Research paper - Chemistry
KETOGENIC DIET : EFFECT ON THE KIDNEY AND LIVER
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Low-carbohydrate ketogenic diets, glucose homeostasis, and
nonalcoholic fatty liver disease
Rebecca C. Schugar and Peter A. Crawford1
Department of Medicine Center for Cardiovascular Research Washington University St. Louis,
MO 63110 USA
Abstract
Purpose of review—Obesity-associated nonalcoholic fatty liver disease (NAFLD) is highly
prevalent, for which weight loss is the generally recommended clinical management. Low-
carbohydrate ketogenic diets have been successful in promoting weight loss, but variations in the
range of metabolic responses to these diets indicate that the effects of altering macronutrient
content are not completely understood. This review focuses on the most recent findings that reveal
the relationship between low-carbohydrate diets and NAFLD in rodent models and humans.
Recent findings—Low-carbohydrate diets have been shown to promote weight loss, decrease
intrahepatic triglyceride content, and improve metabolic parameters of patients with obesity.
These ketogenic diets also provoke weight loss in rodents. However, long-term maintenance on a
ketogenic diet stimulates the development of NAFLD and systemic glucose intolerance in mice.
The relationship between ketogenic diets and systemic insulin resistance in both humans and
rodents remains to be elucidated.
Summary—Because low-carbohydrate ketogenic diets are increasingly employed for treatment
of obesity, NAFLD, and neurological diseases such as epilepsy, understanding the long-term
systemic effects of low-carbohydrate diets is crucial to the development of efficacious and safe
dietary interventions.
Keywords
insulin resistance; ketogenesis; ketolysis; tricarboxylic acid cycle; methionine-choline deficient
diets
Introduction
The incidence of cardiovascular disease attributable to obesity, insulin resistance, and
diabetes has markedly increased [1, 2*]. Insulin resistance is highly correlated with ectopic
lipid accumulation, particularly in the liver. Consequently, the pathogeneses of systemic
insulin resistance and diabetes have been linked to nonalcoholic fatty liver disease
(NAFLD). NAFLD is an independent predictor of cardiovascular disease – a stronger
predictor than peripheral or visceral fat mass [3–4, 5*, 6*]. A critical, but as yet only
preliminarily defined influence over the development of NAFLD is distribution of
macronutrient classes within the diet. Recently, attention has focused on the use of low-
carbohydrate diets and their efficacy in controlling metabolic diseases including NAFLD
[7]. However, while low-carbohydrate diets are effective for weight loss, seizure disorders,
1To whom correspondence should be addressed Peter A. Crawford, MD, PhD Department of Medicine Division of Cardiology
Washington University School of Medicine Campus Box 8086 660 S. Euclid Ave. St. Louis, MO 63110 USA Tel 314-747-3009 eFax
314-219-4589 [email protected]
NIH Public Access
Author Manuscript
Curr Opin Clin Nutr Metab Care. Author manuscript; available in PMC 2013 July 01.
Published in final edited form as:
Curr Opin Clin Nutr Metab Care. 2012 July ; 15(4): 374–380. doi:10.1097/MCO.0b013e3283547157.
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and potentially a host of other neurological diseases, determination of their relationships
with metabolic responses, and fatty liver in particular, remains ongoing. It is because
therapeutic application of low-carbohydrate diets is likely going to increase that further
understanding of the range of metabolic responses observed, and the precise nutritional
determinants of these responses, is so timely. The importance of further understanding the
impact of low-carbohydrate diets is underscored by case reports of humans that reveal
variations in the range of metabolic responses to these diets [8, 9]. This review examines the
metabolic responses of rodents and humans to low-carbohydrate diets, and will elucidate
critical unanswered questions that merit follow-up pre-clinical and clinical evaluation.
NAFLD: epidemiologic and metabolic considerations
The earliest stage of NAFLD is hepatic steatosis, which is defined by intrahepatic
triglyceride (IHTG) concentrations exceeding 55 mg/g liver (5.5\%), or when greater than
5\% of hepatocytes harbor histological evidence of triglyceride storage [6*, 10]. NAFLD
prevalence is 15\% in non-obese patients, but increases in obese (BMI=30.0–39.9 kg/m2) and
extremely obese (BMI≥40.0 kg/m2) patients to 65\% and 85\%, respectively. In addition to its
association with systemic insulin resistance and adverse cardiovascular outcomes, NAFLD
can progress to nonalcoholic steatohepatitis (NASH), which is characterized by steatosis and
signs of hepatocyte injury, hepatic inflammation with collagen deposition, and elevation of
serum alanine aminotransferase (ALT). Approximately 10–29\% of patients with NASH will
develop cirrhosis within 10 years, which can evolve to hepatocellular carcinoma.
Informative reviews on the causes, stages, and implications of NAFLD have been published
recently, but whether NASH can develop independently of a NAFLD stage remains to be
fully explored [6*, 10, 11*].
