INTRODUCTION

1.1 BACKGROUND OF STUDY

Obesity is a medical condition in which excess body fat has accumulated to the extent

that it may have a negative effect on health. People are generally considered obese when

their body mass index (BMI), a measurement obtained by dividing a person's weight by the

square of the person's height, is over 30 kg/m2, with the range 25–30 kg/m2 defined as

overweight. The view that obese people eat little yet gain weight due to a slow metabolism is

not medically supported. On average, obese people have greater energy expenditure than

their normal counterparts due to the energy required to maintain an increased body mass.

Obesity is mostly preventable through a combination of social changes and personal choices.

Changes to diet and exercising are the main treatments. Diet quality can be improved by

reducing the consumption of energy-dense foods, such as those high in fat or sugars, and by

increasing the intake of dietary fiber. Medications can be used, along with a suitable diet, to

reduce appetite or decrease fat absorption. If diet, exercise, and medication are not effective,

a gastric balloon or surgery may be performed to reduce stomach volume or length of the

intestines, leading to feeling full earlier or a reduced ability to absorb nutrients from food,

(Colquitt JL, et al., 2014). Obesity is a leading preventable cause of death worldwide, with

increasing rates in adults and children. In 2015, 600 million adults (12%) and 100 million

children were obese in 195 countries. Obesity is more common in women than men.

Authorities view it as one of the most serious public health problems of the 21st century.

Obesity is stigmatized in much of the modern world (particularly in the Western world),

1
though it was seen as a symbol of wealth and fertility at other times in history and still is in

some parts of the world. In 2013, the American Medical Association classified obesity as a

disease, (Weinstock and Matthew, 2013).

Hypothyroidism, also called underactive thyroid or low thyroid, is a disorder of the

endocrine system in which the thyroid gland does not produce enough thyroid hormone. It

can cause a number of symptoms, such as poor ability to tolerate cold, a feeling of tiredness,

constipation, depression, and weight gain. Occasionally there may be swelling of the front

part of the neck due to goiter, (Hypothyroidism, National Institute of Diabetes and Digestive

and Kidney Diseases, 2016).

Body composition and thyroid hormones appear to be closely related since the latter is

known to be involved in the regulation of basal metabolism and thermogenesis, playing an

important role in lipid and glucose metabolism, food intake and fat oxidation, (Silva JE,

2006). In agreement with this knowledge, it is well known that hypothyroidism causes a

weight increase together with a decrease in basal metabolic rate and thermogenesis, (Rotondi

M, et al., 2009). The causes underlying these alterations are not known although several

theories have been proposed. These include an increased deiodinase activity, decreased

expressions of both TSH and thyroid hormones in adipocytes of obese subjects and high

levels of leptin found in obese subjects, these facts has been interpreted as a defense

mechanism capable of counteracting the accumulation of fat by increasing the energy

expenditure , basal metabolic rate and the total energy expenditure.

2
 Lipid profile or lipid panel is a panel of blood tests that serves as an initial screening

tool for abnormalities in lipids, such as cholesterol and triglycerides. The results of this test

can identify certain genetic diseases and can determine approximate risks for cardiovascular

disease, certain forms of pancreatitis, and other diseases, (National Cholesterol Education

Program, 2002). The lipid profile typically includes: Low-density lipoprotein (LDL), High-

density lipoprotein (HDL), Triglycerides and Total cholesterol.

1.2 AIMS AND OBJECTIVES

The aim of this work is to determine the effects of juglans nigra and urtica dioica on lipid

profile in hypothyroid induced obesity.

The specific objectives include;

1. To induce hypothyroidism using the drug Thiamazole, this will in turn induce obesity.

2. To estimate the serum levels of total cholesterol, high density lipoprotein and

triglyceride.

3. To determine the body mass index of hypothyroid induced obesity.

1.3 JUSTIFICATION
3
 Obesity is increasing at alarming rates in industrialized countries. Too many changes

in the environment seem to have directly contributed to this increase. The first is a reduction

in the energy expenditure required for daily living, as a result of technological

improvements, and the second is an increase in energy intake resulting from the increasing

availability of palatable, low cost, high fat, energy dense food. Obesity increases the

likelihood of various diseases and conditions, particularly cardiovascular diseases, type 2

diabetes, obstructive sleep apnea, certain types of cancer, osteoarthritis and

depression. Obesity is most commonly caused by a combination of excessive food intake,

lack of physical activity, genetic susceptibility, genes, medications, mental disorder, or

endocrine disorders (hypothyroidism). Several Research have been done to evaluate the

levels of lipid profile in obesity and hypothyroidism, and also to determine the relationship

between obesity and hypothyroidism. Pearce, et al (2008), discovered that there are high

levels of lipid profile in hypothyroid patients; hence hypothyroidism can lead to an increase

in lipid profile. Wiseman (1993) also discovered improvement in lipid metabolism after

administration of L-thyroxine therapy to hypothyroid patients, hence increased levels of

thyroid hormones increases lipid metabolism. But no study has been done in this area to

determine the effects of juglans nigra (black walnut) and urtica dioica (nettle leaf) on lipid

profile and body mass index in obesity, therefore this work is done to advice the public on

the effect of black walnut and nettle leaf on obesity.

CHAPTER TWO

4
 LITERETURE REVIEW

2.1 OBESITY

Obesity is a medical condition in which excess body fat has accumulated to the extent

that it may have a negative effect on health. People are generally considered obese when

their body mass index (BMI), a measurement obtained by dividing a person's weight by the

square of the person's height, is over 30 kg/m2, with the range 25–30 kg/m2 defined as

overweight. Obesity increases the likelihood of various diseases and conditions, particularly

cardiovascular diseases, type 2 diabetes, obstructive sleep apnea, certain types of cancer,

osteoarthritis and depression, (Kushner, 2007).

Obesity is increasing at alarming rates in industrialized countries. Two changes in the

environment seem to have directly contributed to this increase. The first is a reduction in the

energy expenditure required for daily living, as a result of technological improvements, and

the second is an increase in energy intake resulting from the increasing availability of

palatable, low cost, high fat, energy dense food.

2.2 Causes of obesity

 The balance between calorie intake and energy expenditure determines a person's

weight. If a person eats more calories than he or she burns (metabolizes), the person

gains weight (the body will store the excess energy as fat). If a person eats fewer

calories than he or she metabolizes, he or she will lose weight. Therefore, the most

5
 common causes of obesity are overeating and physical inactivity. Ultimately, body

weight is the result of genetics, metabolism, environment, behavior, and culture.

 Genetics; A person is more likely to develop obesity if one or both parents are obese.

Genetics also affect hormones involved in fat regulation. For example, one genetic

cause of obesity is leptin deficiency. Leptin is a hormone produced in fat cells and in

the placenta. Leptin controls weight by signaling the brain to eat less when body fat

stores are too high. If, for some reason, the body cannot produce enough leptin or

leptin cannot signal the brain to eat less, this control is lost, and obesity occurs. The

role of leptin replacement as a treatment for obesity is under exploration.

 Over eating; Overeating leads to weight gain, especially if the diet is high in fat.

Foods high in fat or sugar (for example, fast food, fried food, and sweets) have high

energy density (foods that have a lot of calories in a small amount of food).

Epidemiologic studies have shown that diets high in fat contribute to weight gain.

 Frequency of eating; the relationship between frequency of eating (how often you eat)

and weight is somewhat controversial. There are many reports of overweight people

eating less often than people with normal weight. Scientists have observed that people

who eat small meals four or five times daily, have lower cholesterol levels and lower

and/or more stable blood sugar levels than people who eat less frequently (two or

three large meals daily). One possible explanation is that small frequent meals

produce stable insulin levels, whereas large meals cause large spikes of insulin after

meals.
6
 Physical inactivity; sedentary people burn fewer calories than people who are active.

The National Health and Nutrition Examination Survey (NHANES) showed strong

correlations between physical inactivity and weight gain in both sexes.

 Medications; Medications associated with weight gain include certain antidepressants

(medications used in treating depression), anticonvulsants (medications used in

controlling seizures such as carbamazepine, some diabetes medications (medications

used in lowering blood sugar such as insulin, sulfonylureas, and thiazolidinediones),

certain hormones such as oral contraceptives, and most corticosteroids such as

prednisone.

 Psychological factors; for some people, emotions influence eating habits. Many

people eat excessively in response to emotions such as boredom, sadness, stress, or

anger. While most overweight people have no more psychological disturbances than

normal weight people, about 30% of the people who seek treatment for serious weight

problems have difficulties with binge eating.

 Social issues: There is a link between social issues and obesity. Lack of money to

purchase healthy foods or lack of safe places to walk or exercise can increase the risk

of obesity.

 Diseases such as hypothyroidism, insulin resistance, polycystic ovary syndrome, and

Cushing's syndrome are also contributors to obesity. Some diseases, such as Prader-

Willi syndrome, can lead to obesity.

7
2.3 HYPOTHYROIDISM

Hypothyroidism, also called underactive thyroid or low thyroid, is a disorder of the

endocrine system in which the thyroid gland does not produce enough thyroid hormone. It

can cause a number of symptoms, such as poor ability to tolerate cold, a feeling of tiredness,

constipation, depression, and weight gain. Occasionally there may be swelling of the front

part of the neck due to goiter. Untreated hypothyroidism during pregnancy can lead to delays

in growth and intellectual development in the baby or cretinism. Worldwide, too little iodine

in the diet is the most common cause of hypothyroidism. In countries with enough iodine in

the diet, the most common cause of hypothyroidism is the autoimmune condition

Hashimoto's thyroiditis. Less common causes include: previous treatment with radioactive

iodine, injury to the hypothalamus or the anterior pituitary gland, certain medications, a lack

of a functioning thyroid at birth, or previous thyroid surgery. The diagnosis of

hypothyroidism, when suspected, can be confirmed with blood tests measuring thyroid-

stimulating hormone (TSH) and thyroxine levels, (Hypothyroidism, National Institute of

Diabetes and Digestive and Kidney Diseases, 2016).

A. Signs and symptoms of hypothyroidism

People with hypothyroidism often have no or only mild symptoms. Numerous

symptoms and signs are associated with hypothyroidism, and can be related to the

underlying cause, or a direct effect of having not enough thyroid hormones. Hashimoto's

8
thyroiditis may present with the mass effect of a goiter (enlarged thyroid gland),

(Khandelwal D, et al., 2012).

