased stroke volume from increased venous return, but that is not conjectured.  However, at equilibrium, one would expect there to be increased red cell production to maintain the cell to plasma volume ratio, thereby, resulting in adequate oxygen exchange to the tissues.  Whether that is always possible is uncertain because any reduction in the number of functioning nephrons would make the kidney not fully responsive at the Na+ exchange level, and the NT-pro BNP would rise.  This introduces complexity into the investigation, requiring a removal of confounders to establish the effect of anemia.

The other two articles are related studies by the same group in Israel.  They surmised that there was evidence that was being ignored as to the effect of anemia, and the treatment of anemia was essential in addition to other treatments.  They carried out a randomized trial to determine just that, a benefit to treating the anemia.  But they also conjectured that an anemia with a Hb concentration below 12 g/dl has an deleterious effect on the targeted population.  Treatment by intermittent transfusions could potentially provide the added oxygen-carrying capacity, but the fractionation of blood, the potential for transfusion-transmitted disease and transfusion-reactions, combined with the need for the blood for traumatic blood loss made EPO a more favorable alternative to packed RBCs.  The proof-of-concept is told below.  Patients randomized to receive EPO at a lower than standard dose + iron did better.



In this article, Erythropoietin (EPO) and Intravenous Iron (Fe) as Therapeutics for Anemia in Severe and Resistant CHF: The Elevated N-terminal proBNP Biomarker we provides a summary of three articles on the topic and we shading new light on the role that Anemia Hb < 12 g%  plays as a Biomarker for CHF and for

  • prediction of elevated BNP, known as an indicator for the following Clinical Uses:
Clinical Use
  • Rule out congestive heart failure (CHF) in symptomatic individuals
  • Determine prognosis in individuals with CHF or other cardiac disease
  • Maximize therapy in individuals with heart failure by the use of Subcutaneous Erythropoietin (EPO) and Intravenous Iron (Fe)
Evaluation of BNP and NT-proBNP Clinical Performance
Study Sensitivity(%) Specificity(%) PPV(%) NPV(%)
Diagnose impaired LVEF3
BNP 73 77 70 79
NT-proBNP 70 73 61 80
Diagnose LV systolic dysfunction after MI2
BNP 68 69 56 79
NT-proBNP 71 69 56 80
Diagnose LV systolic dysfunction after MI12
BNP 94 40 NG 96
NT-proBNP 94 37 NG 96
Prognosis in newly diagnosed heart failure patients: prediction of mortality/survival1
BNP 98 22 42 94
NT-proBNP 95 37 47 93
Prognosis post myocardial infarction: prediction of mortality2
BNP 86 72 39 96
NT-proBNP 91 72 39 97
Prognosis post myocardial infarction: prediction of heart failure2
BNP 85 73 54 93
NT-proBNP 82 69 50 91
PPV, positive predictive value; NPV, negative predictive value; LVEF, left ventricular ejection fraction; NG, not given.
Reference Range
BNP: < 100 pg/mL13
proBNP, N-terminal: 300 pg/mL
The NT-proBNP reference range is based on EDTA plasma. Other sample types will produce higher values.
Interpretive Information
Symptomatic patients who present with a BNP or NT-proBNP level within the normal reference range are highly unlikely to have CHF. Conversely, an elevated baseline level indicates the need for further cardiac assessment and indicates the patient is at increased risk for future heart failure and mortality.BNP levels increase with age in the general population, with the highest concentrations seen in those greater than 75 years of age.14 Heart failure is unlikely in individuals with a BNP level <100 pg/mL and proBNP level ≤300 pg/mL. Heart failure is very likely in individuals with a BNP level >500 pg/mL and proBNP level ≥450 pg/mL who are <50 years of age, or ≥900 pg/mL for patients ≥50 years of age. Patients in between are either hypertensive or have mild ischemic or valvular disease and should be observed closely.15BNP is increased in CHF, left ventricular hypertrophy, acute myocardial infarction, atrial fibrillation, cardiac amyloidosis, and essential hypertension. Elevations are also observed in right ventricular dysfunction, pulmonary hypertension, acute lung injury, subarachnoid hemorrhage, hypervolemic states, chronic renal failure, and cirrhosis.NT-proBNP levels are increased in CHF, left ventricular dysfunction, myocardial infarction, valvular disease, hypertensive pregnancy, and renal failure, even after hemodialysis.Although levels of BNP and NT-proBNP are similar in normal individuals, NT-proBNP levels are substantially greater than BNP levels in patients with cardiac disease due to increased stability (half-life) of NT-proBNP in circulation. Thus, results from the two tests are not interchangeable.
  1. Cowie MR and Mendez GF. BNP and congestive heart failure. Prog Cardiovasc Dis. 2002;44:293-321.
  2. Richards AM, Nicholls MG, Yandle TG, et al. Plasma N-terminal pro-brain natriuretic peptide and adrenomedullin. New neurohormonal predictors of left ventricular function and prognosis after myocardial infarction. Circulation. 1998:97:1921-1929.
  3. Hammerer-Lercher A, Neubauer E, Muller S, et al. Head-to-head comparison of N-terminal pro-brain natriuretic peptide, brain natriuretic peptide and N-terminal pro-atrial natriuretic peptide in diagnosing left ventricular dysfunction. Clin Chim Acta. 2001;310:193-197.
  4. McDonagh TA, Robb SD, Murdoch DR, et al. Biochemical detection of left-ventricular systolic dysfunction. Lancet. 1998;351:9-13.
  5. Mukoyama Y, Nakao K, Hosoda K, et al. Brain natriuretic peptide as a novel cardiac hormone in humans: Evidence for an exquisite dual natriuretic peptide system, ANP and BNP. J Clin Invest. 1991;87:1402-1412.
  6. Hunt PJ, Richards AM, Nicholls MG, et al. Immunoreactive amino-terminal pro-brain natriuretic peptide (NT-PROBNP): a new marker of cardiac impairment. Clin Endocrinol. 1997;47:287-296.
  7. Davis M, Espiner E, Richards G, et al. Plasma brain natriuretic peptide in assessment of acute dyspnoea. Lancet. 1994;343:440-444.
  8. Kohno M, Horio T, Yokokawa K, et al. Brain natriuretic peptide as a cardiac hormone in essential hypertension. Am J Med. 1992;92:29-34.
  9. Bettencourt P, Ferreira A, Pardal-Oliveira N, et al. Clinical significance of brain natriuretic peptide in patients with postmyocardial infarction. Clin Cardiol. 2000;23:921-927.
  10. Jernberg T, Stridsberg M, Venge P, et al. N-terminal pro brain natriuretic peptide on admission for early risk stratification of patients with chest pain and no ST-segment elevation. J Am Coll Cardiol. 2002;40:437-445.
  11. Richards AM, Troughton RW. Use of natriuretic peptides to guide and monitor heart failure therapy. Clin Chem. 2012;58:62-71.
  12. Pfister R, Scholz M, Wielckens K, et al. The value of natriuretic peptides NT-pro-BNP and BNP for the assessment of left-ventricular volume and function. A prospective study of 150 patients.Dtsch Med Wochenschr. 2002;127:2605-2609.
  13. Siemens ADVIA Centaur® BNP directional insert; 2003.
  14. Redfield MM, Rodeheffer RJ, Jacobsen SJ, et al. Plasma brain natriuretic peptide concentration: impact of age and gender. J Am Coll Cardiol. 2002;40:976-982.
  15. Weber M, Hamm C. Role of B-type natriuretic peptid (BNP) and NT-proBNP in clinical routine.Heart. 2006;92:843-849.


B-type Natriuretic Peptide and proBNP, N-terminal


Anemia as an Independent Predictor of Elevated N-terminal proBNP

Salman A. Haq, MD1, Mohammad E. Alam2, Larry Bernstein, MD, FCAP3,  LB Banko 1, Leonard Y. Lee, MD, FACS4, Barry I. Saul, MD, FACC5, Terrence J. Sacchi, MD, FACC6,  John F. Heitner, MD, FACC7
1Cardiology Fellow,  2  Clinical Chemistry Laboratories, 3 Program Director, Cardiothoracic Surgery, 4 Division of Cardiology,  Department of Medicine, New York Methodist Hospital-Weill Cornell, Brooklyn, NY

(Unpublished manuscript)  Poster Presentation:

Anemia as an Independent Predictor of Elevated N-Terminal proBNP Levels in
Patients without Evidence of Heart Failure and Normal Renal Function.

Haq SA, Alam ME, Bernstein L, Banko LB, Saul BI, Lee LY, Sacchi TJ, Heitner JF.

Table 1.  Patient Characteristics

Variable No Anemia(n=138) Anemia(n=80)
Median Age (years) 63 76
Men (%) 35 33
Creatinine (mg/dl) 0.96 1.04
Hemoglobin (g/dl) 13.7 10.2
LVEF (%) 67 63
Median NT-proBNP (pg/ml) 321.6 1896.0


A series of slide showing the determination of the representation of normal NT-proBNP range
after removal of patient confounders.







N-terminal proBNP (NT-proBNP) has emerged as a primary tool for diagnosing congestive heart failure (CHF). Studies have shown that the level of

  • NT-proBNP is affected by renal insufficiency (RI) and age, independent of the diagnosis of CHF.