Increased IHTG is caused by imbalance between hepatocellular triglyceride formation and
removal. Therefore, multiple hepatocyte pathways play roles, including rate of fatty acid
uptake; esterification of fatty acids into, and lipolysis from, intracellular triacylglycerols
(TAGs); rate of fatty acid oxidation (FAO); rate of TAG secretion as very low-density
lipoproteins (VLDL); and de novo lipogenesis (DNL). Hepatic TAGs are synthesized from
fatty acids that emanate from (i) DNL, (ii) lipolysis of triglycerides stored in adipose tissue,
and/or (iii) from diet-derived fats that are packaged as chylomicrons in intestinal
enterocytes. A significant contributor to increased hepatic triglyceride content in NAFLD is
increased DNL, in which increased hepatocellular carbohydrate is converted to fat. Muscle,
and possibly adipocyte, insulin resistance may contribute to increased hepatocyte
carbohydrate by diversion of glucose to the liver. In addition, magnetic resonance
spectroscopy (MRS) of human livers recently revealed that patients with NAFLD exhibit
augmented delivery of fatty acids, through increased peripheral lipolysis; elevated
hepatocellular flux through the tricarboxylic acid (TCA) cycle; and increased
gluconeogenesis, compared to NAFLD-free subjects [12**].
Classical diet models of NAFLD
A detailed review of rodent dietary models of NAFLD has recently been published [13*].
High-carbohydrate/low-fat, low-carbohydrate/high-fat, and high-carbohydrate/high-fat
formulations have all been studied, as have the contributions of trans, monounsaturated,
polyunsaturated, and chain length of fatty acids [14–18]. The role of nutritional methionine
and choline contents are also important, because seminal rodent experiments have revealed
molecular underpinnings of NAFLD pathogenesis using methionine and choline deficient
(MCD) diets. MCD diets commonly include high sucrose and fat contents, producing
exuberant and rapid (within two weeks) NASH, particularly when carbohydrate content is
highly enriched as either glucose or the particularly lipogenic monosaccharide fructose, as
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opposed to starch [19*, 20*, 21]. Despite robust IHTG and inflammation, MCD models do
not exhibit systemic insulin resistance, and MCD diets induce a catabolic state that includes
muscle wasting.
Choline is a major constituent of plasma and mitochondrial membranes, as well as the
neurotransmitter acetylcholine, and is an essential nutrient that is particularly abundant in
animal proteins. The majority of choline is metabolized via either phosphorylation, to supply
phospholipids for membrane synthesis, or oxidized and ultimately metabolized to S-
adenosyl-L-methionine (SAM), a universal methyl donor [22]. The former pathway has been
cited to support the notion that choline deficiency contributes to NAFLD by reducing VLDL
packaging and secretion. However, choline deficiency also contributes to mitochondrial
dysfunction, and therefore to FAO deficiencies, and has also been linked to increased fatty
acid uptake. In a study conducted to understand the contributions of methionine and choline
deficiency independently, mice were place on a conventional MCD diet, versus methionine
deficient (MD) or choline deficient (CD) diets. Over two weeks, the MD diet reproduced
many of the deleterious effects of the MCD diet including weight loss, hepatocellular injury,
decreased mitochondrial SAM and glutathione, inflammation and fibrosis, whereas choline
deficiency caused only steatosis [23*].
Ketone metabolism and low-carbohydrate (ketogenic) diets
The liver is a major destination for fatty acids derived from lipolysis of adipose stores and
from TAG-derived lipoproteins. Hepatic FAO converts fatty acids to acetyl-CoA, which
condenses with oxaloacetate in the TCA cycle, whose electrons are used in the electron
transport chain to generate high-energy phosphates. During states of high fatty acid
mitochondrial delivery, much FAO-derived acetyl-CoA is diverted from the TCA cycle to
ketogenesis, generating acetoacetate (AcAc) and β-hydroxybutyrate (βOHB). Ketogenesis is
particularly stimulated during low insulin states [24*]. Liver abundantly expresses the key
ketogenic enzyme, mitochondrial HMG-CoA synthase, which is activated by Sirtuin 3-
mediated deacetylation, and whose gene HMGCS2 is negatively regulated through
transcriptional mechanisms downstream of insulin signaling [25, 26]. Because hepatic
mitochondria lack the enzyme necessary for oxidizing ketone bodies, ketones are secreted
and delivered to heart, skeletal muscle and brain, which abundantly express the
mitochondrial matrix enzyme succinyl-CoA:3-oxoacid-CoA transferase (SCOT, encoded by
OXCT1), which is required to convert ketone bodies to acetyl-CoA for terminal oxidation in
the TCA cycle [27]. Thus, hepatic ketogenesis helps maintain TCA cycle homeostasis,
prevents the accumulation of incompletely oxidized fatty acid intermediates, maintains
hepatic redox balance, and supplies extrahepatic organs with energy substrates in glucose-
limiting states that include fasting, poorly-controlled diabetes, and during adherence to low-
carbohydrate, high-fat ketogenic diets (KD). While individuals with NAFLD have also been
reported to exhibit higher circulating ketone concentrations, likely due to increased rates of
FAO, βOHB turnover rates are not increased in individuals with NAFLD [12**]. Increased
TCA flux in NAFLD livers may partially explain normal βOHB turnover rates, but
prospective roles for variation of ketolytic flux through SCOT, and of AcAc turnover,
remain to be determined.