Symptoms include; fatigue, feeling cold, poor memory and concentration, constipation,

weight gain, shortness of breath and abnormal sensation.

Signs include; dry and coarse skin, cool extremities, myxedema, hair loss, slow pulse rate,

and swelling of limbs, (Encyclopedia of Mental Health, 2015).

B. Causes of hypothyroidism

Hypothyroidism is caused by inadequate function of the gland itself (primary

hypothyroidism), inadequate stimulation by thyroid-stimulating hormone from the pituitary

gland (secondary hypothyroidism), or inadequate release of thyrotropin-releasing hormone

from the brain's hypothalamus (tertiary hypothyroidism). Primary hypothyroidism is about a

thousandfold more common than central hypothyroidism, (Bleich S, et al., 2008).

Iodine deficiency is the most common cause of primary hypothyroidism and endemic

goiter worldwide. In areas of the world with sufficient dietary iodine, hypothyroidism is

most commonly caused by the autoimmune disease Hashimoto's thyroiditis (chronic

autoimmune thyroiditis). Hashimoto's may be associated with a goiter. It is characterized by

infiltration of the thyroid gland with T lymphocytes and autoantibodies against specific

thyroid antigens such as thyroid peroxidase, thyroglobulin and the TSH receptor, (Kanazawa

M, et al., 2005).

9
 I. Causes of Primary hypothyroidism; Iodine deficiency (developing countries),

autoimmune thyroiditis, subacute granulomatous thyroiditis, subacute lymphocytic

thyroiditis, postpartum thyroiditis, previous thyroidectomy, previous radioiodine

treatment, previous external beam radiotherapy to the neck.

Medication: lithium-based mood stabilizers, amiodarone, interferon alpha, tyrosine

kinase inhibitors such as sunitinib

II. Causes of Central hypothyroidism; Lesions compressing the pituitary (pituitary

adenoma, craniopharyngioma, meningioma, glioma, Rathke's cleft cyst, metastasis,

empty sella, aneurysm of the internal carotid artery), surgery or radiation to the

pituitary, drugs, injury, vascular disorders (pituitary apoplexy, Sheehan syndrome,

subarachnoid hemorrhage), autoimmune diseases (lymphocytic hypophysitis,

polyglandular disorders), infiltrative diseases (iron overload due to hemochromatosis

or thalassemia, neurosarcoidosis, Langerhans cell histiocytosis), particular inherited

congenital disorders, and infections (tuberculosis, mycoses, syphilis)

III. Congenital hypothyroidism; Thyroid dysgenesis (75%), thyroid dyshormonogenesis

(20%), maternal antibody or radioiodine transfer.

In consumptive hypothyroidism, a high level of type 3 deiodinase inactivates thyroid

hormones and thus leads to hypothyroidism. High levels of type 3 deiodinase generally

occur as the result of a hemangioma. The condition is very rare, (Weber Pasa, et al., 2017).
10
 C. Pathophysiology

The hypothalamus secretes TRH, which stimulates the production of TSH by the

pituitary gland. This, in turn, stimulates the production of thyroxine by the thyroid.

Thyroxine levels decrease TRH and TSH production by a negative feedback process.

Thyroid hormone is required for the normal functioning of numerous tissues in the

body. In healthy individuals, the thyroid gland predominantly secretes thyroxine (T 4), which

is converted, into triiodothyronine (T3) in other organs by the selenium-dependent enzyme

iodothyronine deiodinase, (Goemann, et al., 2011). Triiodothyronine binds to the thyroid

hormone receptor in the nucleus of cells, where it stimulates the turning on of particular

genes and the production of specific proteins. Additionally, the hormone binds to integrin

αvβ3 on the cell membrane, thereby stimulating the sodium–hydrogen ant porter and

processes such as formation of blood vessels and cell growth. In blood, almost all thyroid

hormone (99.97%) is bound to plasma proteins such as thyroxine-binding globulin; only the

free unbound thyroid hormone is biologically active. Overexpression of deiodinase can thus

lead to consumptive hypothyroidism, (Poulain M, et al., 2006).

The thyroid gland is the only source of thyroid hormone in the body; the process

requires iodine and the amino acid tyrosine. Iodine in the bloodstream is taken up by the

gland and incorporated into thyroglobulin molecules. The process is controlled by the

thyroid-stimulating hormone (TSH, thyrotropin), which is secreted by the pituitary. Not

enough iodine, or not enough TSH, can result in decreased production of thyroid hormones,

(Gaitonde, et al., 2012).

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 The hypothalamic–pituitary–thyroid axis plays a key role in maintaining thyroid

hormone levels within normal limits. Production of TSH by the anterior pituitary gland is

stimulated in turn by thyrotropin-releasing hormone (TRH), released from the hypothalamus.

Production of TSH and TRH is decreased by thyroxine by a negative feedback process. Not

enough TRH, which is uncommon, can lead to not enough TSH and thereby to not enough

thyroid hormone production, (Bleich et al., 2008).

Pregnancy leads to marked changes in thyroid hormone physiology. The gland is

increased in size by 10%, thyroxine production is increased by 50%, and iodine

requirements are increased. Many women have normal thyroid function but have

immunological evidence of thyroid autoimmunity (as evidenced by autoantibodies) or are

iodine deficient, and develop evidence of hypothyroidism before or after giving birth,

(Berrington de Gonzalez et al., 2010).

2.4 EFFECT OF THYROID HORMONES ON LIPID METABOLISM

Thyroid hormones induce the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA)

reductase, which is the first step in cholesterol biosynthesis. Moreover, triiodothyronine (T 3)

up regulates LDL receptors by controlling the LDL receptor gene activation. This T 3-

mediated gene activation is done by the direct binding of T 3 to specific thyroid hormone

responsive elements (TREs), (Bakker O, et al., 1998). Furthermore, T3 controls the sterol

regulatory element-binding protein-2 (SREBP-2), which in turn regulates LDL receptor’s

12
gene expression, (Shin DJ and Osborne TF, 2003). T3 has also been associated with

protecting LDL from oxidation, (Faure P, et al., 2004).

Thyroid hormones can influence HDL metabolism by increasing cholesteryl ester

transfer protein (CETP) activity, which exchanges cholesteryl esters from HDL 2 to the very

low density lipoproteins (VLDL) and TGs to the opposite direction, (Lagrost L, 1994). In

addition, thyroid hormones stimulate the lipoprotein lipase (LPL), which catabolizes the TG-

rich lipoproteins, and the hepatic lipase (HL), which hydrolyzes HDL 2 to HDL3 and

contributes to the conversion of intermediate-density lipoproteins (IDL) to LDL and in turn

LDL to small dense LDL (sdLDL), (Kuusi T, et al., 1980). Another effect of T3 is the up-

regulation of apolipoprotein AV (ApoAV), which plays a major role in TG regulation,

(Prieur X, et al., 2005). Indeed, increased levels of ApoAV have been associated with

decreased levels of TGs, (Rensen PC, et al., 2005). Proposed mechanisms for this effect

include the decrease of hepatic VLDL-TG production and the increase of plasma LPL levels

and activity, resulting in increase of lipoprotein remnant generation due to enhanced LPL-

mediated lipolysis of VLDL-TG, (Rensen PC, et al., 2005). Moreover, a greater clearance of

lipoprotein core remnants, caused by increased hepatic uptake due to an enhanced affinity

for the LDL receptor, has also been ascribed to ApoAV, (Rensen PC, et al., 2005).

Beyond their effect on lipid profile thyroid hormones can equally affect a number of

other metabolic parameters related to CVD risk. Indeed, thyroid function can influence

adipocyte metabolism and the production of adipokines. Hyperthyroidism has been

13
associated with increased levels of adiponectin, whereas hypothyroidism is not associated

with significant changes in adiponectin, (Iglesias P and Diez JJ, 2007). Insulin resistance is

also correlated with thyroid function, (Crunkhorn S and Patti ME, 2008). TSH is positively

associated with fasting and postprandial insulin concentration and negatively with insulin

sensitivity, (Fernandez-Real JM, et al., 2006). Moreover, low normal FT4 levels are

significantly associated with increased insulin resistance, (Chubb SA, et al., 2005).

Oxidative stress is also affected by thyroid function with studies however showing

controversial outcomes, (Venditti P and Di Meo S, 2006). Furthermore, endothelial,

(Fernandez-Real JM, et al., 2006) and cardiac function as well as atherosclerosis, (Auer J, et

al., 2003), have been positively associated with thyroid hormone levels. A positive

association between TSH and body mass index (BMI) or waist circumference has also been

described, (De Pergola G, et al., 2007). A large population trial using data from the fourth

and fifth Tromso study showed that this association between TSH and BMI was only

significant in non smokers, (Nyrnes A, et al., 2006).

2.5 THYROID DYSFUNCTION AND BODY WEIGHT

Long before the definition of the metabolic syndrome, alterations in thyroid function

were reported in obese patients. Body composition and thyroid hormones appear to be

closely related since the latter is known to be involved in the regulation of basal metabolism

and thermogenesis, playing an important role in lipid and glucose metabolism, food intake

14
and fat oxidation, (Silva JE, 2006). In agreement with this knowledge, it is well known that

hypothyroidism causes a weight increase together with a decrease in basal metabolic rate

and thermogenesis, (Rotondi M, et al., 2009). Moreover, it has also been reported that there

is an inverse correlation between free thyroxine (fT4) values and body mass index (BMI),

even when fT4 values remain in the normal range, (Knudsen N, et al., 2005). Lately, it has

also been suggested that abnormalities in thyroid function may be secondary to weight

excess. These changes, however, would still be functional, as suggested by their

normalization after weight loss, (Biondi B, 2010).

In various studies on adult obese individuals, thyroid hormone and thyroid-stimulating

hormone (TSH) concentrations have been described as normal, elevated or reduced,

compared to a control group. In obese children, the most common abnormality is

hyperthyrotropinemia, (Reinehr T, 2010).

The causes underlying these alterations are not known although several theories have

been proposed. These include an increased deiodinase activity, as suggested by the increase

in total triiodothyronine (T3) and free T3 (fT3) reported in some subjects. The reported high

conversion rate of T4 to T3 in obese patients has been also interpreted as a defense

mechanism, capable of counteracting the accumulation of fat by increasing the energy

expenditure , basal metabolic rate and the total energy expenditure, being in fact positively

related to the levels of total T3 and fT3. Leptin, a hormone produced by adipocytes, also

alters the activity of deiodinases, thus promoting the conversion of T4 to T3, (Feldt-

Rasmussen U, 2007).