There is some suggestion from recent studies that

  • anemia may also independently affect NT-proBNP levels.


To assess the affect of anemia on NT-proBNP independent of CHF, RI, and age.


We evaluated 746 consecutive patients presenting to the Emergency Department (ED) with shortness of breath and underwent evaluation with serum NT-proBNP.

All patients underwent a trans-thoracic echocardiogram (TTE) and clinical evaluation for CHF. Patients were included in this study if they had a normal TTE (normal systolic function, mitral inflow pattern and left ventricular (LV) wall thickness) and no evidence of CHF based on clinical evaluation. Patients were excluded if they had RI (creatinine > 2 mg/dl) or a diagnosis of sepsis. Anemia was defined using the World Health Organization (W.H.O.) definition of

  • hemoglobin (hgb) < 13 g/dl for males and hgb < 12 g/dl for females.


Of the 746 consecutive patients, 218 patients (138 anemia, 80 no anemia) met the inclusion criteria. There was a markedly significant difference between

  • NT- proBNP levels based on the W.H.O. diagnosis of anemia.

Patients with anemia had a

  • mean NT- proBNP of 4,735 pg/ml compared to 1,230 pg/ml in patients without anemia (p=0.0001).

There was a markedly

  • significant difference in patients who had a hgb > 12 (median 295 pg/ml) when compared to
  • both patients with an hgb of 10.0 to 11.9 (median 2,102 pg/ml; p = 0.0001) and
  • those with a hgb < 10 (median 2,131 pg/ml; p = 0.001).

Linear regression analysis comparing hgb with log NT-proBNP was statistically significant (r = 0.395; p = 0.0001). MANOVA demonstrated that

  • elevated NT- proBNP levels in patients with anemia was independent of age.


This study shows that NT-proBNP is associated with anemia independent of CHF, renal insufficiency, sepsis or age.


B-type natriuretic peptide (BNP) is secreted from the myocardium in response to myocyte stretch. 1-2 BNP is released from the myocytes as a 76 aminoacid N-terminal fragment (NT-proBNP) and a 32-amino acid active hormone (BNP). 3 These peptides have emerged as a primary non-invasive modality for the diagnosis of congestive heart failure (CHF). 4- 7 In addition, these peptides have demonstrated prognostic significance in patients with invasive modality for the diagnosis of

  • congestive heart failure (CHF). 4- 7
  • heart failure 8-9,
  • stable coronary artery disease 10, and
  • in patients with acute coronary syndromes. 11-14

Studies have shown that the level of NT- proBNP is affected by

  • age and renal insufficiency (RI) independent of the diagnosis of CHF. 15,16

There is some suggestion from the literature that

  • anemia may also independently affect NT-proBNP levels. 17-20

Willis et al. demonstrated in a cohort of 209 patients without heart failure that anemia was associated with an elevated NT- proBNP. 17 Similarly, in 217 patients undergoing cardiac catheterization, blood samples were drawn from the descending aorta prior to contrast ventriculography for BNP measurements and

  • anemia was found to be an independent predictor of plasma BNP levels. 18

The objective of this study is to assess the effect of anemia on NT-proBNP independent of CHF, sepsis, age or renal insufficiency.


Patient population

The study population consisted of 746 consecutive patients presenting to the emergency room who underwent NT-proBNP evaluation for the evaluation of dyspnea. Transthoracic echocardiogram (TTE) was available on 595 patients. Patients were included in this study if they had a normal TTE, which was defined as normal systolic function (left ventricular ejection fraction [LVEF] > 45%), normal mitral inflow pattern and normal LV wall thickness. CHF was excluded based on thorough clinical evaluation by the emergency department attending and the attending medical physician. Patients with disease states that may affect the NT- proBNP levels were also excluded:

  1. left ventricular systolic dysfunction (LVEF < 45%),
  2. renal insufficiency defined as a creatinine > 2 mg/dl and
  3. sepsis (defined as positive blood cultures with two or more of the following systemic inflammatory response syndrome (SIRS) criteria: heart rate > 90 beats per minute;
  4. body temperature < 36 (96.8 °F) or > 38 °C (100.4 °F);
  5. hyperventilation (high respiratory rate) > 20 breaths per minute or, on blood gas, a PaCO2 less than 32 mm Hg;
  6. white blood cell count < 4000 cells/mm3 or > 12000 cells/mm³ (< 4 x 109 or > 12 x 109 cells/L), or greater than 10% band forms (immature white blood cells). 21

The study population was then divided into two groups, anemic and non- anemic. Anemia was defined using the world health organization (W.H.O.) definition of hemoglobin (hgb) < 13 g/dl for males and < 12 g/dl for females.The data was also analyzed by dividing the patients into three groups based on hgb levels i.e. hgb > 12, hgb 10 to 11.9 and hgb < 10.

Baseline patient data

Patient’s baseline data including age, gender, ethnicity, hemoglobin (hgb), hematocrit (hct), creatinine, NT- proBNP were recorded from the electronic medical record system in our institution. Chemistry results were performed on the Roche Modular System (Indianapolis, IN), with the NT- proBNP done by chemiluminescence assay. The hemogram was performed on the Beckman Coulter GenS. All TTE’s were performed on Sonos 5500 machine. TTE data collected included LVEF, mitral inflow pattern and LV wall thickness assessment.

Statistical analysis

The results are reported in the means with p < 0.05 as the measure of significance for difference between means. Independent Student’s t-tests were done comparing NT proBNP and anemia. Univariate ANOVAs and multivariate ANOVA (MANOVA) with post hoc tests using the Bonferroni method were used to compare NT- proBNP levels with varying ranges of hgb and age using SPSS 13.0 (SPSS, Chicago, IL). A linear regression analysis was performed using SYSTAT. Calculations included Wilks’Lamda, Pillai trace and Hotelling-Lawley trace. A GOLDMineR® plot was constructed to estimate the effects of age and anemia on NT- proBNP levels. The GOLDMineR® effects plot displays the odds-ratios for predicted NT-proBNP elevation versus the predictor values. Unlike the logistic regression, the ordinal regression, which the plot is derived from, can have polychotomous as well as dichotomous values, as is the case for NT-proBNP.


Of the 746 consecutive patients, 218 patients met the inclusion criteria (fig 1). Baseline characteristics of patients are listed in table 1. The median age for anemic patients was 76 years and 63 years for patients without anemia. One third of patients in both groups were men. The mean hemoglobin for

  • anemic patients was 10.2 g/dl as compared to 13.7 g/dl for non-anemic patients.
  • The mean LVEF of patients with anemia was 64% as compared to 67% for non-anemic patients.

Based on the WHO definition of anemia, 138 patients were determined to be anemic while 80 patients were diagnosed as non-anemic. There was a markedly  significant difference between NT-proBNP levels based on the WHO diagnosis of anemia.

Patients with anemia had a

  • mean NT-proBNP of 4,735 pg/ml compared to 1,230 pg/ml in patients without anemia (p = 0.0001).

Of the 218 patients in the study, 55 patients had a hgb of < 10 g/dl. Analysis using

  • hgb < 10 g/dl for anemia demonstrated a statistically significant difference in the NT-proBNP values.

Patients with a hgb < 10 g/dl had a mean NT- proBNP of 5,130 pg/ml

  • compared to 2,882 pg/ml in patients with a hgb of > 10 g/dl (p = 0.01)

The groups were also divided into three separate categories of hgb for subset analysis:

  • hgb > 12 g/dl,
  • hgb 10 to 11.9 g/dl and
  • hgb < 10 g/dl.

There was a markedly significant difference in

  •  the NT- ProBNP levels of patients who had a hgb > 12 g/dl (median 295 pg/ml) when
  • compared to those with a hgb range of 10.0 g/dl to 11.9 g/dl (median 2,102 pg/ml) (p = 0.0001),

and also a significant difference in

  • NT- proBNP levels of patients with a hgb > 12 g/dl (median 295 pg/ml) when
  • compared to a hgb of < 10 g/dl (median 2,131 pg/ml) (p = 0.001).

However, there was no statistically significant difference in NT-proBNP levels of patients with hgb 10 g/dl to 11.9 g/dl

  • when compared to those with a hgb of < 10 g/dl (p = 1.0).

A scatter plot comparing hgb with log NT-proBNP and fitting of a line to the data by ordinary least squares regression was significant (p = 0.0001) and demonstrated

  • a correlation between anemia and NT-proBNP levels (r = 0.395) (fig. 2).

MANOVA demonstrated that elevated NT- proBNP levels in patients with anemia was independent of age (Wilks’ Lambda [p = 0.0001]). In addition, using GOLDMineR® plots (figure 3a and 3b) with a combination of age and hb scaled as predictors of elevated NT-proBNP,

  • both age and hgb were required as independent predictors.