Ketogenic diets are actively used for weight loss and anticonvulsant therapy, and are
intensively studied as potential adjunctive therapy for brain cancers and neurodegenerative
diseases including Parkinsons and Alzheimers [28–32]. In humans, diets with caloric
contents of up to 75–80\% fat and ≥15\% protein are commonly used for management of
seizure disorders (e.g., 4:1 prescriptions, in which calories are derived from four parts fat,
and one part protein + carbohydrate), and Atkins diets for weight loss typically consist of
60–70\% fat and up to 30\% protein. Anticonvulsant benefits of KDs persist for ≥6 years in
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patients following discontinuation [33]. While the mechanism(s) of KDs salutary
neurological effects have not been fully elucidated, observations from a number of genetic
and acquired epilepsy models in rodents have implicated a dysregulation of the mammalian
target of rapamycin (mTOR) pathway, neuron-astrocyte cross-talk in neurotransmitter
metabolism, and manipulation of hypothalamic hormone signaling [32–36].
Not all patients can tolerate KD regimens, which can be unpalatable, and scrupulous
attention to micronutrient content and overall caloric needs is required. Hyperuricemia,
hypocalcemia, hypomagnesemia, nephrolithiasis, and of course, ketoacidosis can result.
Rare reports implicate the KD in cardiac complications, including cardiomyopathy,
prolonged QTc interval and torsades de pointes arrhythmias [8]. However, this response
appears to be sporadic, as a systemic analysis of 27 children adhering to prescribed KD for
treatment of refractory epilepsy revealed no changes in QTc interval over 12 months [37].
Nonetheless, these data underscore the importance of careful patient monitoring, and
perhaps expansion of monitoring guidelines.
Ketogenic diets and NAFLD in humans and rodents
Promotion of weight loss is often recommended to obese patients with hepatic steatosis, and
is frequently achieved through a reduction of caloric intake. While intensively studied, the
efficacy and safety of shifts in macronutrient content still remain to be determined in a
systematic manner – particularly with respect to effects on NAFLD. A small clinical trial of
18 patients used MRS to measure change in IHTG following two weeks of either a calorie-
restricted diet, or a carbohydrate-restricted diet that was not calorie-restricted, and found that
patients in the carbohydrate-restricted arm exhibited a more profound IHTG reduction,
without any difference in overall weight loss, compared to the calorie restricted arm [38**].
A recent two-year multi-center trial that included over 300 patients enrolled in a
comprehensive lifestyle modification regimen observed similar weight loss achieved by
adherence to low-fat, versus low-carbohydrate diets. The low-carbohydrate diet group
exhibited superior HDL cholesterol profiles [39**]. As expected, urinary ketosis was
markedly more common in the low-carbohydrate group, but the influence of these regimens
on IHTG was not presented. Comparing low-carbohydrate to reduced calorie or low-fat diets
in clinical trials to date, systemic glucose homeostasis has not systematically differed.
Ketogenic diets have been extensively studied in rodents. Using a micronutrient
supplemented KD (Bio-Serv F3666) very high in fat (93.3\%kcal), very low in carbohydrate
(1.8\%), and also reduced in protein (4.7\%), Meratos-Flier and colleagues observed that mice
lose weight, develop ketosis, and induce hepatic gene expression signatures that suggest
reduced DNL and increased FAO [40*]. To explore the relationships among KD, IHTG, and
NAFLD, Garbow et al. maintained C57BL/6J mice for 12 weeks on either (i) Bio-Serv
F3666 KD; (ii) a high-fat (40\%kcal) `Western diet (WD) also enriched in sucrose (40\%); or
(iii) a standard low-fat (13\%kcal) polysaccharide-rich chow control diet [41**]. The KD is
reduced in protein content due to the fact that induction of ketosis in rodents requires
restriction of not only carbohydrates but also protein [42]. Mice fed the KD for 12 weeks
were lean, euglycemic, ketotic, and hypoinsulinemic, but were glucose intolerant, and
exhibited NAFLD. MRS revealed that KD-fed mice accumulate hepatic lipid within 3 weeks
after initiation of the diet, and the hepatic gene expression signature for DNL (encoded
mediators of SREBP-1c, FAS, ACC1, SCD1) was suppressed compared to livers of chow-
fed controls. In contrast, mice fed the WD ultimately accumulate higher IHTG than KD-fed
animals, but do so much more slowly, and as expected due to the high sucrose content,
induce mediators of DNL. Intriguingly, unlike steatotic livers of WD-fed mice, livers of
KD-fed mice developed hepatic endoplasmic reticulum (ER)-stress, inflammation,
macrophage accumulation, and hepatocellular injury, and only KD-fed mice exhibited
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elevated serum ALT concentrations. A number of non-mutually exclusive mechanisms may
account for the murine hepatic phenotypes observed with this KD. First is the prospective
influence of choline and methionine deficiencies. While choline-replete rodent diets are
supplemented to contain ~2.5 g/kg, BioServ F3666 KD is not supplemented, and therefore
contains only 200–300 mg/kg naturally-derived from the fat sources. Methionine content in
this KD, derived from casein, is also reduced, at 2.2 g/kg, whereas methionine-replete diets
contain ~4 g/kg. A second prospective contributor to the NAFLD signatures in KD-fed mice
is overall reduction of protein: classical studies indicate that diets containing <14\% protein
retard normal growth, reproduction, and lactation in mice [43]. A third prospective influence
is cellular injury though ER stress-inducing membrane remodeling in periportal hepatocytes
that receive more fat than can be oxidized or exported via VLDL secretion. Fourth, ceramide
production within macrophages or hepatocytes, favored by high intracellular concentrations
of saturated fatty acids, may also trigger the inflammasome, whose biomarkers were
selectively elevated in livers of KD-fed mice. Finally, splice variants of the insulin-
sensitizing nuclear receptor transcription factor PPARγ may exhibit distinct activities in
different steatotic contexts. While the `adipocyte isoform form, PPARγ2, was induced in
livers by WD, the `macrophage isoform PPARγ1 was selectively induced in livers of KD-
fed mice.