15
 Another mechanism claimed to explain the high values of T3 and fT3 has been related

to the fact that the expressions of both TSH and thyroid hormones are reduced in adipocytes

of obese subjects as compared to individuals of normal weight. This would prompt

decreased tissue responsiveness to circulating thyroid hormones and would also explain the

consequent increased compensatory secretion of TSH and fT3 in an attempt to force the state

of peripheral resistance, (Myers MG Jr, et al., 2010).

Another potential cause of increased blood concentration of TSH may be the high

levels of leptin, found in obese subjects. The main action of leptin is to report centrally the

amount of fat, leading to a decrease in appetite and food intake. In case of obesity, increased

leptin is considered as an evidence of “leptin resistance”, (Myers MG Jr, et al., 2010). In

addition to this action, leptin has also been shown to stimulate centrally the transcription of

pro thyrotropin-releasing hormone (TRH) and consequently also that of TRH and TSH. This

increase in TSH, and therefore in T3, could be interpreted as a defense mechanism of the

body against weight gain. In agreement with this interpretation, we observe the opposite in

anorexia nervosa, in which low levels of fT3 and TSH are interpreted as signs of a

physiological adaptation to reduce metabolic energy expenditure. Moreover, TSH receptors

are also localized in adipose tissue and thus TSH may directly stimulate the production of

leptin by adipocytes. Another explanation might be the impaired feedback due to a lowered

number of T3 receptors in the hypothalamus, (Burman KD, et al., 1980).

A further explanation could be the inflammatory state that characterizes obesity. It is

well recognized that in obesity, the adipose tissue secretes a distinct quantity of

16
inflammatory cytokines, and some of these, such as tumor necrosis factor-a, (TNF-a)

interleukin-1 (IL-1) and interleukin-6 (IL-6), escape into the general circulation provoking

systemic symptoms. The secretion of these cytokines, which have been proven to inhibit

sodium/iodide symporter (NIS) mRNA expression and iodide uptake activity in Fisher rat

thyroid cell line (FRTL-5) and human thyroid cells, might therefore explain the

compensatory raised TSH level in obese individuals. This would also explain the tissue

resistance to TSH and additionally its reversibility after weight loss, (Ajjan RA, et al,. 1998).

2.6 EFFECTS OF THYROID DYSFUNCTION ON LIPID PARAMETERS

Although decreased thyroid function is accompanied by reduced activity of HMG-

CoA reductase, TC and LDL-C levels are increased in patients with overt hypothyroidism,

(Pearce EN, et al., 2008). This is due to the decreased LDL-receptors’ activity, resulting in

decreased catabolism of LDL and IDL, (Walton KW, et al., 1965). Moreover, a decrease in

LPL activity is found in overt hypothyroidism, decreasing the clearance of TG-rich

lipoproteins, (Nikkila EA and Kekki M, 1972). Therefore, overt hypothyroid patients may

also present with elevated TG levels associated with increased levels of VLDL and

occasionally fasting chylomicronemia, (Al-Tonsi AA, et al., 2004). The VLDL and IDL

particles in hypothyroidism are rich in cholesterol and apolipoprotein E, thus resembling β-

VLDL particles of type III hyperlipoproteinemia. Therefore, the full-blown clinical

17
syndrome of the type III hyperlipoproteinemia may develop in patients homozygous for the

apolipoprotein E2 allele if they become hypothyroid.

Hypothyroid patients may also exhibit elevated levels of HDL-C, (Pearce EN, et al.,

2008), mainly due to increased concentration of HDL 2 particles. Indeed, due to a reduction

of HL activity a decrease in HDL2 catabolism is observed, (Lam KS, et al., 1986). Moreover,

decreased activity of the CETP results in reduced transfer of cholesteryl esters from HDL to

VLDL, thus increasing HDL-C levels, (Dullaart RP, et al., 1990). Hypothyroid patients have

increased lipoprotein (a) [LP (a)] levels (Tzotzas T, et al., 2000), which are associated with

increased CVD risk, (Klausen IC, et al., 1992).

Administration of substitution therapy with L-thyroxine significantly improves lipid

metabolism abnormalities. A period of 4-6 weeks of thyroxin replacement therapy is usually

needed to correct dyslipidemia in overt hypothyroidism. In general, changes in serum

lipoproteins in hypothyroid patients are correlated with changes in free T 4 (FT4), (Wiseman

SA, et al., 1993). A study in newly-diagnosed hypothyroid patients (n=60) showed a

decrease in serum TC and LDL-C levels after thyroxine treatment, (Abbas JM, et al., 2008).

However, when the effects of substitution therapy on qualitative lipid profile were assessed

no change in LDL particle size was seen, (Abbas JM, et al., 2008). A more dramatic

reduction of TC levels has been observed in hypothyroid patients with higher baseline TSH

levels (Elder J, et al., 1990)

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 Serum HDL-C levels tend to decrease with thyroid replacement, but this is a less

consistent finding (Verdugo C, et al., 1987) Serum Lp(a) levels also tend to decrease with

restoration of euthyroidism. Moreover, a decrease in CIMT has been observed after

thyroxine treatment in hypothyroid patients (Nagasaki T, et al., 2003). The presence of overt

hypothyroidism in patients with dyslipidemia is not rare. We found that 2.8% of the patients

who visited our outpatient lipid clinic with dyslipidemia had elevated levels of TSH and

reduced levels of FT4, (Tsimihodimos V, et al., 1999). After restoration of euthyroidism with

levothyroxine therapy, a significant decrease of serum levels of TC and LDL-C,

apolipoprotein B (ApoB) and Lp(a) was observed, while levels of HDL-C, TGs and

apolipoprotein AI (ApoAI) were not significantly changed, (Tsimihodimos V, et al., 1999).

Superimposed dyslipidemia should be taken into account in cases of failure of substitution

therapy to normalize the lipid profile despite the restoration of euthyroidism (Duntas LH,

2002).

Hypothyroidism is one of the most common causes of secondary dyslipidemia.

Therefore, before starting hypolipidemic therapy, the evaluation of thyroid function is

needed. Thyroid failure is associated with increased levels of creatinine kinase (CK), (Beyer

IW, et al., 1998). Statin therapy may substantially increase levels of CK. A study examined

the effects of accidentally starting statin therapy in patients with undiagnosed

hypothyroidism (n=9). These patients had significantly higher CK levels (1095 U/L)

compared with untreated hypothyroid patients matched for freeT 4 levels (n=18; CK=395;

19
p<0.05), (Beyer IW, et al., 1998).Therefore, it is imperative to firstly correct thyroid

dysfunction with thyroxine substitution therapy and then treat the underlying dyslipidemia

with statins, (Beyer IW, et al., 1998).

2.7 MANAGEMENT OF OBESITY

Management of obesity can include lifestyle changes, medications, or surgery. The main

treatment for obesity consists of dieting and physical exercise, (Lau DC, et al., 2007). Diet

programs may produce weight loss over the short term, (Strychar I, 2006). But maintaining

this weight loss is frequently difficult and often requires making exercise and a lower calorie

diet a permanent part of an individual's lifestyle, (Shick SM, et al., 1998). Success rates of

long-term weight loss maintenance with lifestyle changes are low, ranging from 2 to 20%.

(Wing, et al., 2005) Dietary and lifestyle changes are effective in limiting excessive weight

gain in pregnancy and improve outcomes for both the mother and the child. The National

Institutes of Health recommend a weight loss goal of 5% to 10% of the person's current

weight over six months, (Colquitt, et al., 2014).

One medication, orlistat, is current widely available and approved for long term use.

Weight loss however is modest with an average of 2.9 kg (6.4 lb) at 1 to 4 years and there is

little information on how these drugs affect longer-term complications of obesity. Its use is

associated with high rates of gastrointestinal side effects, (Rucker D, et al., 2007).

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2.8 DIAGNOSIS OF OBESITY

1) BODY MASS INDEX

The body mass index (BMI) or Quetelet index is a value derived from the mass (weight) and

height of an individual. The BMI is defined as the body mass divided by the square of the

body height, and is universally expressed in units of kg/m 2, resulting from mass in kilograms

and height in meters. BMI provides a simple numeric measure of a person's thickness or

thinness, allowing health professionals to discuss weight problems more objectively with

their patients. BMI was designed to be used as a simple means of classifying average

sedentary (physically inactive) populations, with an average body composition. (Physical

status: the use and interpretation of anthropometry, 2007). For these individuals, the current

value recommendations are as follow: a BMI from 18.5 up to 25 kg/m2 may indicate optimal

weight, a BMI lower than 18.5 suggests the person is underweight, a number from 25 up to

30 may indicate the person is overweight, and a number from 30 upwards suggests the

person is obese, (Assessing Your Weight and Health Risk, 2014). Lean athletes often have a

high muscle to fat ratio and therefore a BMI that is misleadingly high relative to their body

fat percentage, (Defining obesity, NHS, 2014). The WHO regards a BMI of less than 18.5 as

underweight and may indicate malnutrition, an eating disorder, or other health problems,

while a BMI equal to or greater than 25 is considered overweight and above 30 is considered

obese, (BMI Classification, Global Database on Body Mass Index, 2012).

21
These ranges of BMI values are valid only as statistical categories.

Category BMI (kg/m2)

From to

Very severely underweight 0 15

Severely underweight 15 16

Underweight 16 18.5

Normal (healthy weight) 18.5 25

Overweight 25 30

Obese Class I (Moderately obese) 30 35

Obese Class II (Severely obese) 35 40

Obese Class III (Very severely obese) 40 45

Obese Class IV (Morbidly Obese) 45 50

Obese Class V (Super Obese) 50 60

Obese Class VI (Hyper Obese) 60

(BMI Classification, Global Database on Body Mass Index, 2012).

2.9 LIPID PROFILE

22
Lipid profile or lipid panel is a panel of blood tests that serves as an initial screening tool for

abnormalities in lipids, such as cholesterol and triglycerides. The results of this test can

identify certain genetic diseases and can determine approximate risks for cardiovascular

disease, certain forms of pancreatitis, and other diseases, (National Cholesterol Education

Program, 2002).