What about the effect of anemia? The GOLDminer analysis of ordinal regression was carried out in a database from which renal insufficiency and CHF were removed. The anemia would appear to have an independent effect on renal insufficiency. Figure 4 is a boxplot comparison of NT – proBNP, the age normalized function NKLog (NT- proBNP)/eGFR formed from taking 1000*Log(NT- proBNP) divided by the MDRD at eGFR exceeding 60 ml/min/m2 and exceeding 30 ml/min/m2. The transformed variable substantially makes the test independent of age and renal function. The boxplot shows the medians, 97.5, 75, 25 and 2.5 percentiles. There appears to be no significance in the NKLog(NT pro-BNP)/MDRD plot. Table II compares the NT-proBNP by WHO criteria at eGFR 45, 60 and 75 ml/mln/m2 using the t-test with unequal variance assumed, and the Kolmogorov-Smirnov test for nonparametric measures of significance. The significance at 60 ml/min/m2 is marginal and nonexistent at 75 ml/min/m2. This suggests that the contribution from renal function at above 60 ml/min2 can be ignored. This is consistent with the findings using the smaller, trimmed database, but there is an interaction between

  •  anemia, and
  •  eGFR at levels below 60 ml/min/m2


The findings in this study indicate that

  • anemia was associated with elevated NT-proBNP levels independent of CHF, renal insufficiency, sepsis or age.

These findings have been demonstrated with NT-proBNP in only one previous study. Wallis et al. demonstrated that after adjusting for age, sex, BMI, GFR, LVH and valvular disease;

  1. only age,
  2. valvular disease and
  3. low hemoglobin

were significantly associated with increased NT-proBNP. 18.

In our study, CHF was excluded based on both a normal TTE and a thorough clinical evaluation. In the only other study directly looking at NT- proBNP levels in anemic patients without heart failure

  • only 25% of patients had TTEs, with one patient having an LVEF of 40%. 17

BNP, the active molecule released after cleavage along with NT- proBNP, has also been studied in relation to blood hemoglobin levels. 18 In 263 patients undergoing cardiac catheterization  blood samples were drawn from the descending aorta prior to contrast ventriculography to determine the value of BNP. Anemia was present in 217 patients. Multivariate linear regression model adjusting for

  1.  age,
  2.  gender,
  3.  body mass index,
  4.  history of myocardial infarction,
  5.  estimated creatinine clearance, and
  6.  LVEF
  • found hgb to be an independent predictor of BNP levels.

In our study, patients with anemia were slightly older than those without anemia. However, both MANOVA and GOLDMineR® plot demonstrated that

  • elevated NT-proBNP levels in patients with anemia was independent of age.

Other studies have found that BNP is dependent on renal insufficiency and age. Raymond et al. randomly selected patients to complete questionnaires regarding CHF and

  1. then underwent pulse and blood pressure measurements,
  2.  electrocardiogram (ECG),
  3.  echocardiography and
  4.  blood sampling. 15

A total of 672 subjects were screened and 130 were determined to be normal, defined as

  • no CHF or ischemic heart disease,
  • normal LVEF,
  • no hypertension,
  • diabetes mellitus,
  • lung disease, and
  • not on any cardiovascular drugs.

They found

  1. older age,
  2. increasing dyspnea,
  3. high plasma creatinine and a
  4. LVEF < 45%

to be independently associated with an elevated NT-proBNP plasma level by multiple linear regression analysis. In another study, McCullough et al. evaluated the patients from the Breathing Not Properly Multinational Study

  • looking at the relationship between BNP and renal function in CHF. 16

Patients were excluded if they were on hemodialysis or had a estimated glomerular filteration rate (eGFR) of < 15 ml/min. They found that the BNP levels correlated significantly with the eGFR, especially in patients without CHF, suggesting

  1. chronic increased blood volume and
  2. increased left ventricular wall tension as a possible cause. 16

Our study was designed to exclude patients with known diseases such as CHF and renal insufficiency in order to demonstrate

  • the independent effect of anemia on elevated NT-proBNP levels.

The mechanism for elevated NT-proBNP levels in patients with anemia is unknown. Some possible mechanisms that have been reported in the literature include

  • hemodilution secondary to fluid retention in patients with CHF 18,
  • decreased oxygen carrying capacity with accompanying tissue hypoxia which
  • stimulates the cardio-renal compensatory mechanism leading to increased release of NT-proBNP. 17

The findings from our study suggest that

  •  NT-proBNP values should not be interpreted in isolation of hemoglobin levels and
  • should be integrated with other important clinical findings for the diagnosis of CHF.

Further studies are warranted

  1.  to assess the relationship between anemia and plasma natriuretic peptides, and
  2. possibly modify the NT-proBNP cutoff points for diagnosing acutely decompensated CHF in patients with anemia.


This study shows that elevated NT-proBNP levels are associated with anemia independent of

  •   CHF,
  •  renal insufficiency,
  •  sepsis and
  •  age.

NT-proBNP levels should be interpreted with caution in patients who have anemia.


1. Brunea BG, Piazza LA, de Bold AJ. BNP gene expression is specifically modulated by stretch and ET-1 in a new model of isolated rat atria.Am J Physiol  1997; 273:H2678-86.

2. Wiese S, Breyer T, Dragu A, et al. Gene expression of brain natriuretic peptide  in isolated atrial and ventricular human myocardium: influence of angiotensin II and diastolic fiber length. Circ 2000; 102:3074-79.

3. de Lemos JA, McGuire DK, Drazner MH. B-type natriuretic peptide in cardiovascular disease. Lancet 2003; 362:316-22.

4.   Dao Q, Krishnaswamy P, Kazanegra R, et al. Utility of B-type natriuretic  peptide in the diagnosis of congestive heart failure in an urgent care setting. J Am  Coll Cardiol 2001; 37:379-85.

5. Morrison LK, Harrison A, Krishnaswamy P, Kazanegra R, Clopton P, Maisel A. Utility of rapid natriuretic peptide assay in differentiating congestive heart failure from lung  disease in patients presenting with dyspnea.
J Am Coll Cardiol  2003; 39:202-09.

6.  Maisel AS, Krishnaswamy P, Nowak RM, et al.  Rapid measurement of B-type natriuretic peptide in the emergency diagnosis of heart failure. N Engl J Med 2002; 347:161-67.

7. Januzzi JL, Camargo CA, Anwaruddin S, et al. The N-terminal Pro-BNP investigation of dyspnea in the emergency department (PRIDE) study. Am J  Cardiol 2005; 95:948-954.

8.  Tsutamoto T, Wada A, Meada K, et al.   Attenuation of compensation of  endogenous cardiac natriuretic peptide system  in chronic heart failure: prognostic role  of plasma  brain natriuretic peptide concentration in patients with chronic  symptomatic  left ventricular dysfunction.
Circulation 1997; 96(2): 509-16.

9.  Anand IS, Fisher LD, Chiang YT, et al. Changes in brain natriuretic peptide and norepinephrine over time and mortality and morbidity in the Valsartan Heart Failure Trial (Val-HEFT). Circulation 2003; 107:1278-1283.

10. Omland T, Richards AM, Wergeland R and Vik-Mo H. B-type natriuretic peptide and long term survival in patients with stable coronary artery disease.
Am J Cardiol 2005; 95:24-28.

11. Omland T, Aakvaag A, Bonarjee VV. et al. Plasma brain natriuretic peptide as an indicator of left ventricular systolic dysfunction and long term prognosis after acute myocardial infarction. Comparison with plasma atrial natriuretic peptide and N-terminal proatrial natriuretic peptide.
Circulation 1996; 93:1963-1969.

12. de Lemos JA, Morrow DA, Bently JH, et al. The prognostic value of B-type natriuretic peptide in patients with acute coronary syndromes. N Engl J Med 2001; 345:1014-1021.

13. Richards AM, Nicholls MG, Espiner EA, et al. B-type natriuretic peptides and  ejection fraction for prognosis after myocardial infarction. Circulation 2003; 107:2786-2792.

14. Sabatine MS, Morrow DA, de Lemos JA, et al.  Multimarker approach to risk  stratification in non-ST elevation acute coronary syndromes: simultaneous  assessment of troponin I, C-reactive protein and B-type natriuretic peptide.
Circulation 2002; 105:1760-1763.

15. Raymond I, Groenning BA, Hildebrandt PR, Nilsson JC, Baumann M, Trawinski   J, Pedersen F.  The influence of age, sex andother variables on the plasma level of N-terminal pro brain natriureticpeptide in a large sample of the general  population. Heart 2003; 89:745-751.