Hepatic fibroblast growth factor 21 (FGF21) has emerged as a key regulator of hepatic
metabolism, glucose, and fatty acid oxidative flux, and insulin sensitivity [44, 45]. KD
feeding of Fgf21−/− mice leads to weight gain, reduced ketosis, and hepatic steatosis relative
to KD-fed wild type controls in two weeks, consistent with impaired ability of the liver to
oxidize fatty acids in the absence of FGF21 [46]. Indeed, serum FGF21 concentrations are
elevated in patients with diagnosed NAFLD, and obesity is an FGF21-resistant state [47,
48]. Fgf21 mRNA is markedly induced in livers of mice fed either KD or WD for 12 weeks
[41**].
Insulin resistance and ketogenic diets in rodentsM
KD-fed mice develop systemic glucose intolerance, and their livers exhibit ER stress,
steatosis, cellular injury, and macrophage accumulation. However, indices of insulin
resistance were not observed [41**]. Compared to chow-fed and WD-fed mice, KD-fed
mice exhibited reduced homeostatic model assessment of insulin resistance (HOMA-IR)
values, normal insulin-induced hepatic and skeletal muscle Akt phosphorylation, and
increased whole-body glucose disposal by insulin tolerance test (ITT). Badman et al. also
observed insulin-sensitizing effects of 7 weeks of this KD on obese leptin-deficient ob/ob
mice: while neither obesity nor IHTG of ob/ob mice was ameliorated by feeding the KD, the
diet did improve glucose intolerance, hyperinsulinema, and glucose disposal by ITT [49]. In
apparent contradistinction, Shulman and colleagues observed a 350\% increase in hepatic
diacylglycerol content, PKCε activation, and decreased insulin signaling at the level of
insulin receptor substrate IRS-2 tyrosine phosphorylation in wild-type mice fed this KD for
5 weeks [50*]. Furthermore, hyperinsulinemic-euglycemic clamp studies revealed
impairment of insulin-mediated suppression of glucose production by livers of KD-fed wild-
type mice. The apparent discordance of these findings by this latter group of investigators
may be explained by two factors. First, liver and muscle may exhibit distinct phenotypes in
KD-fed mice. Although KD-induced hepatic insulin resistance was observed in the clamp
studies, impairment of insulin-stimulated peripheral glucose disposal in KD-fed mice was
much more subtle. Therefore, enhanced systemic response to insulin (by ITT) in KD-fed
mice likely reflects augmentation of insulin-mediated peripheral glucose disposal, relative to
the chow group, that overrides any prospective impairment of insulin-mediated suppression
of hepatic glucose production that may exist in KD-fed mice. Second, hyperinsulinemic-
euglycemic clamp studies in mice have proven controversial, due to variations of (i)
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methods that normalize hepatic glucose output, (ii) insulin dosing regimens among
investigators, and (iii) responses of humans versus anesthetized mice – especially those fed a
KD, which creates an unusual hypoinsulinemic-euglycemic metabolic state, accompanied by
markedly increased IHTG [51*, 52].
Important conclusions emerge from the heretofore performed studies of KD-fed mice. First,
the relevance of the NAFLD signatures in KD-fed mice to human NAFLD pathophysiology
remains to be determined. While it is intriguing that increased IHTG, inflammation, and ER
stress may be dissociable from systemic insulin resistance, this constellation of findings is
not unique in that it has been observed with MCD diets in rodents, which have also been
criticized as less relevant to human NAFLD. Second, the combination of hypoinsulinemia,
euglycemia, glucose intolerance, and enhanced insulin responsiveness by ITT all suggest
that glucose-stimulated insulin secretion by pancreatic β-cells could be abnormal in KD-fed
mice. Finally, while the use of this low-protein, very high-fat, low-carbohydrate diet is not
directly relevant to macronutrient distributions ingested by humans, it does provide an
important foundation to elucidate the range of metabolic responses that occur in states
relevant to human physiology.