The lipid profile typically includes:

 Low-density lipoprotein (LDL)

 High-density lipoprotein (HDL)

 Triglycerides

 Total cholesterol

Using these values, a laboratory may also calculate:

 Very low-density lipoprotein (VLDL)

 Cholesterol:HDL ratio

23
A. Cholesterol is an organic molecule. It is a sterol, (Razin S and Tully JG May 1970), a

type of lipid molecule, and is biosynthesized by all animal cells, because it is an

essential structural component of all animal cell membranes and is essential to

maintain both membrane structural integrity and fluidity. Cholesterol allows animal

cells to function without a cell wall (which in other species protects membrane

integrity and cell viability); this allows animal cells to change shape rapidly. In

addition to its importance for animal cell structure, cholesterol also serves as a

precursor for the biosynthesis of steroid hormones, bile acid and vitamin D,

(Hanukoglu I, 1992). Cholesterol is the principal sterol synthesized by all animals. In

vertebrates, hepatic cells typically produce the greatest amounts. It is absent among

prokaryotes (bacteria and archaea), although there are some exceptions, such as

Mycoplasma, which require cholesterol for growth, (Razin S and Tully JG, 1970).

B. A triglyceride is an ester derived from glycerol and three fatty acids (from tri- and

glyceride), (Nomenclature of Lipids, 2007).Triglycerides are the main constituents of

body fat in humans and other animals, as well as vegetable fat, (Nelson, et al.,

2000).They are also present in the blood to enable the bidirectional transference of

adipose fat and blood glucose from the liver, and are a major component of human

skin oils, (Lampe, et al., 1983). There are many different types of triglyceride, with

the main division between saturated and unsaturated types. Saturated fats are

24
 "saturated" with hydrogen — all available places where hydrogen atoms could be

bonded to carbon atoms are occupied. These have a higher melting point and are more

likely to be solid at room temperature. Unsaturated fats have double bonds between

some of the carbon atoms, reducing the number of places where hydrogen atoms can

bond to carbon atoms. These have a lower melting point and are more likely to be

liquid at room temperature, (Lampe, et al., 1983).

C. High-density lipoproteins (HDL) are one of the five major groups of lipoproteins,

(LDL and HDL: Bad and Good Cholesterol, 2017). Lipoproteins are complex particles

composed of multiple proteins which transport all fat molecules (lipids) around the

body within the water outside cells. They are typically composed of 80-100 proteins

per particle (organized by one, two or three ApoA; more as the particles enlarge

picking up and carrying more fat molecules) and transporting up to hundreds of fat

molecules per particle. Increasing concentrations of HDL particles are strongly

associated with decreasing accumulation of atherosclerosis within the walls of

arteries. This is important because atherosclerosis eventually results in sudden plaque

ruptures, cardiovascular disease, stroke and other vascular diseases. HDL particles are

sometimes referred to as "good cholesterol" because they can transport fat molecules

out of artery walls, reduce macrophage accumulation, and thus help prevent or even

regress atherosclerosis, but studies have shown that HDL-lacking mice still have the

25
 ability to transport cholesterol to bile, suggesting that there are alternative

mechanisms for cholesterol removal, (Betteridge, et al., 2008).

D. Low-density lipoprotein (LDL) is one of the five major groups of lipoprotein which

transport all fat molecules around the body in the extracellular water, (LDL and HDL:

Bad and Good Cholesterol, 2017). These groups, from least dense, compared to

surrounding water (largest particles) to most dense (smallest particles), are

chylomicrons (aka ULDL by the overall density naming convention), very low-

density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density

lipoprotein and high-density lipoprotein (HDL). LDL delivers fat molecules to the

cells and can drive the progression of atherosclerosis if they become oxidized within

the walls of arteries. LDL particles pose a risk for cardiovascular disease when they

invade the endothelium and become oxidized, since the oxidized forms are more

easily retained by the proteoglycans. A complex set of biochemical reactions regulates

the oxidation of LDL particles, chiefly stimulated by presence of necrotic cell debris

and free radicals in the endothelium. Increased concentrations of LDL particles is

strongly associated with the development of atherosclerosis over time, (Glagov, et al.,

1987)

26
2.9.1 Juglans nigra

Juglans nigra, the eastern black walnut, is a species of deciduous tree in the walnut

family, Juglandaceae, native to eastern North America. It grows mostly in riparian zones,

from southern Ontario, west to southeast South Dakota, south to Georgia, northern Florida

and southwest to central Texas. Wild trees in the upper Ottawa Valley may be an isolated

native population or may have derived from planted trees.

Black walnut is an important tree commercially, as the wood is a deep brown color

and easily worked. The fruits, walnuts, are cultivated for their distinctive and desirable taste.

Often, trees are grown for both lumber and walnuts simultaneously and many cultivars have

been developed for improved quality nuts or wood. Black walnut is currently under

pressure from the thousand cankers disease that is causing decline of walnuts in some areas.

Black walnut is also allelopathic, which means that it releases chemicals from roots and

other tissues that harm other organisms and give the tree a competitive advantage; this is

often undesirable as it can harm garden plants and grasses.

27
Figure 1; black walnut

(Whittemore, et al., 2004)

The fruit production tends to occur irregularly with some years producing larger crops

than others. Fruiting may begin when the tree is 4–6 years old, but large crops take 20 years.

Total life span of J. nigra is about 130 years. Black walnut does not leaf out until late spring

when the soil has warmed and all frost danger is past. Like other trees of the order Fagales,

such as oaks, hickories, chestnuts, and birches, it is monoecious, with wind-pollinated

catkins. Male and female flowers are in separate spikes, and the female flowers typically

appear before the male on a single tree (dichogamy). As a consequence, self-pollination is

unlikely. However, individual trees usually are not self-sterile; if they are not pollinated by

neighboring trees, they may set self-fertilized seeds. For maximum seed germination, the

seeds should be cold-moist stratified for 3–4 months, although the exact time depends on the

seed source. The seedlings emerge in April or May and typically grow 90 cm (35 in) their

first year and even more in the 2nd year. Black walnut often loses its leaves earlier than

other deciduous trees growing in the same area after having a growing period of 115–135

days, (Williams, et al., 1990).

28
 Black walnut has a strong taproot, which makes the seedlings resilient, but difficult to

transplant. It is more resistant to frost than the English or Persian walnut, but thrives best in

the warmer regions of fertile, lowland soils with high water tables, although it will also grow

in drier soils, but much more slowly, (Dir and Michael 1990). Some soils preferred by black

walnut include Alfisol and Entisol soil types. Walnut grows best on sandy loam, loam, or silt

loam type soils but will also grow well on silty clay loam soils. It prefers these soils due to

the fact that these soils hold large quantities of water, which the tree draws from during dry

periods. Visually, black walnut is similar to the butternut (Juglans cinerea) in leaf shape,

and the range also overlaps significantly. The fruits are quite different, and their presence

makes an identification easy, as black walnut fruits are round and butternuts are more oval-

oblong shaped. When a fruit is not available, two species can be differentiated based on the

leaf scars, or the place where the leaf meets the stem: butternut has a leaf scar with a flat

upper edge and with a velvety ridge above that flat part, but black walnut has an indented

leaf scar with no hairy ridge. (Whittemore, et al., 2004)

A. Black Walnut Uses

Black walnut tincture has historically been known for anti-fungal, anti-helminthic

(parasite killing), anti-viral and anti-bacterial effects. Some herbalists use them as part of

anti-cancer protocols, such as Dr. Hulda Clark's 21 Day Cancer Cure Program.

Baseline of Health Foundation notes that:

29
 Before vitamins and minerals were commonly used, herbalists were known to use

black walnut for a variety of conditions including easing scrofula, ulcers, wounds, and

rickets, scurvy and as a gargle. In more recent times, Russian military hospitals also used the

nut as a cleansing and quick healing medication for wounds and ulcers.

The black walnut hull’s tannin content is thought to help shrink the sweat glands and reduce

excessive sweating. Other uses include:

 lowering blood pressure and cholesterol levels

 menorrhagia (heavy menstrual bleeding) and diarrhea

 aiding digestion

 helping relieve colic, heartburn and flatulence

 stimulating bile flow

 easing pain in spleen

 balancing blood sugar levels

 warding off heart disease

 combating malaria

 helping with syphilis

 helping with skin conditions such as boils and acne

30
 B. CHEMICAL COMPOSITION

 VITAMINS AND MINERALS

 Ca     0.89%      Fe     0.00021

 P     0.77      Mn     182 Mg/lb

 K     0.87      Cu     tr

 Na     tr      Zn     tr

 Cl     0.14      Iodine     142 Mg/lb

 Mg     0.12      Sulfur     0.088

(Marcela L and Martinez, 2008).

2.9.4 Urtica dioica

Urtica dioica, often called common nettle, stinging nettle (although not all plants of this

species sting) or nettle leaf, is a herbaceous perennial flowering plant in the family

Urticaceae. It is native to Europe, Asia, northern Africa, and North America, and introduced

elsewhere. (Burning and Stinging Nettle, 2013). The species is divided into six subspecies,

five of which have many hollow stinging hairs called trichomes on the leaves and stems,

which act like hypodermic needles, injecting histamine and other chemicals that produce a

31
stinging sensation upon contact ("contact urticaria"). The plant has a long history of use as a

source for traditional medicine, food, tea, and textile raw material in ancient societies.

Figure 2; Nettle leaf

(Adisesh, et al., 2013)

A. Nettle sting mechanism and treatment

Urtica dioica produces its inflammatory effect on skin (stinging, burning sensation often

called "contact urticaria") both by impaling the skin via spicules – causing mechanical

irritation – and by biochemical irritants, such as histamine, serotonin, and choline, among

other chemicals, (Michael and Greenberg 2003).

Anti-itch drugs, usually in the form of creams containing antihistamines or hydrocortisone,

may provide relief from nettle dermatitis. In Great Britain, the use of dock leaves on nettle

stings is an established folk remedy, and revolves around the sap released from rubbing the

leaf over affected areas of skin, which provides a cooling sensation. Docks and nettles

regularly grow in the vicinity of each other due to both plants favoring the same soil

32
conditions, and this may have aided the dock's popularity as a treatment for nettle stings.

(Adisesh, et al., 2013)

B. Stinging Nettle Therapeutic Benefits and Claims.

 Stinging nettle contains amines, flavonoids, lignans, minerals (calcium, potassium,

iron, and silicon) and vitamins A, B2, C and K, organic acids, scopoletin (isolated

from the flowers), plant sterols, polysaccharides, lectins, and tannins.