16. McCollough PA, Duc P, Omland T, McCord J, Nowak RM, Hollander JE, et al. B-type natriuretic peptide and renal function in the diagnosis of heartfailure:  an analysis from the  Breathing Not Properly Multinational Study.
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17. Willis MS, Lee ES, Grenache DG. Effect of anemia on plasma concentrations of  NT-proBNP.
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20. Wu AH, Omland T, Wold KC, McCord J, Nowak RM, et al. Relationship  of B-type natriuretic peptide and anemia  in patients withand without heart failure:  A substudy from the Breathing Not Properly(BNP) Multinational Study.
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Table Legends

Table I. Clinical characteristics of the study population

Table II. Comparison of NT- proBNP means under WHO criteria at different GFR

Table I
Variable No Anemia(n=80) Anemia(n=138)
Median age (years) 63 76
    Men (%) 27 (34) 47 (34)
    Women (%) 53 (66) 91 (66)
Weight (kg) 82.9 80.1
Chest Pain 21 (26) 3 (2)
Hemoglobin (g/dl) 13.7 10.2
Hematocrit (%) 40.5 30.5
Mean Corpuscular Volume 97 87
Creatinine (mg/dl) 0.99 1.07
Median NT-proBNP (pg/ml) 321 1896
Medical History
    HTN (%) 12 (15) 51 (37)
    Prior MI (%) 11 (14) 5 (4)
    ACS (%) 16 (20) 3 (2)
    CAD (%) 2 (1) 3 (2)
     DM (%) 18 (22) 11 (8)
   Clopidogrel 58 (72) 15 (11)
   Beta Blockers 68 (85) 27 (20)
   Ace Inhibitors 45 (56) 18 (13)
   Statins 57 (71) 17 (12)
   Calcium Channel Blocker 17 (21) 8 (6)
LVEF (%) 67 64

HTN: Hypertension CAD: Coronary Artery Disease
MI: Myocardial Infarction DM: Diabetes Mellitus
ACS: Acute Coronary Syndrome LVEF: Left Ventricular Ejection Fraction

Table II
GFR WHO Mean P (F) N NPar
> 45 0 3267 0.022 (4.33) 661
1 4681
> 60* 0 2593 0.031 (5.11) 456 0.018
1 4145
> 60r 0 786 0.203 (3.63) 303 0.08
1 3880
> 75 0 2773 > 0.80 320 0.043
1 3048

*AF, valve disease and elevated troponin T included
r AF, valve disease and elevated troponin T removed


FIGURE 1. Study population flow chart. (see poster)
FIGURE 2. Relationship between proBNP and hemoglobin. (see above)
FIGURE 3. NT-proBNP levels in relation to anemia (see above)

Supplementary Material

Table based on LatentGOLD Statistical Innovations, Inc., Belmont, MA., 2000: Jeroen Vermunt & Jay Magidson)

4-Cluster Model

Number of cases                                   408
Number of parameters (Npar)             24

Chi-squared Statistics
Degrees of freedom (df)                          71                     p-value
L-squared (L²)                                    80.2033                    0.21
X-squared                                            80.8313                     0.20
Cressie-Read                                        76.6761                     0.30
BIC (based on L²)                          -346.5966
AIC3 (based on L²)                        -132.7967
CAIC (based on L²)                       -417.5966

Model for Clusters
 Intercept                Cluster1      Cluster2     Cluster3     Cluster4     Wald     p-value
————–           0.1544           0.1434        0.0115        -0.3093     1.1981     0.75
Cluster Size           0.2870          0.2838       0.2487          0.1805

< 1.5                       0.0843           0.2457       0.0006          0.0084
1.6-2.5                   0.6179            0.6458       0.0709          0.2809
2.5-3.5                  0.2848           0.1067         0.5319          0.5883
> 3.5                      0.0130           0.0018         0.3966         0.1224
> 90                     0.1341             0.7919         0.0063         0.6106
61-90                  0.6019            0.2040          0.1633         0.3713
41-60                  0.2099            0.0041          0.3317         0.0175
< 41                     0.0542            0.0001         0.4987        0.0006
under 51           0.0668           0.5646          0.0568        0.0954
51-70                 0.3462            0.3602          0.3271         0.3880
over 70             0.5870            0.0752          0.6161         0.5166
No anemia      0.7518             0.6556          0.2041         0.0998
Anemia            0.2482             0.3444          0.7959         0.9002

———          Cluster1          Cluster2      Cluster3      Cluster4
Overall           0.2870            0.2838         0.2487        0.1805

< 1.5                0.2492              0.7379           0.0013         0.0116
1.6-2.5            0.4163               0.4243           0.0427        0.1167
2.6-3.5           0.2296               0.0887          0.3723        0.3095
> 3.5              0.0328                0.0023          0.7982        0.1666
> 90              0.1001                0.5998           0.0043        0.2958
61-90           0.5198                 0.1716           0.1136         0.1950
41-60           0.3860                 0.0055          0.5847         0.0238
< 41             0.1205                  0.0002          0.8785         0.0008
< 51            0.0720                 0.7458           0.0910          0.0912
51-70         0.3036                 0.3084           0.2013          0.1867
over 70     0.3773                  0.0409          0.3633           0.2186
No anemia 0.4589              0.3957           0.1076           0.0378
Anemia     0.1342                 0.1844            0.3742           0.3073

Hemoglobin on NT proBNP 3


The effect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study

Donald S Silverberg, MDa; Dov Wexler, MDa; David Sheps, MDa; Miriam Blum, MDa; Gad Keren, MDa; Ron Baruch, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Shoshana Steinbruch, RNa; Itzhak Shapira, MDa; Shlomo Laniado, MDa; Adrian Iaina, MDa

J Am Coll Cardiol. 2001;37(7):1775-1780. doi:10.1016/S0735-1097(01)01248-7


This is a randomized controlled study of anemic patients with severe congestive heart failure (CHF) to assess the effect of correction of the anemia on cardiac and renal function and hospitalization.


Although mild anemia occurs frequently in patients with CHF, there is very little information about the effect of correcting it with erythropoietin (EPO) and intravenous iron.


Thirty-two patients with moderate to severe CHF (New York Heart Association [NYHA] class III to IV)
who had a left ventricular ejection fraction (LVEF) of 40% despite maximally tolerated doses of CHF medications and
  • whose hemoglobin (Hb) levels were persistently between 10.0 and 11.5 g% were randomized into two groups.
Group A (16 patients) received subcutaneous EPO and IV iron to increase the level of Hb to at least 12.5 g%. In Group B (16 patients) the anemia was not treated. The doses of all the CHF medications were maintained at the maximally tolerated levels except for oral and intravenous (IV) furosemide, whose doses were increased or decreased according to the clinical need.


Over a mean of 8.2 +/- 2.6 months,
  • four patients in Group B and none in Group A died of CHF-related illnesses.
  • The mean NYHA class improved by 42.1% in A and worsened by 11.4% in B.
  • The LVEF increased by 5.5% in A and decreased by 5.4% in B.
  • The serum creatinine did not change in A and increased by 28.6% in B.
  • The need for oral and IV furosemide decreased by 51.3% and 91.3% respectively in A and increased by 28.5% and 28.0% respectively in B.
  • The number of days spent in hospital compared with the same period of time before entering the study decreased by 79.0% in A and increased by 57.6% in B.


When anemia in CHF is treated with EPO and IV iron, a marked improvement in cardiac and patient function is seen,
  • associated with less hospitalization and renal impairment and less need for diuretics. (J Am Coll Cardiol 2001;37:1775– 80)

Anemia of any cause is known to be capable of causing congestive heart failure (CHF) (1). In patients hospitalized with CHF the 

  • mean hemoglobin (Hb) is about 12 g% (2,3),

which is considered the lower limit of normal in adults (4). Thus, anemia appears to be

common in CHF. Recently, in 142 patients in our special CHF outpatient clinic, we found that

  • as the CHF worsened, the mean Hb concentration decreased, from 13.7 g% in mild CHF (New York Heart Association [NYHA] class I) to 10.9 g% in severe CHF (NYHA 4), and
  • the prevalence of a Hb 12 g% increased from 9.1% in patients with NYHA 1 to 79.1% in those with NYHA 4 (5).
The Framingham Study has shown that anemia is an
  • independent risk factor for the production of CHF (6).
Despite this association of CHF with anemia,
  • its role is not mentioned in the 1999 U.S. guidelines for the diagnosis and treatment of CHF (7), and
  • many studies consider anemia to be only a rare contributing cause of hospitalization for CHF (8,9).
Recently, we performed a study in which the anemia of severe CHF that was resistant to maximally tolerated doses of standard medications
  • was corrected with a combination of subcutaneous (sc) erythropoietin (EPO) and intravenous iron (IV Fe) (5).
We have found this combination to be safe, effective and additive
  • in the correction of the anemia of chronic renal failure (CRF) in both
  • the predialysis period (10) and the dialysis period (11).
The IV Fe appears to be more effective than oral iron (12,13). In our previous study of CHF patients (5), the treatment resulted in
  • improved cardiac function,
  • improved NYHA functional class,
  • increased glomerular filtration rate,
  • a marked reduction in the need for diuretics and
  • a 92% reduction in the hospitalization rate
compared with a similar time period before the intervention. In the light of these positive results, a prospective randomized study was undertaken
  • to determine the effects of the correction of anemia in severe symptomatic CHF resistant to maximally tolerated CHF medication.

Abbreviations and Acronyms

CABG coronary artery bypass graft
CHF congestive heart failure
CRF chronic renal failure
EPO erythropoietin
%Fe Sat percent iron saturation
GFR glomerular filtration rate
Hb hemoglobin
Hct hematocrit
IU international units
IV intravenous
LVEF left ventricular ejection fraction
NYHA New York Heart Association
PA pulmonary artery
sc subcutaneous
SOLVD Studies Of Left Ventricular Dysfunction


Patients.Thirty-two patients with CHF were studied. Before the study, the patients were treated for least six months in the CHF clinic with

  • maximally tolerated doses of angiotensin-converting enzyme inhibitors, the beta-blockers bisoprolol or carvedilol, aldospirone, long-acting nitrates, digoxin and oral and intravenous (IV) furosemide.