Conclusions
KDs are prescribed with increasing frequency for NAFLD, obesity, and neurological
disease, and while they have beneficial attributes, their metabolic effects are not yet
completely understood, and patient responses to these diets can be variable. Recent studies
have provided insight into the contribution of macronutrient content on liver health and
demonstrate the influences of shifting macronutrient class distributions. Therefore, future
studies of low-carbohydrate diets in rodents and humans must take into consideration
additional factors including the effects of low overall protein, choline and methionine
content, plus the saturation and length of dietary fatty acids. Achieving macro- and
micronutrient balance will be essential to developing efficacious diets that promote weight
loss while maintaining systemic health.
Acknowledgments
The authors thank David Cotter and Jessica Flowers (Harlan Teklad) for helpful discussions. This work was
supported in part by grants from the NIH (R01-DK091538) and the Washington University Diabetic Cardiovascular
Disease Center (to P.A.C.) and from the American Heart Association pre-doctoral Midwest affiliate (to R.C.S.).
Funding sources: to P.A.C.: NIH R01-DK091538, and the Washington University Diabetic Cardiovascular Disease
Center
to R.C.S.: American Heart Association pre-doctoral award, Midwest affiliate
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nutrients
Article
Very Low-Calorie Ketogenic Diet: A Safe and
Effective Tool for Weight Loss in Patients with
Obesity and Mild Kidney Failure
Adriano Bruci 1, Dario Tuccinardi 2 , Rossella Tozzi 3 , Angela Balena 3, Silvia Santucci 1,
Riccardo Frontani 1, Stefania Mariani 3, Sabrina Basciani 3, Giovanni Spera 3, Lucio Gnessi 3,
Carla Lubrano 3,† and Mikiko Watanabe 3,*,†
1 Nephrology and Dialysis Unit, Santa Maria alla Gruccia Hospital, 52025 Arezzo, Italy;
[email protected] (A.B.); [email protected] (S.S.); [email protected] (R.F.)
2 Department of Endocrinology and Diabetes, University Campus Bio-Medico of Rome, 00128 Rome, Italy;
[email protected]
3 Department of Experimental Medicine, Section of Medical Pathophysiology, Food Science and
Endocrinology, Sapienza University of Rome, 00161 Rome, Italy; [email protected] (R.T.);
[email protected] (A.B.); [email protected] (S.M.); [email protected] (S.B.);
[email protected] (G.S.); [email protected] (L.G.); [email protected] (C.L.)
* Correspondence: [email protected]it; Tel.: +39-06-499-70716
† These authors contributed equally.
Received: 20 December 2019; Accepted: 23 January 2020; Published: 27 January 2020
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Abstract: Very low-calorie ketogenic diets (VLCKD) are an effective and increasingly used tool for
weight loss. Traditionally considered high protein, ketogenic diets are often looked at with concern
by clinicians due to the potential harm they pose to kidney function. We herein evaluated the efficacy
and safety of a VLCKD in patients with obesity and mild kidney failure. A prospective observational
real-life study was conducted on ninety-two patients following a VLCKD for approximately 3 months.
Thirty-eight had mild kidney failure and fifty-four had no renal condition and were therefore
designated as control. Anthropometric parameters, bioelectrical impedance and biochemistry data
were collected before and at the end of the dietary intervention. The average weight loss was
nearly 20\% of initial weight, with a significant reduction in fat mass. We report an improvement of
metabolic parameters and no clinically relevant variation regarding liver and kidney function. Upon
stratification based on kidney function, no differences in the efficacy and safety outcomes were found.
Interestingly, 27.7\% of patients with mild renal failure reported normalization of glomerular filtrate
after dietary intervention. We conclude that, when conducted under the supervision of healthcare
professionals, a VLCKD is an effective and safe treatment for weight loss in patients with obesity,
including those affected by mild kidney failure.
Keywords: chronic kidney disease; high protein diet; very low-calorie diet; VLCD; VLCKD; very low
energy diet; safety; kidney function; renal function; low carbohydrate diet
1. Introduction
The epidemic of obesity and its comorbidities represents an increasingly worrisome medical
and economic burden according to WHO reports [1]. Several strategies are available for weight loss
and maintenance, such as the modification of lifestyle (diet and physical activity), pharmacology and
surgery [2–9]. Many dietary patterns have been proposed throughout the years, and although several
authors tried to determine what was best, it is now acknowledged that there is no optimal choice for
Nutrients 2020, 12, 333; doi:10.3390/nu12020333 www.mdpi.com/journal/nutrients
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Nutrients 2020, 12, 333 2 of 10
each patient both efficacy- and safety-wise, and the treatment should be tailored to the needs, habits,
and clinical condition [10].