 Used for hundreds of years as an herbal remedy, the herb is best known for its ability

to ease the pain in the muscles and joints caused by arthritis and gout.

 Stinging nettle is used as a diuretic and laxative. Various extracts of stinging nettle

have shown to be effective in treating diarrhea, edema and urinary disorders, as well

as prostate diseases.

 In several European countries, the herb has become a preferred treatment for early

stages of benign enlargement of the prostate gland, often called benign prostatic

hyperplasia (BPH).

 As a treatment of BPH stinging nettle is often used in combination with other herbs

like saw palmetto (Serenoa repens), pygeum (Pygeum africanum) and pumpkin

(Cucurbita pepo).

33
 Used directly on the hair, stinging nettle is thought to add shine, and prevent oily hair

and dandruff.

 It is also believed to be effective in treating or preventing baldness, as well as getting

rid of head lice.

 Stinging nettle has shown promise in reducing sneezing and itching as results from

hay fever. This use as an herbal remedy for hay fever is successful due to the nettles

ability to reduce the body’s production of histamines in relation to the allergen.

 Used as a medicinal herb to treat respiratory issues such

as asthma, bronchitis, sinusitis and allergies.

 Stinging nettle is thought to be an effective histamine blocker as well as an

inflammation reducer, and it has been used to treat allergic rhinitis without the side

effects of popular allergy medications.

 Taken internally, it may be effective against ulcers, intestinal inflammation,

and hemorrhoids.

 Stinging nettle contains 3, 4-divanillyltetrahydrofuran, which is used by bodybuilders

to increase free testosterone, (Adisesh, et al., 2013).

C. Action on hyperlipidemia and atherosclerosis

Daily administration of aqueous extract of Urtica dioica at 150 mg/kg for 30 days, either

as part of a normal or high fat diet, caused a reduction in serum lipids and lipoproteins.

34
 Significant decreases in cholesterol and LDL/HDL ratio (Low Density/High Density

Lipoproteins) were observed, (Daher et al., 2006). Similarly, administration of an

ethanolic extract to hypercholesterolemic rats, using doses of 100 mg/kg and 300 mg/kg,

was responsible for the decreased of cholesterol. (Nassiri-Asl et al., 2009).

D. Possible Side Effects and Interactions of Stinging Nettle

The potentially harmful effects of using stinging nettle as an herbal remedy are rather

numerous. The herb can interfere with blood thinners such as Warfarin, Clopidogrel, and

Aspirin. It lowers blood pressure, which could increase the strength of the effects of the

following medications; ACE inhibitors, Beta blockers, and Calcium channel blockers and

more. Stinging nettle is a natural herb used as a diuretic and increases the risk of dehydration

when taken with Furosemide and Hydrochlorothiazide. This herb raises the risk of

hypoglycemia as well. Stinging nettle should not be used by pregnant or nursing women.

Those with diabetes should also avoid the use it, (Adisesh, et al., 2013).

35
 CHAPTER THREE

MATERIALS AND PROCEDURE

3.1 STUDY AREA

This study was conducted in Madonna University Teaching Hospital (MUTH), Elele, Rivers

State.

3.2 PLANT MATERIAL

Uncooked Black walnut (juglans nigra) and nettle leaf (urtica dioca) was purchased from

Adonte market in Aniocha-south LGA, Delta State, and identified at the Department of

Biochemistry, Madonna University, Elele Rivers State.

3.3 PREPARATION OF EXTRACT

Uncooked Black walnut and nettle leaf were air dried at room temperature for two weeks,

after which they were grounded to smaller particle sizes using electronic blender and boiled

separately at 1000C. They were allowed to cool at 250C and then packaged in a clean glass-

air tight container and stored at room temperature, from which daily ration was taken,

measured and administered intraperitoneally using a gavage (feeding tube).

36
3.4 PREPARATION AND ADMINISTRATION OF DRUG

Thiamazole was purchased from BDH pharmacy representatively at Asaba in Delta state,

Nigeria. The drug was dissolved in water in the ratio 5mg per 5ml of water, packaged in a

clean glass air tight container and stored at room temperature, from which daily ration was

taken, measured and administered intraperitoneally using a gavage (feeding tube).

3.5 ANIMAL HANDLING

Female wistar albino rats weighing 138-188g were purchased from the animal center of

Madonna University Elele. Each of the animals were housed individually in an animal cage

with wire mesh and saw dust lining, and they were kept in a conducive room inside the

animal house, the animals were allowed to acclimatize for 2 weeks during which time food

and water were given ad libitum. After two weeks, they were numbered and grouped into 5

groups (A-E), 6 rats in each group, drug and food was administered accordingly.

3.6 EXPERIMENTAL DESIGN

Five equal groups each of six rats were housed individually in cages covered with wire mesh

at room temperature, and kept under normal healthy conditions. All the rat’s weights were

determined after every two weeks and recorded.

Group A: Received food and water ad libitum throughout the experiment.

Group B: Received in addition to food and water, 5mg of Thiamazole intraperitoneally,

everyday from day 14 to day 42.

37
Groups C: Received in addition to food and water, 5mg of Thiamazole intraperitoneally

everyday from day 14 to day 28, and were given 5mg/kg body weight of juglans nigra

(black walnut) intraperitoneally everyday from day 29 to day 42.

Group D: Received in addition to food and water, 5mg of Thiamazole intraperitoneally

everyday from day 14 to day 28, and were given 5mg/kg body weight of urtica dioica (nettle

leaf) intraperitoneally everyday from day 29 to day 42.

Group E: Received in addition to food and water, 5mg of Thiamazole intraperitoneally

everyday from day 14 to day 28, and were given 5mg/kg body weight of juglans nigra

(black walnut) and urtica dioica (nettle leaf) intraperitoneally everyday from day 29 to day

42.

3.7 SAMPLE COLLECTION

At the end of the experimental period, the animals were sacrificed, blood sample was

collected via cardiac puncture, Each sample of blood was collected into a plain test tubes

labeled accordingly, and kept at room temperature to clot and retract, after which it was

dislodged and centrifuged for 10 minutes at 3000r.p.m. Serum samples were separated and

used for determination of different biochemical parameters.

38
3.8 MEASUREMENT OF BMI

The weights and heights of the rats were recorded at day 1, 14, 28 and 42 respectively, and

their body mass index was calculated.

BMI = Weight (g)/ Height2 (cm2)

This is expressed in g/cm2

3.9 LABORATORY PROCEDURES

All reagents were commercially purchased and the manufacturers’ SOP strictly followed.

A. DETERMINATION OF SERUM TOTAL CHOLESTEROL

Enzymatic end‐point method (Trinder et al 1969) was used as modified by Randox

Laboratories, UK. LOT No: 429660; Expiry date: 2019- 10- 28.

PRINCIPLE

REACTION

cholesteryl ester hydrolase

Cholesteryl ester + H2O ------------------------------------>cholesterol + fatty acid
cholesterol oxidase
Cholesterol + O2 -------------------------------> cholest-4-en-3-one + H2O2
Peroxidase
2H2O2+4-aminophenazone + phenol -------------> 4-(p-benzoquinonemonoimino)-phenazone + 4 H2O

39
Cholesterol is determined after enzymatic hydrolysis and oxidation. The indicator is formed

from hydrogen peroxide and 4-aminoantipyrine in the presence of phenol and peroxidase.

PROCEDURE

Three dry test tubes labeled test, standard and blank were placed in a test tube rack, using

automatic micropipette, 10µl of distilled water was pipette into the blank tube, 10µl of

cholesterol standard pipette into the test tube labeled standard while 10µl sample was pipette

into the sample test tube, 1000µl of cholesterol reagent was then added to all the tubes. The

tubes were mixed thoroughly after each addition and incubated at 37ºc for 10minutes colour

development. The pinkish colour obtained was read colorimetrically at 500nm wavelength,

the spectrophotometer was zeroed with the blank, and then standard and test absorbances

were recorded appropriately.

Calculation: When using a standard

Test concentration= Absorbance of test x concentration of standard

Absorbance of standard

B. DETERMINATION OF SERUM TRIGLYCERIDE

Enzymatic end‐point method (Koditscheck et al 1969) was used as modified by

Randox Laboratories, UK. LOT No: 442428; Expiry date: 2020- 11- 18.

40
Principle:
REACTION
Triglycerides + 3H2O --------------------> glycerol + fatty acids
lipase

Glycerol + ATP -------------------------> glycerol-3-phosphate + ADP

Glycerokinase

Glycerol-3-phosphate + O2 -----------------------------------> dihydroxyacetone phosphate + H2O2

glycerophosphate oxidase

H2O2 + 4-aminophenazone + 4-chlorophenol -----------------> 4-(p-benzoquinone-monoimino)-phenazone +

2H2O + HCl. peroxidase

The triglycerides are determined after enzymatic hydrolysis with lipases; the indicator is
quinoneimine from hydrogen perioxide, 4-amino phenazone and 4-chlorophenol under the
catalytic influence of perioxidases to form the colour measured colorimetrically at 540nm
wavelength.

Procedure: Three dry test tubes labeled test, standard and blank were placed in a test tube

rack; using an automatic micropipette, 10µl of distilled water was pipette into the blank tube,

10µl of triglycerides standard pipette into the standard tube while 10µl of sample was pipette

into the sample tube, 1000µl of the triglyceride reagent was then added to all the tubes.

The tubes were mixed thoroughly after each addition and incubated for 10minutes at 20-

25ºc, the absorbance of sample (A sample) and standard (A standard) were measured against

the reagent blank within 60minutes at 500nm, the spectrophotometer was zeroed with the

sample blank.

41
Calculation: When using a standard

Triglyceride concentration= Absorbance of test x concentration of standard

Absorbance of standard

C. DETERMINATION OF SERUM HIGH DENSITY LIPOPROTEIN

Precipitation method (Abell by et al 1952) was used as modified by Randox

laboratories, UK. LOT No: 259460.

PRINCIPLE

Low density lipoprotein (LDL) and VLDL and chylomicron fractions are precipitated

quantitatively by addition of phototungstic assay in presence of magnesium ion, after

centrifugation, the HDL fraction which remain in the supernatant is determined.