In some patients these agents could not be given because of contraindications and in others they had to be stopped because of side effects. Despite this maximal treatment

  • the patients still had severe CHF  (NYHA classIII), with  fatigue and/or shortness of breath  on even mild exertion or at rest.  All had levels of
  • Hb in the range of 10 to 11.5 g%  on at least three consecutive visits over a three-week period.
  • All had a LVEF of 40%.

Secondary causes of anemia including hypothyroidism, and folic acid and vitamin B12 deficiency were ruled out and

  • there was no clinical evidence of gastrointestinal bleeding.

The patients were randomized consecutively into two groups:

  • Group A, 16 patients, was treated with sc EPO and IV Fe to achieve a target Hb of at least 12.5 g%.
  • Group B, 16 patients, did not receive the EPO and IV Fe.

Treatment protocol for correction of anemia.

All patients in Group A received the combination of sc EPO and IV Fe. The EPO was given once a week at a starting dose of 4,000 international units (IU) per week  and
the dose was increased  to two  or  three  times a week or decreased to once every few weeks as  necessary

  • to achieve and maintain a target Hb of 12.5 g%.

The IV Fe (Venofer-Vifor International, Switzerland), a ferric sucrose product, was given in a dose of 200 mg IV in 150 ml saline over 60 min every two weeks

  • until the serum ferritin reached 400 g/l or
  • the %Fe saturation (%Fe Sat is serum iron/total iron binding capacity 100) reached 40% or
  • the Hb reached 12.5g%. 

The IV Fe was then given at longer intervals as needed to maintain these levels.


Visits to the clinic were at two- to three week intervals depending on the patient’s status. This was the same frequency of visits to the CHF clinic as before then,

  • potassium and ferritin and %Fe Sat were performed on every visit.
  • blood pressure was measured by an electronic device on every visit.
  • LVEF was measured initially and at four- to six-month intervals by MUGA radioisotope ventriculography.

This technique measures

  • the amount of blood in the ventricle at the end of systole and at the end of diastole, thus giving
  • a very accurate assessment of the ejection fraction.

It has been shown to be an accurate and reproducible method of measuring the ejection fraction (14).  Hospital records were reviewed at the end of the intervention period to compare

  • the number of days hospitalized during the study with 
  • the number of days hospitalized during a similar period 
    • when the patients were treated in the CHF clinic before the initial randomization and entry into the study.

Clinic records were reviewed to evaluate the types and doses of CHF medications used before and during the study. The mean follow-up for patients was 8.2 +/-  2.7 months (range 5 to 12 months).  The study was done with the approval of the local ethics committee.Statistical analysis.

An analysis of variance with repeated measures (over time) was performed to compare the two study groups (control vs. treatment) and

  • to assess time trend and the interactions between the two factors.
  • A separate analysis was carried out for each of the outcome parameters.
  • The Mann-Whitney test was used to compare the change in NYHA class between two groups.

All the statistical analysis was performed by SPSS (version 10).


The mean age in Group A (EPO and Fe) was 75.3 +/-  14.6 years and in group B was 72.2 +/-  9.9 years. There were 11 and 12 men in Groups A and B, respectively.
Before the study the two groups were similar in
  1. cardiac function,
  2. comorbidities,
  3. laboratory investigations and
  4. medications
  • (Tables 1, 2 and 3), except for IV furosemide (Table 3),
which was higher in the treatment group. The mean NYHA class of Group A before the study was 3.8  0.4 and was 3.5  0.5 in Group B. The contributing factors to CHF in Groups A and B, respectively, are seen in Table 1 and were similar.
Table 1. Medical Conditions and Contributing Factors to Congestive Heart Failure in the 16 Patients Treated for the Anemia and in the 16 Controls

Table 1 medical conditions heart failure anemia

Table 2. The Effect of Correction of Anemia by Intravenous Iron and Erythropoietin Therapy on Various Parameters in 16 Patients in the Treatment (A) and 16 in the Control (B) Group

Table 2 medications to treat heart failure anemia

p values are given for analysis of variance with repeated measures and for independent t tests for comparison of baseline levels between the two groups.
BP  blood pressure; Fe Sat  iron saturation; Hb  hemoglobin; IV  intravenous; NS  not stated; Std Dev.  standard deviation.

The main contributing factors to CHF were considered to be

  • ischemic heart disease (IHD) in 11 and 10 patients respectively,
  • hypertension in two and two patients,
  • valvular heart disease in twoand two patients, and
  • idiopathic cardiomyopathy in one and two patients, respectively.

A significant change after treatment was observed in the two groups in the following parameters:

  • IV furosemide,
  • days in hospital,
  • Hb,
  • ejection fraction,
  • serum creatinine and
  • serum ferritin.
In addition, the interaction between the study group and time trend was significant in all measurements except for blood pressure and %Fe Sat. This interaction indicates that
  • the change over time was significantly different in the two groups.
Table 3. The Effect of Correction of Anemia by Intravenous Iron and Erythropoietin Therapy on Various Parameters in 16 Patients in the Treatment (A) and 16 in the Control (B) Group

Table 3  CHF aneia EPO

p values are given for analysis of variance with repeated measures and for independent t tests for comparison of baseline levels between the two groups.
BP  blood pressure; Fe Sat  iron saturation; Hb  hemoglobin; IV  intravenous; NS  not stated; Std Dev.  standard deviation.

We find in the comparisons of Tables 2 and 3:

  1. before treatment the level of oral furosemide was higher in the control group (136.2 mg/day) compared with the treatment group (132.2 mg/day).
  2. after treatment, while the dose of oral furosemide of the treated patients was reduced  to 64.4 mg/day
  • the dose of the nontreated patients was increased to 175 mg/day.

The same results of improvement in the treated group and deterioration in the control group are expressed in the following parameters:

  1. IV furosemide, days in hospital,
  2. Hb,
  3. ejection fraction and
  4. serum creatinine.

The NYHA class was

  • 3.8 +/- 0.4 before treatment and 2.2 +/- 0.7 after treatment in Group A  (delta mean = – 1.6) and
  • 3.5 +/-  0.7 before treatment and 3.9 +/- 0.3 after treatment in Group B. (delta mean = 0.4)

The improvement in NYHA class was significantly higher (p < 0.0001) in the treatment group compared with the control group (Table 4).

Table 4. Changes from Baseline to Final New York Heart Association (NYHA) Class
Initial minus final

Table 4  changes from NYHA baseline  CHF anemia

The improvement in NYHA class was statistically higher (p <  0.0001) in the treatment group compared with control.

There were no deaths in Group A and four deaths in Group B.

Case 1: A 71-year-old woman with severe mitral insufficiency and severe pulmonary hypertension  (a pulmonary artery [PA] pressure of 75 mm Hg) had persistent NYHA 4 CHF  and died during mitral valve surgery  seven months after onset of the study. She was hospitalized for 21 days  in the seven months before randomization and for 28 days  during the seven months after randomization.

Case 2:

A 62-year-old man with a longstanding history of hypertension complicated by IHD, coronary artery bypass graft (CABG) and atrial fibrillation had persistent NYHA 4 CHF  and a PA pressure of 35 mm Hg,  and died from pneumonia and septic shock eight months after onset of the study. He was hospitalized for seven days in the eight months before randomization and for 21 days during the eight months  after

Case 3:
A 74-year old man with IHD, CABG, chronic obstructive pulmonary disease, a history of heavy smoking and diabetes had persistent NYHA 4 CHF and a PA pressure of  28 mm Hg, and died of pulmonary  edema and cardiogenic shock nine months after onset of the study. He was hospitalized for 14 days in the nine months before  randomization and for 41 days during the nine months after randomization.

Case 4:
A 74-year-old man with a history of IHD, CABG, diabetes, dyslipidemia, hypertension and atrial fibrillation, had persistent NYHA 4 CHF and a PA pressure of 30 mm Hg,  and died of pneumonia and septic shock   six months after onset of the study. He was hospitalized for five days in the six months before randomization and for 16 days during the nine months after randomization.


 Main findings.

The main finding of the present study is that the correction of

  • even mild anemia in patients with symptoms of very severe CHF despite being on maximally tolerated drug therapy
  • resulted in a significant improvement in their cardiac function and NYHA functional class.

There  was also a large

  • reduction in the number of days of  hospitalization compared with a similar period before the  intervention.
  • all this was achieved despite a marked reduction in the dose of oral and IV furosemide.

In the group in whom the anemia was not treated, four  patients died during the study. In all four cases

  • the CHF was unremitting and contributed to the deaths. 

In addition,  for the group as a whole, 

  • the LVEF, the NYHA class and  the renal function worsened.

There was also need for

  • increased oral and IV furosemide as well as increased  hospitalization.

Study limitations.

The major limitations of this study are

  1. the smallness of the sample size and
  2. the fact that randomization and treatment were not done in a blinded fashion.

Nevertheless, the two groups were almost identical in

  1. cardiac, renal and anemia status;
  2. in the types and doses of medication they were taking before and during the intervention and
  3. in the number of hospitalization days before the intervention.

Although the results of this study, like those of  our previous uncontrolled study (5), suggest that

  • anemia may play an important role in the mortality and morbidity of  CHF,
  • a far larger double-blinded controlled study should be carried out to verify our findings.

Anemia as a risk factor for hospitalization and death in CHF.