According to the thrifty gene theory, obesity and its complications are due to the change in food
type and availability. In fact, insulin resistance has been linked to the lack of fasting and fullness
succession, leading to a reduced ability to safeguard glucose for the most important functions, such as
cerebral activity and reproduction [11]. Based on this, dietary interventions mimicking fasting periods
have been proposed in order to rescue abilities that were lost throughout the ages. Significantly reduced
dietary carbohydrates (less than 50 g/day) lead to ketones synthesis [12]. Although historically linked
to diabetic acidosis, ketones may be present in small quantities in many physiological conditions,
such as after an overnight fast, subsequent to strenuous physical activity or in response to a protein-rich
meal. Ketone bodies are then utilized as fuel by many extra-hepatic tissues, such as the central nervous
system, skeletal muscle, and the heart [13].
Very Low-Calorie Ketogenic Diets (VLCKDs), dietary interventions falling into the fasting
mimicking ones, are characterized by a very low carbohydrate content (<20 g/daily), 1–1.5 g protein/Kg
ideal body weight, 15–30 g fat/daily and about 500–800 caloric intake/daily [14]. To favor compliance,
VLCKDs are often delivered through meal replacements modelling a Mediterranean diet. Among
the advantages of a VLCKD are the rapid weight loss obtained, satiety induction and muscle mass
preservation, all of these resulting in increased compliance [15]. VLCKDs are currently recommended as
an effective and feasible dietary intervention in subjects with obesity [16]. However, due to the relative
abundance of proteins compared to carbohydrates and fats, VLCKDs are often regarded as possibly
damaging kidney function, and are usually not recommended in subjects with reduced filtration.
A systematic review investigating renal outcomes reported that the kidney seems little affected
by Very Low Calorie Diets, although the assessed studies only included adults with normal kidney
function, and the diets were quite heterogeneous in macronutrient ratio, making results difficult to
interpret [17]. Little evidence is instead available relative to the safety profile in patients with kidney
function impairment, where the only study investigating a VLCKD by enrolling subjects with normal
kidney function together with mild failure did not stratify based on it and therefore reports partial but
promising results regarding renal safety [18]. A recent literature revision reported an improvement in
renal function parameters upon weight loss in diabetic patients [19]. Taken together, available evidence
possibly suggests that a VLCKD, with the profound weight loss usually obtained, might be an effective
tool to manage patients with obesity and mild kidney failure.
We therefore herein evaluated the effect of a VLCKD in terms of weight loss, improvement of
metabolic syndrome markers and safety outcomes in a population with mild kidney failure and healthy
control subjects.
2. Materials and Methods
2.1. Study Design and Population
This was a real life observational prospective study conducted at the High Specialization Centre
for the Care of Obesity, Sapienza University of Rome. Patients with obesity accessing the Centre had
medical history collected, physical exam and laboratory work performed (hematology, biochemistry)
as part of the routine all patients accessing the center undergo for initial evaluation. Those willing
to undergo a VLCKD for weight loss purposes were enrolled, so long as they were in absence of
contraindications according to national guidelines, such as known hypersensitivity to one or more
components used in the meal replacement products; history of cardiac, cerebrovascular, or major
gastrointestinal diseases; psychiatric disturbances; diagnosis of insulin-dependent diabetes mellitus
(IDDM); pregnancy; lactation; CKD with an estimated glomerular filtration rate (eGFR) <60 [16].
Based on renal function, the patients were stratified in two groups: MCKD (Mild Chronic Kidney
Disease) with an eGFR between 60 and 89 mL/min/1.73m2, and NKF (Normal Kidney Function)
subjects, with an eGFR ≥ 90 mL/min/1.73 m2.
Nutrients 2020, 12, 333 3 of 10
The study protocol was approved by the Medical Ethical Committee of Sapienza University
of Rome (ref. CE5475). The study was conducted in accordance with the Declaration of Helsinki
(1964) and subsequent amendments. All participants provided written consent before starting their
participation in the study.
2.2. Diet Protocol
All patients underwent a VLCKD with the use of replacement meals following a protocol consisting
in 5 steps (New Penta, Cuneo, Italy). During the first two steps, net carbohydrate intake was set between
20 and 50 g/day. Ketosis was confirmed every week with the use of commercially available urine reagent
strips (Ketur-Test, Roche Diagnostics, Switzerland). Protein and lipid intake were approximately
1–1.4 gr/kg of ideal body weight/day and 15–30 g/day, respectively. Recommended water intake was
at least 2 lt/day. Total caloric intake was between 450 and 800/day based on calculated ideal body
weight. To avoid micronutrients deficiency, mineral and vitamin supplements were recommended
throughout the dietary intervention as per current guidelines [16]. During the first step, only meal
replacements and a set amount and quality of vegetables were allowed, and during the second step
one meal consisted of a protein dish, with one less replacement meal provided. In the subsequent
phases, caloric intake gradually increased, and a step-by-step carbohydrate reintroduction was carried
out. The mean duration of the whole protocol was 14.9 ± 8.5 weeks, steps 1 and 2 covering the first
half and steps 3 to 5 the second half.
2.3. Anthropometric Parameters
Body weight and height were obtained in fasting subjects wearing light clothing and no shoes
with an empty bladder. The same calibrated scale and stadiometer were used for all patients. Waist
circumference was measured in the same instance at the midpoint between the lower rib margin and
the iliac crest, the patients had their waist uncovered and were asked to stand with their feet close
together and their weight equally distributed on each leg. Systolic and diastolic blood pressure were
measured at baseline and at the end of the intervention.