REACTION

(1) ApoB containing lipoproteins + α-cyclodextrin + Mg+2 + dextran SO4 ---> soluble non-
reactive complexes with apoB-containing lipoproteins

(2) HDL-cholesteryl esters PEG-cholesteryl esterase > HDL-unesterified cholesterol + fatty

acid

(3) Unesterified chol + O2 PEG-cholesterol oxidase > cholestenone + H2O2

(4) H2O2 + 5-aminophenazone + N-ethyl-N-(3-methylphenyl)-N’_succinyl ethylene

diamine + H2O + H+ peroxidase > qunoneimine dye + H2O

42
PROCEDURE

In two centrifuge tubes labeled macro and semi micro, 500µl of serum reagent standard was

pipetted into the centrifuge tube labeled ‘macro’. 200µl of serum and reagent standard was

pipette into the centrifuge tube labeled ‘semi micro’. 1000µl of precipitant (R1) was pipette

into the centrifuge tube labeled ‘macro’ and 500µl of diluted precipitant (R1) was pipette

into the centrifuge tube labeled ‘semi micro’. Mix and allow sitting for 10minutes at room

temperature. Then centrifuge for 10minutes at 400rpm, 2minutes at 12000rpm to obtain the

supernatant. Three dry test tubes labeled reagent blank, standard and sample were placed in a

test tube rack; using a micropipette, 100µl of distilled water was pipette into the reagent

blank, 100µl of supernatant (HDL precipitant) was pipette into the sample test tube, another

100µl of standard was pipette into standard test tube and 1000µl of cholesterol reagent was

added to all the test tubes respectively.

The test tubes were mixed thoroughly and incubated for 10minutes at 20-25ºc; the

absorbance of the sample and standard were measured against the reagent blank within

60minutes at 500nm.

Calculation: When using a standard

Test concentration= Absorbance of test x concentration of standard

Absorbance of standard

43
3.9.1 STATISTICAL ANALYSIS

The results were expressed as mean ± S.D. The data were analyzed using the statistical

package for social sciences (SPSS; version20) for windows 7. Independent students t-test

was used to compare means, and values were considered significant at p<0.05 and non-

significant at p>0.05.

44
 CHAPTER FOUR

RESULTS

Results obtained from this study where expressed in mean±standard deviation.

KEY

DAY 1 – Day 13 First day of experiment

DAY 14 – Day 27 Administration of Thiamazole

DAY 28 – Day 41 Administration of Urtica dioica and Juglans nigra extract

DAY 42 – day of sacrifice

p˂0.05 – significant

p˃0.05 – not significant

45
TABLE 4.1: MEAN VALUE OF WEIGHT (g) OF ALL THE ANIMALS TREATED
THROUGHOUT THE EXPERIMENT.

GROUP A GROUP B GROUP C GROUP D GROUP E

DAY 1 159.67±22.80 171.83±15.73 171.5±8.41 158.5±7.87 170.83±12.02

DAY 14 161.0±22.70 174.17±14.96 173.5±8.85 160.5±8.43 172.67±11.69

DAY 28 167.33±24.48 179.67±12.29 188.16±12.9 173.17±8.3 185.33±9.77

DAY 42 171.5±23.40 187.83±11.44 178.5±9.20 162.0±8.1 171.67±20.07

Table 4.1 shows that there was significant increase (P˂0.05) in the weight of all the groups

(167.33±24.48, 179.67±12.29, 188.16±12.9, 173.17±8.3 and 185.33±9.77 respectively) after

28 days. There was also a significant reduction (P˂0.05) in the weight of group C, D and E

(178.5±9.20, 162.0±8.1 and 171.67±20.07 respectively) after 42 days, however group B

significantly increased (P˂0.05) after day 42 (187.83±11.44).

TABLE 4.2: MEAN VALUE OF BODY MASS INDEX, BMI (g/cm2), OF THE ANIMALS
TREATED THROUGHOUT THE EXPERIMENT

46
 GROUP A GROUP B GROUP C GROUP D GROUP E
DAY 1 0.54±0.03 0.55±0.53 0.54±0.05 0.56±0.05 0.53±0.03

DAY 14 0.54±0.03 0.56±0.22 0.54±0.05 0.55±0.05 0.53±0.03

DAY 28 0.54±0.04 0.59±0.02 0.57±0.06 0.59±0.04 0.58±0.04

DAY 42 0.53±0.04 0.60±0.03 0.54±0.05 0.54±0.05 0.51±0.04

Table 4.2 shows that there was a significant increase (P˂0.05) in the BMI of group B, C, D

and E (0.59±0.02, 0.57±0.06, 0.59±0.04 and 0.58±0.04 respectively) after 28 days. Groups

C, D and E however show significant decrease (P˂0.05) of BMI (0.54±0.05, 0.54±0.05 and

0.51±0.04 respectively) after 42 days.

TABLE 4.3: MEAN VALUES OF TOTAL CHOLESTEROL, HIGH DENSITY LIPOPROTIEN AND
TRIGLYCERIDE CONCENTRATION (mg/dl) OF ALL THE ANIMALS TREATED THROUGHOUT THE
EXPERIMENT

GROUP A GROUP B GROUP C GROUP D GROUP E P-VALUE

TOTAL

47
CHOLESTEROL 40.0±1.59 99.14±9.90 67.26±33.01 34.00±7.90 59.99±37.86 P˂0.05

HIGH DENSITY
63.69±40.7 95.86±21.6 42.44±14.05 24.41±8.30 33.74±11.29 P˂0.05
LIPOPROTEIN

TRIGLYCERIDE
64.64±9.69 87.10±15.82 54.47±5.68 98.90±24.09 58.29±16.61 P˂0.05

Table 4.3 shows that the total cholesterol level was found to be significantly high (P˂0.05),

in group B (99.14±9.90) when compared with group C, D and E (67.26±33.01, 34.00±7.90

and 59.99±37.86 respectively). High density lipoprotein (HDL) level was also found to be

significantly high (P˂0.05) in group B (95.86±21.6), when compared with groups A, C, D

and E (63.69±40.7, 42.44±14.05, 24.41±8.30 and 33.74±11.29 respectively). Triglyceride

level was also significantly high (P˂0.05) in group B and D (87.10±15.82 and 98.90±24.09

respectively), when compared with group A, C and E (64.64±9.69, 54.47±5.68 and

58.29±16.61 respectively).

4.3 Multiple comparism of the levels of Total Cholesterol, HDL, Triglyceride and BMI of
all the groups.

TOTAL HDL TRIGLYCERIDE BMI

CHOLESTEROL

GAVsGB* GAVsGB GAVsGB GAVsGB*

48
 GCVsGB GCVsGB* GCVsGB* GCVsGB*

GDVsGB* GDVsGB* GDVsGB GDVsGB*

GEVsGB GEVsGB* GEVsGB GEVsGB*

KEY

GAVsGB – Group A Vs Group B

GCVsGB – Group C Vs Group B

GDVsGB – Group D Vs Group B

GEVsGB – Group E Vs Group B

* INDICATES SIGNIFICANT DIFFERENCE (P˂0.05)

Table 4.4 shows that the total cholesterol level of group B (99.14±9.90) is significantly

higher (P˂0.05) than that of group A (40.0±1.59) and group D (34.00±7.90). High density

lipoprotein level of group B (95.86±21.6) is significantly higher (P˂0.05) than that of C, D,

and E (42.44±14.05, 24.41±8.30 and 33.74±11.29 respectively). Triglyceride however shows

significant difference between group B and group C (87.10±15.82 and 54.47±5.68

respectively). BMI of group B (0.60±0.03) was also significantly higher (P˂0.05) than that

of all other groups (0.53±0.04, 0.54±0.05, 0.54±0.05 and 0.51±0.04 respectively)

49
 CHAPTER FIVE

DISCUSSION AND CONCLUSION

5.1 DISCUSSION

This study was done to confirm if hypothyroidism can increase the levels of lipid

profile and to determine the effect of black walnut and nettle leaf on obesity induced by

hypothyroidism.

Result showed that the total cholesterol, high density lipoprotein, triglyceride and

BMI levels of the hypothyroid animals (99.14±9.90, 95.86±21.6, 87.10±15.82 and 0.60±0.03
50
respectively) was found to be significantly higher ( P˂0.05) than that of the normal animals

(40.0±1.59, 63.69±40.7, 64.64±9.69 and 0.53±0.04 respectively). Similar results were seen in a

study carried out by Pearce et al, (2008), on the levels of lipid profile in hypothyroid

patients. They found out that there are increased levels of total cholesterol, triglycerides and

HDL in patients with overt and subclinical hypothyroidism.

The result also shows a significant decrease (P˂0.05) of HDL, triglyceride and BMI

levels (42.44±14.05, 54.47±5.68 and 0.54±0.05 respectively) of the animals that were treated

with black walnut when compared to the hypothyroid animals ( 95.86±21.6, 87.10±15.82 and

0.60±0.03 respectively). The result also shows a non significant decrease (p value= 0.362) of

the total cholesterol level of the animals treated with walnut ( 67.26±33.01), when compared

with the hypothyroid animals (99.14±9.90). Marcela and Martinez (2008) found out that there

is high iodine content in black walnut, so this reduction could be as a result of this high

iodine content of black walnut, since Wiseman (1993) reviewed that iodine supplement

reduces lipid profile.

The results also show a significant decrease (P˂0.05) in the levels of total cholesterol,

HDL and BMI (34.00±7.90, 24.41±8.30 and 0.54±0.05 respectively) of the animals treated

with nettle leaf when compared with the hypothyroid animals ( 99.14±9.90, 95.86±21.6 and

0.60±0.03 respectively). This is in line with the work carried out by Daher CF et al, (2006),

they discovered significant decrease in total cholesterol, HDL and triglyceride level after

daily administration of aqueous extract of Urtica dioica (nettle leaf).

51
 This study also shows significant reduction (P˂0.05) of HDL and BMI levels

(33.74±11.29 and 0.51±0.04 respectively) of the animals that was treated with both black

walnut and nettle leaf, when compared with the hypothyroid group (95.86±21.6 and

0.60±0.03 respectively). It also shows a non significant decrease (p˃0.05) of total cholesterol

and triglyceride levels (59.99±37.86 and 58.29±16.61 respectively) of the animals that was

treated with both black walnut and nettle leaf, when compared with the hypothyroid group

(99.14±9.90 and 87.10±15.82 respectively).

These reductions in lipid profile and BMI could be as a result of the high iodine

content of black walnut and nettle leaf, as reviewed by Marcela and Martinez (2008). This

increase in iodine concentration will therefore increase the synthesis of thyroid hormones,

which will increase the body’s metabolic rate, thereby reducing the lipid profile level and

BMI.