Our results are consistent with a recent analysis of 91,316 patients hospitalized with CHF (15). Anemia was found to be a stronger predictor of

  • the need for early rehospitalization than  was hypertension,  IHD or the presence of a previous CABG.  

A recent analysis of the Studies Of Left Ventricular Dysfunction (SOLVD) (16) showed that

  • the level of hematocrit (Hct) was an independent risk factor for mortality.

During a mean follow-up of 33 months the mortality was

  • 22%, 27% and 34% for those with a Hct of 40, 35 to 40 and 35 respectively.

The striking response of our patients to

  • correction of mild anemia suggests that the failing heart may be very susceptible to anemia.

It has, in fact, been found in both animal (17) and human studies (17–19) that

  • the damaged heart is more vulnerable to anemia and/or ischemia than is the normal heart.

These stimuli may result in a more marked reduction in cardiac function than occurs in the normal heart and may explain why,  in our study,

  • the patients were so resistant to high doses of CHF medications and
  • responded so well when the anemia was treated.

Our findings about the importance of anemia in CHF are not surprising when one considers that, in dialysis patients,

  • anemia has been shown to be associated with an increased prevalence and incidence of CHF (20) and that
  • correction of anemia in these patients is associated with improved
    • cardiac function (21,22),
    • less mortality (23,24) and
    • fewer hospitalizations (23,25).

Effect of improvement of CHF on CRF.

Congestive heart failure can cause progressive renal failure (26,27). Renal ischemia is found very early on

  • in patients with cardiac dysfunction (28,29), and
  • chronic ischemia may cause progression of renal failure (30). Indeed, the development of
  • CHF in patients with essential hypertension has been found to be one of the most powerful predictors of
  • the eventual development of end-stage renal disease (31).

Patients who develop CHF after a myocardial infarction experience a

  • fall in the glomerular filtration rate (GFR) of about 1 ml/min/month if the CHF is not treated (32).

In another recent analysis of the SOLVD study, treating the CHF with

  • both angiotensin-converting enzyme inhibitors and beta-blockers resulted in better preservation of the renal function than did
  • angiotensin-converting enzyme inhibitors alone (26),
suggesting that the more aggressive the treatment of the CHF, the better the renal function is preserved. In the present study, as in our previous one (5), we found that the deterioration of GFR was prevented with
  • successful treatment of the CHF, including correction of the anemia, whereas
  • the renal function worsened when the CHF remained severe

All these findings suggest that early detection and treatment of CHF and systolic and/or diastolic dysfunction from whatever cause could prevent

  • the deterioration not only of the cardiac function
  • but of the renal function as well.

This finding has very broad implications in the prevention of CRFbecause most patients with advanced CRF have

  • either clinical evidence of CHF or at least some degree of systolic dysfunction (33).

Systolic and/or diastolic dysfunction can occur early on in many  conditions, such as

  • essential hypertension (34),
  • renal disease of any cause (35,36) or
  • IHD, especially after a myocardial infarction (37).

The early detection and adequate treatment of this cardiac dysfunction, including correction of the anemia, could prevent this cardiorenal insufficiency. To detect cardiac dysfunction early on, one would need  at least an echocardiogram and MUGA radio-nucleotide ventriculography. These tests should probably be done not only in patients with signs and symptoms of CHF,   but in all patients where CHF or systolic  and/or diastolic dysfunction are suspected, such as those with a history of heart disease or suggestive changes of ischemia or hypertrophy on the electrocardiogram, or in patients with hypertension or renal disease.

Other positive cardiovascular effects of EPO treatment.

Another possible explanation for the improved cardiac function in this study may be the direct effect that EPO itself has on improving cardiac muscle function (38,39) and myocardial cell growth (39,40) unrelated to its  effect of the anemia. In fact EPO may be  crucial in the formation of the heart muscle in utero (40). It may also improve  endothelial function (41).  Erythropoietin may be superior to blood transfusions  not only  because adverse reactions to EPO are infrequent, but also because

  • EPO causes the production and release of young cells from the bone marrow into the blood.

These cells have an oxygen dissociation curve that is shifted to the right of the normal curve, causing the release of

  • much greater amounts of oxygen into the tissues than occurs normally (42).

On the other hand, transfused blood consists of older red cells with an oxygen dissociation curve that is

  • shifted to the left, causing the release of much less oxygen into the tissues than occurs normally (42).

The combination of IV Fe and EPO. The use of IV Fe along with EPO has been found to have an additive effect, 

  • increasing the Hb even more than would occur with EPO alone while at the same time
  • allowing the dose of EPO to be reduced (10 –13).
  • The lower dose of EPO will be cost-saving and also reduce the chances of hypertension developing (43).
 We used iron sucrose (Venofer) as our IV Fe medication because, in our experience, it is extremely well tolerated (10,11) and  
  • has not been  associated  with any serious side effects in more than 1,200 patients over six years.

Implications of treatment of anemia in CHF. The correction of anemia is not a substitute for the well-documented effective therapy of CHF but seems to be  an important, if not vital,  addition to the therapy. It is surprising, therefore,  that judging from  the  literature  on CHF,

  • such an obvious treatment for improving CHF is so rarely considered.

We believe that correction of the anemia will have an important role to play in

  • the amelioration of cardiorenal insufficiency, and that this improvement will have
  • significant economic  implications as well.


The authors thank Rina Issaky, Miriam Epstein, Hava Ehrenfeld and Hava Rapaport for their secretarial assistance.
Reprint requests and correspondence: Dr. D. S. Silverberg, Department of Nephrology, Tel Aviv Medical Center, Weizman 6, Tel Aviv, 64239, Israel.


The use of subcutaneous erythropoietin and intravenous iron for the treatment of the anemia of severe, resistant congestive heart failure improves cardiac and renal function and functional cardiac class, and markedly reduces hospitalizations

Donald S Silverberg, MDa; Dov Wexler, MDa; Miriam Blum, MDa; Gad Keren, MDa; David Sheps, MDa; Eyal Leibovitch, MDa; David Brosh, MDa; Shlomo Laniado, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Itzhak Shapira, MDa; Dov Gavish, MDa; Ron Baruch, MDa; Bella Koifman, MDa; Carl Kaplan, MDa; Shoshana Steinbruch, RNa; Adrian Iaina, MDa

J Am Coll Cardiol. 2000;35(7):1737-1744. doi:10.1016/S0735-1097(00)00613-6


This study evaluated the prevalence and severity of anemia in patients with congestive heart failure (CHF) and

  • the effect of its correction on cardiac and renal function and hospitalization.


The prevalence and significance of mild anemia in patients with CHF is uncertain, and the role of erythropoietin with intravenous iron supplementation in treating this anemia is unknown.


In a retrospective study, the records of the 142 patients in our CHF clinic were reviewed to find
  • the prevalence and severity of anemia (hemoglobin [Hb]12 g).
In an intervention study, 26 of these patients, despite maximally tolerated therapy of CHF for at least six months, still had had severe CHF and were also anemic. They were treated with
  • subcutaneous erythropoietin and intravenous iron sufficient to increase the Hb to 12 g%.
The doses of the CHF medications, except for diuretics, were not changed during the intervention period.


The prevalence of anemia in the 142 patients increased with the severity of CHF,
  • reaching 79.1% in those with New York Heart Association class IV.
In the intervention study, the anemia of the 26 patients was treated for a mean of 7.2 5.5 months.
  • The mean Hb level and mean left ventricular ejection fraction increased significantly.
  • The mean number of hospitalizations fell by 91.9% compared with a similar period before the study.
  • The New York Heart Association class fell significantly,
  • as did the doses of oral and intravenous furosemide.
  • The rate of fall of the glomerular filtration rate slowed with the treatment.


Anemia is very common in CHF and its successful treatment is associated with a significant improvement in
  • cardiac function,
  • functional class,
  • renal function and
  • in a marked fall in the need for diuretics and hospitalization.
Abbreviations and Acronyms
ACE Angiotensin-converting enzyme
CHF congestive heart failure
COPD chronic obstructive pulmonary disease
CRF chronic renal failure
CVA cerebrovascular accident
EPO erythropoietin
Fe iron
g% grams Hb /100 ml blood
GFR glomerular filtration rate
Hb hemoglobin
Hct hematocrit
IV intravenous
LVEF left ventricular ejection fraction
LVH left ventriculr hypertrophy
NYHA New York Heart Association
%Fe Sat percent iron saturation
sc subcutaneous
TNF tumor becrosis factor
ACE Angiotensin-converting enzyme
CHF congestive heart failure
COPD chronic obstructive pulmonary disease
CRF chronic renal failure
CVA cerebrovascular accident
EPO erythropoietin
Fe iron
g% grams Hb /100 ml blood
GFR glomerular filtration rate
Hb hemoglobin
Hct hematocrit
IV intravenous
LVEF left ventricular ejection fraction
LVH left ventriculr hypertrophy
NYHA New York Heart Association
%Fe Sat percent iron saturation
sc subcutaneous
TNF tumor becrosis factor

The mean hemoglobin (Hb) in patients with congestive heart failure (CHF) is about 12 g Hb per 100 ml blood (g%) (1–3), which is considered to be the lower limit of normal in adult men and postmenopausal women (4). Thus, many patients with CHF are anemic, and