2.4. Bioelectrical Impedance Analysis
Bioelectrical Impedance Analysis (BIA) was performed to evaluate indices of body composition
(BIO 101 equipment, Akern s.r.l, Pontassieve, Italy): with Fat, Fat Free and Skeletal Muscle Mass (FM,
FFM, and MM, respectively) expressed in kg, and Total Body, Intra Cellular and Extra Cellular Water
(TBW, ECW and ICW, respectively) expressed in lt.
2.5. Biochemistry
Blood samples were collected by venipuncture between 8 a.m. and 10 a.m. after an overnight fast.
Samples were then transferred to the local laboratory and handled according to the local standards of
practice. Insulin, Glucose, Glycosilated Haemoglobin A1C (HbA1C), lipid profile, electrolytes, uric acid,
liver enzymes, and renal function were measured. Glomerular Filtration Rate (GFR) was calculated
with the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation, that performs
better than other equations at higher GFR, with less bias and greater accuracy, especially when actual
GFR is >60 mL/min per 1.73 m2 [20].
Nutrients 2020, 12, 333 4 of 10
2.6. Data Management and Statistical Methods
Data are expressed as mean ± standard deviation (SD) for continuous variables and as percentage
for dichotomous variables. Normality was assessed with the Shapiro–Wilk test and variables were
Log transformed when the distribution was non-normal. Within-group analysis was performed by
dependent sample Student t-test. Between group differences were assessed by general linear mixed
model analysis of end of diet variables of the NKF and MCKD groups. The variables of group, baseline
values, age and gender were included in the model as fixed effects. Differences were considered
statistically significant when p < .05. Statistical analysis was performed using GraphPad Prism Version
6.00 for Windows, GraphPad Software, San Diego California USA and SPSS Statistics for Windows,
Version 25.0, Armonk, NY, USA: IBM Corp.
3. Results
3.1. VLCKD Is Confirmed as a Safe and Effective Tool for Weight Loss and Metabolic Improvement in Subjects
with Obesity.
Ninety-two patients with obesity (23 men and 69 women) were consecutively evaluated. The mean
age was 51.3 ± 12.2 years; the mean BMI was 33.8 ± 5.8 kg/m2. Baseline characteristics are summarized
in Table 1. After the dietary intervention, body weight and BMI were significantly decreased, and BIA
data showed this was predominantly caused by a significant reduction in fat mass, with a minor yet
significant and expected loss in muscle mass. Of note, 1.1\% of patients were deemed as non-responders,
with <5\% weight loss achieved; 11\% reached a weight loss between 5\% and 10\%; the majority of
patients lost 10 to 20\% initial weight (63.7\%), with 24.2\% reaching a weight loss equal to or exceeding
20\%. A reduction in Total, Intra and Extra cellular water was also observed, consistent with the
known diuretic effect of ketogenic diets [21]. Both systolic and diastolic blood pressure improved
over time, and 33.3\% of those on antihypertensive medication had it reduced or discontinued due to
the blood pressure lowering. Glucose metabolism evaluation showed a frank improvement, with a
significant decrease in both fasting glycaemia and HbA1c. Lipid metabolism assessment demonstrated
a significant reduction in total cholesterol and triglycerides levels, and no significant modification in
HDL and LDL levels. Uric acid and ferritin were also significantly decreased (Table 1).
Liver and kidney function evaluation showed no significant changes, other than a small but
significant increase in Blood Urea Nitrogen (BUN) levels. The mild but significant dehydration of the
subjects at the end of the diet might justify the small increase in albumin and BUN levels. Calcium and
phosphate levels showed a slight increase within normal ranges (9.41 ± 0.43 vs. 9.57 ± 0.44 mg/dL,
p-value < 0.0001; and 3.53 ± 0.51 vs. 3.70 ± 0.46, p-value 0.005, respectively), whereas sodium and
potassium were stable, as was PTH (Table 1). Finally, no clinical signs of gout, kidney stones or
gallbladder disease were reported by patients throughout the dietary intervention, as assessed during
follow up visits.
The following minor adverse events were reported by some patients: constipation, diarrhea,
abdominal cramping, nausea, fatigue, hunger, and dizziness, but none were deemed intolerable and,
most often, were resolved within the first few days of the dietary intervention. No standardized
questionnaire was collected to assess side effects.
Nutrients 2020, 12, 333 5 of 10
Table 1. Baseline and end of the diet population characteristics.