5.2 CONCLUSION

Hypothyroidism induced by Thiamazole significantly increased the levels of lipid

profile and body mass index (BMI), leading to obesity. Black walnut significantly decreased

the levels of triglyceride, HDL and BMI, while nettle leaf significantly reduced the levels of

total cholesterol, HDL and BMI. However both black walnut (juglans nigra) and nettle leaf

(urtica dioica) decreased the levels of lipid profile and BMI, thereby reducing obesity.

52
5.3 RECOMMENDATION

Black walnut and nettle leaf can be recomme Obese and hypothyroid patients can be

taking as a therapy to increase the synthesis of thyroid hormones, which will then increase

their body’s metabolic rate. I also recommend the screening for hypothyroidism as part of

the screening tests for obese patients.

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63
 APPENDIX I

REAGENTS

1. REAGENTS

The reagents were provided by Randox Laboratory. Initial concentrations of solutions

were used in this research work.

 Cholesterol Reagent from Randox Laboratory, UK.

Contents Initial concentrations of solutions

Enzyme reagent
4-Aminoantipyrine 0.03mmol/l
Phenol 6mmol/l

64
Peroxidase ≥0.5m/m
Cholesterol esterase ≥0.15m/ml
Cholesterol oxidase ≥0.1m/ml
Standard 5.17mmol/l
Pipes buffer 100mmol/l: ph=6.8

 Triglyceride Reagent from Randox Laboratory, UK.

Contents Concentration on the test

Buffer
Pipes buffer 40mmol/l, ph 7.6
4-Chloro-phenol 5.5mmol/l
Magnesium 17.5mmol/l
Enzyme reagent
4-aminophenazone 0.5mmol/l
ATP 1.0mmol/l
Lipase ≥150μ/ml
Glycerol kinases ≥0.04μ/l
Glycerol-3-phosphate oxidase ≥1.5μ/l
Peroxidase ≥0.5μ/l

65
 Standard 2.29mmol/l

 Preparation of Working Reagent

15ml of R1a was added to R1b.

 High Density Lipoprotein Reagent from Randox Laboratory, UK.

Contents Initial concentration of solution

Phosphotungstic 0.55mmol/l
Magnesium 25mmol/l

 Preparation of Working Reagent

1. 20ml of distilled water was added to 80ml of HDL precipitant.

2. The prepared reagent was used up within 5 days and was stored at 2- 8°C.

2. Raw data

TABLE 1: ABSORBANCE AND CONCENTRATION OF TOTAL CHOLESTEROL

ABSORBANCE CONCENTRATION
Mg/dl
GROUP A
1 0.049 41.81
2 0.047 40.11
3 0.048 40.97
4 0.045 38.41
5 0.046 39.26
6 0.050 42.67
GROUP B
1 0.115 98.15
2 0.106 90.47
3 0.101 86.20
4 0.127 108.39
5 0.117 99.85
6 0.131 111.80
GROUP C

66
 1 0.062 53.77
2 0.070 59.74
3 0.019 36.22
4 0.103 87.91
5 0.145 123.75
6 0.026 42.18
GROUP D
1 0.043 36.70
2 0.035 29.87
3 0.026 22.19
4 0.040 34.14
5 0.041 34.99
6 0.054 46.09
GROUP E
1 0.007 5.853
2 0.022 18.78
3 0.106 90.47
4 0.101 86.07
5 0.082 69.98
6 0.104 88.76
Absorbance of standard—0.232
Concentration of standard—198Mg/dl

TABLE 2: ABSORBANCE AND CONCENTRATION OF HIGH DENSITY LIPOPROTEIN

ABSORBANCE CONCENTRATIO
N
Mg/dl
GROUP A
1 0.085 47.14
2 0.053 29.39
3 0.023 12.75
4 0.135 74.87
5 0.200 110.92
6 0.193 107.04
GROUP B
1 0.230 127.56
2 0.172 95.39
3 0.200 110.92
4 0.154 85.41
5 0.117 64.89
6 0.164 90.96
GROUP C
1 0.106 58.79

67
 2 0.106 58.79
3 0.052 28.84
4 0.012 26.66
5 0.078 43.26
6 0.062 38.27
GROUP D
1 0.045 24.96
2 0.047 26.10
3 0.022 12.20
4 0.065 36.05
5 0.052 28.84
6 0.033 18.30
GROUP E
1 0.037 20.52
2 0.051 28.29
3 0.092 51.03
4 0.073 40.49
5 0.045 24.96
6 0.067 37.16
Absorbance of standard—0.357
Concentration of standard—198mg/dl

TABLE 3: ABSORBANCE AND CONCENTRATION OF TRIGLYCERIDE

ABSORBANCE CONCENTRATION
Mg/dl
GROUP A
1 0.063 75.98
2 0.047 56.68
3 0.064 77.19
4 0.052 62.72
5 0.045 54.62
6 0.050 60.65
GROUP B
1 0.085 102.52
2 0.062 72.36
3 0.060 72.72
4 0.065 78.39
5 0.091 109.75
6 0.072 86.84

68
 GROUP C
1 0.050 60.30
2 0.039 47.04
3 0.043 51.86
4 0.046 55.48
5 0.042 50.65
6 0.051 61.51
GROUP D
1 0.110 132.67
2 0.082 98.89
3 0.067 80.81
4 0.061 73.57
5 0.102 123.02
6 0.070 84.42
GROUP E
1 0.031 37.39
2 0.042 50.65
3 0.060 72.36
4 0.066 79.60
5 0.037 44.62
6 0.054 65.13
Absorbance of standard—0.165
Concentration of standard—199mg/dl

TABLE 4: BODY MASS INDEX

WEIGHT HEIGHT HEIGHT2 BODY MASS

(g) (cm) (cm2) INDEX (BMI)
(g/cm2)
GROUP A
1 152 16.4 268.96 0.56
2 154 16.6 275.56 0.55
3 214 21.4 457.96 0.47
4 198 19.2 368.64 0.53
5 178 17.8 316.84 0.56
6 165 16.9 285.61 0.58
GROUP B
1 196 18.9 357.21 0.55
2 148 16.8 282.24 0.52
3 189 17.8 316.84 0.60
4 182 17.5 306.25 0.59
5 195 18.2 331.24 0.59

69
 6 195 18.1 327.61 0.60
GROUP C
1 182 18.4 338.56 0.54
2 168 19.3 372.49 0.45
3 192 18.0 324.0 0.59
4 181 18.3 334.89 0.54
5 173 18.0 324.0 0.53
6 180 17.5 306.25 0.58
GROUP D
1 134 17.8 316.84 0.42
2 156 17.8 316.84 0.49
3 174 17.0 289.0 0.61
4 170 17.6 309.76 0.55
5 168 16.5 272.25 0.62
6 160 17.2 295.84 0.54
GROUP E
1 190 18.7 349.69 0.54
2 134 17.0 289.0 0.46
3 176 18.6 345.96 0.50
4 187 18.4 338.56 0.55
5 173 18.0 324.0 0.53
6 170 19.0 361.0 0.47

APPENDIX 11

Oneway
[DataSet2]

Descriptives

N Mean Std. Std. Error 95% Confidence Interval for Minimum Maxim
Deviation Mean um

Lower Bound Upper Bound

TOTAL_CHOL. CONTROL 6 40.5383 1.59342 .65051 38.8661 42.2105 38.41 42.67

HYPOTHYROID 6 99.1433 9.89884 4.04118 88.7551 109.5315 86.20 111.80

HYPOTHYROID 6 67.2617 33.01079 13.47660 32.6190 101.9044 36.22 123.75

ET WALNUT

HYPOTHYROID 6 33.9967 7.88658 3.21968 25.7202 42.2731 22.19 46.09

ET NETTLE

70
 HYPOTHYROID 6 59.9850 37.85793 15.45543 20.2555 99.7145 5.85 90.47
ET WALNUT ET
NETTLE

Total 30 60.1850 31.76771 5.79996 48.3227 72.0473 5.85 123.75

CONTROL 6 63.6850 40.69438 16.61341 20.9789 106.3911 12.75 110.92
HYPOTHYROID 6 95.8550 21.56046 8.80202 73.2287 118.4813 64.89 127.56
HYPOTHYROID 6 42.4350 14.04759 5.73491 27.6930 57.1770 26.66 58.79
ET WALNUT
HYPOTHYROID 6 24.4083 8.30288 3.38964 15.6950 33.1217 12.20 36.05
HDL_C
ET NETTLE
HYPOTHYROID 6 33.7417 11.29391 4.61072 21.8894 45.5939 20.52 51.03
ET WALNUT ET
NETTLE
Total 30 52.0250 33.24376 6.06945 39.6116 64.4384 12.20 127.56
CONTROL 6 64.6400 9.68986 3.95587 54.4711 74.8089 54.62 77.19

HYPOTHYROID 6 87.0967 15.81634 6.45699 70.4984 103.6949 72.36 109.75

HYPOTHYROID 6 54.4733 5.67767 2.31790 48.5150 60.4317 47.04 61.51

ET WALNUT

HYPOTHYROID 6 98.8967 24.08616 9.83313 73.6198 124.1735 73.57 132.67

TG
ET NETTLE

HYPOTHYROID 6 58.2917 16.61875 6.78458 40.8514 75.7320 37.39 79.60

ET WALNUT ET
NETTLE

Total 30 72.6797 22.85854 4.17338 64.1441 81.2152 37.39 132.67

ANOVA

Sum of Squares Df Mean Square F Sig.