  • this anemia has been shown to worsen as the severity of the CHF progresses (5,6).
Severe anemia of any cause can produce CHF, and treatment of the anemia can improve it (7). In patients with chronic renal failure (CRF) who are anemic,
  • treatment of the anemia with erythropoietin (EPO) has improved many of the abnormalities seen in CHF,
  • reducing left ventricular hypertrophy (LVH) (8 –10),
  • preventing left ventricular dilation (11) and,
    • in those with reduced cardiac function, increasing the left ventricular ejection fraction (LVEF)(8 –10),
    • the stroke volume (12) and
    • the cardiac output (12).
In view of the high prevalence of anemia in CHF, it is surprising that we could find no studies in which EPO was used in the treatment of the anemia of CHF, and the use of EPO is not included in U.S. Public Health Service guide-lines of treatment of the anemia of CHF (13). In fact, anemia has been considered
  • only a rare contributing factor to the worsening of CHF, estimated as contributing to
  • no more than 0% to 1.5% of all cases (14 –16).
Perhaps for this reason, recent guidelines for the prevention and treatment of CHF do not mention treatment of anemia at all (17). If successful treatment of anemia could improve cardiac function and patient function in CHF,
  • this would have profound implications, because,
  • despite all the advances made in the treatment of CHF, it is still a major and steadily increasing cause of hospitalizations, morbidity and mortality (18 –20).
The purpose of this study is to examine
  • the prevalence of anemia (Hb 12 g%) in patients with different levels of severity of CHF and
  • to assess the effect of correction of this anemia in severe CHF patients
  • resistant to maximally tolerated doses of CHF medication.
A combination of subcutaneous (SC) EPO and intravenous (IV) iron (Fe) was used. We have found this combination to be additive in improving the anemia of CRF (21,22).



The medical records of the 142 CHF patients being treated in our special outpatient clinic devoted to CHF were reviewed to determine the prevalence and severity of anemia and CRF in these patients. These patients were referred to the clinic either from general practice or from the various wards in the hospital.

Intervention study.

Despite at least six months of treatment in the CHF clinic,
  • 26 of the above patients had persistent, severe CHF (New York Heart Association [NYHA] class III),
  • had a Hb level of 12 g% and were on
    • angiotensin-converting enzyme [ACE] inhibitors, the 
    • alpha-beta-blocker carvedilol,
    • long-acting nitrates,
    • digoxin, 
    • aldactone and
    • oral and IV furosemide.

These 26 patients participated in an intervention study. The mean age was 71.76  8.12 years. There were 21 men and 5 women. They  all had a

  • LVEF below 35%,
  • persistent fatigue and
  • shortness

    of breath on mild to moderate exertion and often at rest, and had

  • required hospitalizations at least once during their follow-up in the CHF clinic for pulmonary edema.
In 18 of the 26 patients, the CHF was associated with ischemic heart disease either
  • alone in four patients, or
  • with hypertension in six,
  • diabetes in four,
  • the two together in three, or with
  • valvular heart disease in one.
Of the remaining eight patients,
  • four had valvular heart disease alone and
  • four had essential hypertension alone.
Secondary causes of anemia including
  • gastrointestinal blood loss (as assessed by history and by three negative stool occult blood examinations),
  • folic acid and vitamin B12 deficiency and
  • hypothyroidism were ruled out.
Routine gastrointestinal endoscopy was not carried out. The study passed an ethics committee.
Table 1. Initial Characteristics of the 142 Patients With CHF Seen in the CHF Clinic
Age, yearsMale/female,  %Associated conditionsDiabetesHypertensionDyslipidemiaSmoking

Main cardiac diagnosis
Ischemic heart disease

Dilated CMP

Valvular heart disease


LVEF,  %

Left atrial area (n 15 cm2)

Pulmonary artery pressure  (15 mm Hg)

Previous hospitalizations/year

Serum Na, mEq/liter

Serum creatinine, mg%

Hemoglobin, g%

70.1 +/- 11.1










32.5 +/- 12.2

31.3  +/- 10.3

43.1  +/-14.9

3.2  +/- 1.5

139.8  +/- 4.0

1.6   +/-  1.1

11.9   +/- 1.5

CMP  cardiomyopathy; LVEF  left ventricular ejection fraction; NYHA  New York Heart Association class.

Correction of the anemia.

All patients received the combination of SC EPO and IV Fe. The EPO was given once a week at a starting dose of 2,000 IU per week subcutaneously, and the dose was increased or decreased as necessary to achieve and maintain a target Hb of 12 g%. The IV Fe (Venofer-Vifor International, St. Gallen, Switzerland), a ferric sucrose product, was given in a dose of 200 mg IV in 150 ml saline over 60 min every week until the serum ferritin reached 400  g/liter or the percent Fe saturation (%Fe Sat: serum iron/total iron binding capacity   100) reached 40% or until the Hb reached 12 g%. The IV Fe was then given at longer intervals as needed to maintain these levels.

Medication dose.

Except for oral and IV furosemide therapy, the doses of all the other CHF medications, which were used in the maximum tolerated doses before the intervention, were kept unchanged during the intervention period.

Duration of the study.

The study lasted for a mean of 7.2  5.5 months (range four to 15 months).


Visits were at weekly intervals initially and then at two- to three-week intervals depending on the patient’s status. This was the same frequency of visits to the CHF clinic as before the intervention study.
  • A complete blood count, serum creatinine, serum ferritin and % Fe Sat were performed on every visit.
  •  An electronic device measured the blood pressure on every visit.
  • The LVEF was measured by a multiple gated ventricular angiography heart scan initially and at four- to six-month intervals.
Hospital records were reviewed to compare the number of hospitalizations during the time the patients were treated for the anemia with the number of hospitalizations
  • during a similar period of time that they were treated in the CHF clinic 
    before the anemia was treated.
Clinic records were reviewed to evaluate the types and doses of CHF medications used 
before and during the study.

Period of time that they were treated in the CHF clinic before the anemia was treated.

Clinic records were reviewed to evaluate the types and doses of CHF medications used before and during the study.  The glomerular filtration rate (GFR) was calculated from the serum creatinine by the formula: 1/serum creatinine in mg% x 100 GFR in ml/min. The rate of change of the GFR before and during the intervention period was calculated by comparing the change in GFR per month in the year before the intervention with that during the intervention.

Statistical analysis.

Mean standard deviation was calculated. One-way analysis of variance (ANOVA) was performed to compare parameter levels between the four NYHA groups. Hochberg’s method of multiple comparisons (23) was used for pair-wise comparison between two groups. A p value of less than 0.05 was considered statistically significant. In the intervention study, the significance of the difference between the initial values and those at the end of the study for the individual parameters in the 26 treated patients was assessed by paired student’s t test; p < 0.05 was considered statistically significant. All the statistical analysis was performed by the SPSS program (Version 9, Chicago, Illinois).


CHF: the whole study group.

The clinical, biochemical and hematological characteristics of the 142 patients seen in the clinic are shown in Tables 1 and 2.

  • Sixty-seven patients (47%) had severe CHF as judged by a NYHA class of IV (Table 2).
  • Seventy- nine of the 142 patients (55.6%) were anemic (Hb  12 g%).

The mean Hb level fell progressively from 13.73 +/- 0.83 g% in class I NYHA to 10.90 +/- 1.70 g% in class IV NYHA (p  0.01). The percentage of patients with Hb  12 g% increased from 9.1% in class I to 79.1% in class IV.
Fifty eight patients (40.8%) had CRF as defined as a serum creatinine  1.5 mg%. The mean serum creatinine increased from 1.18 +/_  0.38 mg% in class I NYHA, to 2.0 +/-    1.89 mg% in class IV NYHA, p  0.001. The percentage of patients with an elevated serum creatinine ( 1.5 mg%)      increased from 18.2% in class I to 58.2% in class IV.

The mean ejection fraction fell from 37.67 +/-  15.74% in class I to 27.72 +/-  9.68% (p  0.005) in class IV.

Table 2. LVEF and Biochemical and Hematological Parameters by NYHA Class in 142 Patients With CHF 
NYHA Class I II III IV  Significantly Different Pairs*

 *p  0.05 at least between the two groups by pair-wise comparison between groups.

†p  0.05 at least between the groups by ANOVA.

No. of patients





(total 142) (%)

    (7.7)    (18.3)    (26.8)    (47.2)

Hb, g%†

13.73 (0.83)

13.38 (1.26)

11.95 (1.48)

10.90 (1.70) 

1–3, 1–4, 2–3, 2–4

Serum creatinine,





1–2, 1–3, 1–4


    (0.38)     (0.29)      (0.38)     (1.89)

LVEF, %†

37.67 (15.74)

32.88 (12.41)

32.02 (10.99)

27.72 (9.68)

1–4, 2–4

Hb 12 g%,  (%)


5 (19.2) 

20 (52.6) 

53 (79.1)

Serum creatinine

    2      5     12     39

1.5 mg%,  (%) 





The intervention study: medications.

The percentage of patients receiving each CHF medication before and after the intervention period and the reasons for not receiving  them are seen in Table 3.

Table 3. Number (%) of the 26 Patients Taking Each Type of Medication Before and During the Intervention Period and the Reason Why the Medication Was Not Used

Medication    No. of Patients  (%)         Reason for Not Receiving the Medications (No. of Patients)
BP  blood pressure; CRF  chronic renal failure; IV  intravenous.

The main reason for not receiving:

  • 1) ACE inhibitors was the presence of reduced renal function;
  • 2) carvedilol was the presence of chronic obstructive pulmonary disease (COPD);
  • 3) nitrates was low blood pressure and aortic stenosis and
  • 4) aldactone was hyperkalemia.
Table 4. Mean Dose of Each Medication Initially and at the End of the Intervention Period in the 26 Patients

                                       No. of Patients                                 Initial Dose ^                 Final Dose^
Carvedilol (mg/day)                      20                                                        26.9 15.5                                   28.8 14.5
Captopril (mg/day)                          7                                                        69.6 40.0                                 70.7 40.4
Enalapril (mg/day)                        13                                                        25.7 12.5                                   26.9 12.6
Digoxin (mg/day)                          25                                                       0.10 0.07                                    0.10 0.07
Aldactone (mg/day)                       19                                                        61.2 49.2                                   59.9 47.1
Long-acting nitrates                      23                                                        53.2 13.2                                   54.1 14.4
Oral furosemide (mg/day)           26                                                      200.9 120.4                                78.3 41.3*
IV furosemide (mg/month)         26                                                      164.7 178.9                                  19.8 47.0*
*p  0.05 at least vs. before by paired Student’s t test.
^  +/-

The mean doses of the medications are shown in Table 4. 

The mean dose of oral furosemide was 200.9 +/-  120.4 mg/day before and 78.3 +/-  41.3 mg/day after the intervention (p   0.05). The dose of IV furosemide was 164.7 +/-  19.8,  178.9 mg/month before and  7.0 mg/month after the intervention (p  0.05).  

The doses of the other CHF medications were almost identical in the two periods.

Clinical results.

There were three deaths during the intervention period. An 83-year-old man died after eight months of respiratory failure after many years of COPD, a 65-year-old man at eight months of a CVA with subsequent pneumonia and septic shock and a 70-year-old man at four months of septicemia related to an empyema that developed after aortic valve replacement.
Three patients, a 76-year-old man, an 85-year-old woman and a 72-year-old man, required chronic hemodialysis after six, 16 and 18 months, respectively. The serum creatinines of these three patients at onset of the anemia treatment were 4.2, 3.5 and 3.6 mg%, respectively. All three had improvement in their NYHA status but
  • their uremia worsened as the renal function deteriorated, demanding the initiation of dialysis.

In no cases, however, was pulmonary congestion an indication for starting dialysis.

Functional results (Table 5).

During the treatment period, the NYHA class fell from a mean of 3.66 +/- 0.47 to 2.66 +/- 0.70 (p 0.05), and
  • 24 had some improvement in their functional class.
The mean LVEF increased from 27.7 +/- 4.8 to 35.4  +/- 7.6% (p 0.001), an increase of 27.8%.
Compared with a similar period of time before the onset of the anemia treatment, the mean number of hospitalizations fell from 2.72 +/-  1.21 to 0.22 +/-  0.65 per patient (p   0.05)a decrease of 91.9%.
No significant changes were found in the mean systolic/diastolic blood pressure.

Hematological results (Table 5).

  • The mean hematocrit (Hct) increased from 30.14 +/- 3.12%) to 35.9  +/- 4.22% (p < 0.001).
  • The mean Hb increased from 10.16 +/- 0.95 g%) to 12.10 +/-  1.21 g% (p <  0.001).
  • The mean serum ferritin increased from 177.07 +/-  113.80  g/liter to 346.73 +/- 207.40 g/liter (p  0.005).
  • The mean serum Fe increased from 60.4 +/- 19.0 g% to 74 +/- .80  20.7 g% (p  0.005). 
  • The mean %Fe Sat increased from 20.05   6.04% to 26.14 =/- 5.23% (p  0.005).
  • The mean dose of EPO used throughout the treatment period was 5,227  +/- 455 IU per week, and
  • the mean dose of IV Fe used was 185.1 +/- 57.1 mg per month.
In four of the patients, the target Hb of 12 g% was maintained despite stopping the EPO for at least four months.

Renal results (Table 5).

The changes in serum creatinine were not significant. The estimated creatinine clearance fell at a rate of 0.95 + 1.31 ml/min/month before the onset of treatment of the anemia and increased at a rate of 0.85 + 2.77 ml/min/month during the treatment period.
Table 5. The Hematological and Clinical Data of the 26 CHF Patients at Onset and at the End of the Intervention Period

————–                                         Initial ^                                    Final^
Hematocrit, vol%                              30.14 3.12                            35.90 4.22*
Hemoglobin, g%                                10.16 0.95                              2.10 1.21*
Serum ferritin, g/liter                    177.07 113.80                       346.73  207.40*
Serum iron, g%                                  60.4 19.0                               74.8  20.7*
% iron saturation                              20.5 6.04                               26.14 5.23*
Serum creatinine, mg%                   2.59 0.77                                 2.73 1.55
LVEF, %                                              27.7 4.8                                   35.4  7.6*
No. hospitalizations/patient          2.72 1.21                                 0.22   0.65*
Systolic BP, mm Hg                       127.1 19.4                                128.9  26.4
Diastolic BP, mm Hg                       73.9 9.9                                   74.0   12.7
NYHA (0–4)                                     3.66 0.47                                2.66 0.70*
*p  0.05 at least vs before by paired Student’s t test.     ^ +/-
BP  blood pressure; LVEF  left ventricular ejection fraction; NYHA  New York Heart Association.


The main findings in the present study are that anemia is common in CHF patients and becomes progressively more prevalent and severe as CHF progresses. In addition, for patients with resistant CHF, the treatment of the associated anemia causes a marked improvement in their

  1. functional status,
  2. ejection fraction and
  3. GFR.
        • All these changes were associated with a markedly
            • reduced need for hospitalization and
            • for oral and IV furosemide.

The effect of anemia on the ischemic myocardium.

We used the IV Fe together with EPO to avoid the Fe deficiency caused by the use of EPO alone (38,39).
The Fe deficiency will cause

  • a resistance to EPO therapy and
  • increase the need for higher and higher doses to maintain the Hb level (39,40).

These high doses will not only be expensive but may increase the blood pressure excessively (41). The IV Fe reduces the dose of EPO needed to correct the anemia, because

  • the combination of SC EPO and IV Fe has been shown to have an additive effect on correction of the anemia of CRF (21,22,39,42).

Oral Fe, however, has no such additive effect (39,42). The relatively low dose of EPO needed to control the anemia in our study may explain why

  • the blood pressure did not increase significantly in any patient.

We used Venofer, an Fe sucrose product, as our IV Fe supplement because, in our experience (21,22,43), it has very few side effects and, indeed, no side effects with its use were encountered in this study.

The Effect of Anemia Correction on Renal Function.

Congestive heart failure is often associated with some degree of CRF (1–3,27–29), and

  • this is most likely due to renal vasoconstriction and ischemia (28,29).

When the anemia is treated and the cardiac function improves,

  • an increase in renal blood flow and glomerular filtration is seen (7,28).

In the present study, renal function decreased as the CHF functional class worsened (Table 2). The rate of deterioration of renal function was slower during the intervention period. Treatment of anemia in CRF has been associated with

  • a rate of progression of the CRF that is either unchanged (30) or is slowed (31–33).

It is possible, therefore, that adequate treatment of the anemia in CHF may, in the long term, help slow down the progression of CRF.

Possible Adverse Effects of Correction of the Anemia.

There has been concern, in view of the recent Amgen study (34), that correction of the Hct to a mean 42% in hemodialysis patients might increase cardiovascular events in those receiving EPO compared with those maintained at a Hct of 30%. Although there is much uncertainty about how to interpret this study (35), there is a substantial body of evidence that shows

  • correction of the anemia up to a Hb of 12 g% (Hct 36%) in CRF on dialysis is safe and desirable (35–38), and
  • results in a reduction in mortality, morbidity and in the number and length of hospitalizations.

The same likely holds true for the anemia of CHF with or without associated CRF. Certainly, our patients’ symptoms were strikingly improved, as was their cardiac function (LVEF) and need for hospitalization and diuretics. It remains to be established

  • if correction of the anemia up to a normal Hb level of 14 g% might be necessary in order to further improve the patient’s clinical state.

The Role of Fe Deficiency and its Treatment in the Anemia of CHF.

We used the IV Fe together with EPO to avoid the Fe deficiency caused by the use of EPO alone (38,39). The Fe deficiency will cause

  • a resistance to EPO therapy and increase the need for higher and higher doses to maintain the Hb level (39,40).

These high doses will not only be expensive but may

  • increase the blood pressure excessively (41).

The IV Fe reduces the dose of EPO needed to correct the anemia, because the combination of SC EPO and IV Fe has been shown to have an additive effect on correction of the anemia of CRF (21,22,39,42). Oral Fe,  however, has no such additive effect (39,42). The relatively low dose of EPO needed to control the anemia in our study may explain

  • why the blood pressure did not increase significantly in any patient.

We used Venofer, an Fe sucrose product, as our IV Fe supplement because, in our experience (21,22,43), it has very few side effects and, indeed, no side effects with its use were encountered in this study.

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