Between Groups 15838.148 4 3959.537 7.372 .000

TOTAL_CHOL. Within Groups 13428.295 25 537.132

Total 29266.442 29
Between Groups 19475.719 4 4868.930 9.681 .000
HDL_C Within Groups 12573.558 25 502.942
Total 32049.277 29
TG Between Groups 8989.814 4 2247.454 9.117 .000

71
 Within Groups 6163.059 25 246.522

Total 15152.873 29

Post Hoc Tests

Multiple Comparisons
Dunnett T3

Dependent (I) ID (J) ID Mean Std. Sig. 95% Confidence

Variable Difference Error Interval
(I-J) Lower Upper
Bound Bound

TOTAL_CHOL. CONTROL HYPOTHYROID -58.60500 *

4.09320 .000 -75.8752 -41.3348

HYPOTHYROID -26.72333 13.4922 .499 -84.7785 31.3318

ET WALNUT 9

HYPOTHYROID 6.54167 3.28474 .489 -7.1593 20.2427

ET NETTLE

HYPOTHYROID -19.44667 15.4691 .861 -86.0403 47.1469

ET WALNUT ET 2
NETTLE

72
 CONTROL 58.60500* 4.09320 .000 41.3348 75.8752

HYPOTHYROID 31.88167 14.0694 .362 -24.8807 88.6440

ET WALNUT 7

HYPOTHYROID 65.14667 *
5.16696 .000 47.0365 83.2568
HYPOTHYROID
ET NETTLE

HYPOTHYROID 39.15833 15.9750 .299 -26.1895 104.5061

ET WALNUT ET 3
NETTLE

26.72333 13.4922 .499 -31.3318 84.7785

CONTROL
9

-31.88167 14.0694 .362 -88.6440 24.8807

HYPOTHYROID
7
HYPOTHYROID ET WALNUT HYPOTHYROID 33.26500 13.8558 .318 -23.8539 90.3839
ET NETTLE 7

HYPOTHYROID 7.27667 20.5058 1.000 -64.1106 78.6640

ET WALNUT ET 3
NETTLE

CONTROL -6.54167 3.28474 .489 -20.2427 7.1593

HYPOTHYROID -65.14667 *
5.16696 .000 -83.2568 -47.0365
HYPOTHYROID -33.26500 13.8558 .318 -90.3839 23.8539
HYPOTHYROID ET NETTLE ET WALNUT 7
HYPOTHYROID -25.98833 15.7872 .666 -91.7126 39.7359
ET WALNUT ET 4
NETTLE
19.44667 15.4691 .861 -47.1469 86.0403
CONTROL
2
-39.15833 15.9750 .299 - 26.1895
HYPOTHYROID
HYPOTHYROID ET WALNUT 3 104.5061
ET NETTLE HYPOTHYROID -7.27667 20.5058 1.000 -78.6640 64.1106
ET WALNUT 3
HYPOTHYROID 25.98833 15.7872 .666 -39.7359 91.7126
ET NETTLE 4
HDL_C CONTROL -32.17000 18.8010 .624 - 37.6278
HYPOTHYROID
9 101.9678
HYPOTHYROID 21.25000 17.5754 .887 -48.4618 90.9618
ET WALNUT 0
HYPOTHYROID 39.27667 16.9556 .351 -31.4047 109.9580
ET NETTLE 8

73
 HYPOTHYROID 29.94333 17.2413 .616 -40.1840 100.0707
ET WALNUT ET 5
NETTLE
32.17000 18.8010 .624 -37.6278 101.9678
CONTROL
9
HYPOTHYROID 53.42000 *
10.5054 .006 15.6851 91.1549
ET WALNUT 6
HYPOTHYROID HYPOTHYROID 71.44667 *
9.43214 .002 34.5963 108.2970
ET NETTLE
HYPOTHYROID 62.11333* 9.93652 .003 25.1546 99.0721
ET WALNUT ET
NETTLE
-21.25000 17.5754 .887 -90.9618 48.4618
CONTROL
0
-53.42000 *
10.5054 .006 -91.1549 -15.6851
HYPOTHYROID
6
HYPOTHYROID ET WALNUT HYPOTHYROID 18.02667 6.66174 .185 -6.2638 42.3171
ET NETTLE
HYPOTHYROID 8.69333 7.35853 .907 -17.0772 34.4638
ET WALNUT ET
NETTLE
-39.27667 16.9556 .351 - 31.4047
CONTROL
8 109.9580
-71.44667 *
9.43214 .002 - -34.5963
HYPOTHYROID
108.2970
HYPOTHYROID ET NETTLE HYPOTHYROID -18.02667 6.66174 .185 -42.3171 6.2638
ET WALNUT
HYPOTHYROID -9.33333 5.72262 .669 -29.5612 10.8945
ET WALNUT ET
NETTLE
-29.94333 17.2413 .616 - 40.1840
CONTROL
5 100.0707
HYPOTHYROID -62.11333 *
9.93652 .003 -99.0721 -25.1546
HYPOTHYROID ET WALNUT
HYPOTHYROID -8.69333 7.35853 .907 -34.4638 17.0772
ET NETTLE
ET WALNUT
HYPOTHYROID 9.33333 5.72262 .669 -10.8945 29.5612
ET NETTLE
HYPOTHYROID -22.45667 7.57243 .127 -49.9112 4.9979

HYPOTHYROID 10.16667 4.58493 .353 -6.5745 26.9079

TG CONTROL ET WALNUT

HYPOTHYROID -34.25667 10.5990 .109 -75.3986 6.8853

74
 HYPOTHYROID 6.34833 7.85362 .989 -22.3508 35.0475
ET WALNUT ET
NETTLE

CONTROL 22.45667 7.57243 .127 -4.9979 49.9112

HYPOTHYROID 32.62333 *
6.86042 .021 5.5537 59.6930
ET WALNUT

HYPOTHYROID -11.80000 11.7636 .960 -54.0061 30.4061

HYPOTHYROID
ET NETTLE 4

HYPOTHYROID 28.80500 9.36607 .093 -3.6882 61.2982

ET WALNUT ET
NETTLE

CONTROL -10.16667 4.58493 .353 -26.9079 6.5745

HYPOTHYROID -32.62333 *
6.86042 .021 -59.6930 -5.5537

HYPOTHYROID -44.42333* 10.1026 .038 -86.1142 -2.7324

HYPOTHYROID ET WALNUT ET NETTLE 3

HYPOTHYROID -3.81833 7.16960 .999 -32.2945 24.6578

ET WALNUT ET
NETTLE

34.25667 10.5990 .109 -6.8853 75.3986

CONTROL
3

11.80000 11.7636 .960 -30.4061 54.0061

HYPOTHYROID
4
HYPOTHYROID ET NETTLE HYPOTHYROID 44.42333* 10.1026 .038 2.7324 86.1142
ET WALNUT 3

HYPOTHYROID 40.60500 11.9465 .064 -1.9621 83.1721

ET WALNUT ET 9
NETTLE

CONTROL -6.34833 7.85362 .989 -35.0475 22.3508

HYPOTHYROID -28.80500 9.36607 .093 -61.2982 3.6882

HYPOTHYROID ET WALNUT HYPOTHYROID 3.81833 7.16960 .999 -24.6578 32.2945
ET NETTLE ET WALNUT

HYPOTHYROID -40.60500 11.9465 .064 -83.1721 1.9621

ET NETTLE 9

*. The mean difference is significant at the 0.05 level.

75
T-Test

Group Statistics

ID N Mean Std. Deviation Std. Error Mean

CONTROL 6 40.5383 1.59342 .65051

TOTAL_CHOL. HYPOTHYROID ET 6 67.2617 33.01079 13.47660
WALNUT
CONTROL 6 63.6850 40.69438 16.61341
HDL_C HYPOTHYROID ET 6 42.4350 14.04759 5.73491
WALNUT
CONTROL 6 64.6400 9.68986 3.95587
TG HYPOTHYROID ET 6 54.4733 5.67767 2.31790
WALNUT

ONEWAY BMI BY ID
/STATISTICS DESCRIPTIVES
/PLOT MEANS
/MISSING ANALYSIS
/POSTHOC=T3 ALPHA(0.05).

Oneway

Descriptives

BMI

N Mean Std. Std. 95% Confidence Interval for Minimum Maximum

Deviation Error Mean

76
 Lower Bound Upper Bound

CONTROL 6 .5417 .03869 .01579 .5011 .5823 .47 .58

HYPOTHYROID 6 .5750 .03271 .01335 .5407 .6093 .52 .60
HYPOTHYROID ET WALNUT 6 .5383 .04956 .02023 .4863 .5903 .45 .59
HYPOTHYROID ET NETTLE 6 .5383 .07521 .03070 .4594 .6173 .42 .62
HYPOTHYROID ET WALNUT 6 .5083 .03764 .01537 .4688 .5478 .46 .55
ET NETTLE
Total 30 .5403 .05048 .00922 .5215 .5592 .42 .62

ANOVA
BMI

Sum of Squares df Mean Square F Sig.

Between Groups .013 4 .003 1.386 .267

Within Groups .060 25 .002
Total .074 29

Post Hoc Tests

Multiple Comparisons
Dependent Variable: BMI
Dunnett T3

(I) ID (J) ID Mean Std. Error Sig. 95% Confidence Interval

Difference Lower Bound Upper
(I-J) Bound

HYPOTHYROID -.03333 .02068 0.048 -.1055 .0388

HYPOTHYROID ET WALNUT .00333 .02567 1.000 -.0868 .0935

CONTROL HYPOTHYROID ET NETTLE .00333 .03453 1.000 -.1255 .1322

HYPOTHYROID ET WALNUT .03333 .02204 0.741 -.0431 .1098

ET NETTLE
HYPOTHYROID CONTROL .03333 .02068 0.048 -.0388 .1055

77
 HYPOTHYROID ET WALNUT .03667 .02424 0.740 -.0503 .1236
HYPOTHYROID ET NETTLE .03667 .03348 0.930 -.0917 .1651
HYPOTHYROID ET WALNUT .06667 .02036 0.069 -.0042 .1376
ET NETTLE
CONTROL -.00333 .02567 1.000 -.0935 .0868
HYPOTHYROID -.03667 .02424 0.050 -.1236 .0503
HYPOTHYROID ET WALNUT HYPOTHYROID ET NETTLE .00000 .03677 1.000 -.1319 .1319
HYPOTHYROID ET WALNUT .03000 .02541 0.907 -.0595 .1195
ET NETTLE
CONTROL -.00333 .03453 1.000 -.1322 .1255
HYPOTHYROID -.03667 .03348 0.048 -.1651 .0917
HYPOTHYROID ET NETTLE HYPOTHYROID ET WALNUT .00000 .03677 1.000 -.1319 .1319
HYPOTHYROID ET WALNUT .03000 .03433 0.981 -.0987 .1587
ET NETTLE
CONTROL -.03333 .02204 0.741 -.1098 .0431

HYPOTHYROID ET WALNUT HYPOTHYROID -.06667 .02036 0.049 -.1376 .0042

ET NETTLE HYPOTHYROID ET WALNUT -.03000 .02541 0.907 -.1195 .0595

HYPOTHYROID ET NETTLE -.03000 .03433 0.981 -.1587 .0987

78