Baseline End of Diet p
n 92 92
Female gender n (\%) 69 (65) 69 (65)
HTN treatment n (\%) 39 (42.9)
Age (years) 51.27 ± 12.20
Weight (Kg) 92.40 ± 18.31 76.82 ± 14.95 <0.0001
BMI (kg/m2) 33.85 ± 5.84 28.21 ± 4.90 <0.0001
Fat Mass (Kg) 35.63 ± 9.93 24.40 ± 9.00 <0.0001
Fat Free Mass (Kg) 56.77 ± 13.40 52.42 ± 10.89 <0.0001
Skeletal Muscle Mass (Kg) 37.27 ± 9.57 33.93 ± 8.10 <0.0001
TBW (Lt) 42.44 ± 9.85 39.20 ± 8.11 <0.0001
ECW (Lt) 19.62 ± 4.47 18.57 ± 3.96 <0.0001
ICW (Lt) 22.80 ± 5.79 20.74 ± 4.95 <0.0001
SBP (mmHg) 137.1 ± 12.2 132.2 ± 9.2 <0.0001
DBP (mmHg) 81.5 ± 6.7 77.4 ± 4.6 <0.0001
Creatinine (mg/dL) 0.79 ± 0.17 0.78 ± 0.17 0.139
e GFR (ml/min/1.73m2) 94.46 ± 18.75 95.75 ± 18.52 0.32
BUN (g/L) 0.36 ± 0.10 0.39 ± 0.09 <0.0001
Glucose (mg/dL) 95.32 ± 13.26 88.25 ± 10.24 0.002
HbA1c (\%) 5.65 ± 0.81 5.33 ± 0.39 <0.0001
AST (mg/dL) 25.38 ± 16.11 20.83 ± 6.12 0.233
ALT (mg/dL) 32.08 ± 26.42 23.44 ± 12.60 0.138
Total cholesterol (mg/dL) 206.91 ± 45.65 184.46 ± 41.17 0.004
LDL (mg/dL) 120.17 ± 42.92 117.38 ± 38.65 0.388
HDL (mg/dL) 55.97 ± 18.42 51.69 ± 11.37 0.141
Triglycerides (mg/dL) 156.44 ± 90.87 102.62 ± 35.71 0.003
Total Protein (g/dL) 7.07 ± 0.38 7.06 ± 0.39 0.915
Albumin (g/dL) 4.14 ± 0.25 4.18 ± 0.28 0.035
Uric Acid (mg/dL) 5.23 ± 1.04 4.83 ± 1.11 <0.0001
Calcium (mg/dL) 9.41 ± 0.43 9.57 ± 0.44 <0.0001
Phosphorus (mg/dL) 3.53 ± 0.51 3.70 ± 0.46 0.005
Sodium (mmol/L) 141.13 ± 2.45 141.51 ± 2.07 0.155
Potassium (mmol/L) 4.53 ± 0.40 4.55 ± 0.40 0.244
PTHi (ng/L) 36.16 ± 10.65 33.66 ± 12.16 0.384
Ferritin (ug/L) 112.34 ± 125.29 101.69 ± 67.72 0.027
Urinary protein (mg/dL) 12.43 ± 9.50 11.03 ± 7.15 0.207
Data shown as means ± standard deviation (SD) of the mean. The p value is from a dependent sample t-test. HTN,
hypertension; BMI, Body Mass Index; TBW, Total Body Water; ECW, Extra Cellular Water; ICW, Intra Cellular Water;
SBP, Systolic Blood Pressure; DBP, Diastolic Blood Pressure; eGFR, estimated Glomerular Filtration Rate; BUN,
Blood Urea Nitrogen; LDL, Low Density Lipoprotein; HDL, High Density Lipoprotein. Significant p values are
highlighted in bold.
3.2. Mild Impairment in Kidney Function Does Not Mediate a Difference in Safety and Efficacy Outcomes
of a VLCKD
Based on renal function, the patients were stratified into two groups: 38 MCKD (Mild Chronic
Kidney Disease) subjects had a glomerular filtration volume (GFV) between 60 and 89 mL/min,
corresponding to Stage 2 Chronic Kidney Disease, and 54 NKF (Normal Kidney Function) subjects had
a GFV equal to or higher than 90 mL/min. MCKD subjects were then compared to NKF. Unsurprisingly,
the MCKD group had a mean age that was higher than that of NKF, but the groups were not different
relative to anthropometric parameters other than fat mass, significantly lower in the MCKD, albumin
(lower in NKF), and sodium, again lower in NKF. No significant differences in anthropometric
parameters (body weight, BMI), blood pressure or biochemical parameters (glucose metabolism and
lipid profile, electrolytes, uric acid, hepatic enzymes) were found between the two groups over time
(Table 2). Moreover, no between-group difference was observed regarding the percentage of patients
reaching 5\%, 5–10\%, 10–20\% or over 20\% weight loss. Of note, 27.7\% of patients with MCKD reported
an improvement of renal function at the end of the dietary intervention leading to an eGFR ≥90.
Nutrients 2020, 12, 333 6 of 10
Table 2. Differences between NKF and MCKD before and after treatment.
NKF (e GFR ≥ 90) MCKD (eGFR 60–89)
Variable Baseline End of Diet p Baseline End of Diet p p *
N 54 NA 38 NA
Female gender n (\%) 43 (80) NA 22 (68) NA 0.22
HTN treatment n (\%) 20 (43.5) NA 19 (44.2) NA
Diet length (weeks) 15.33 ± 7.98 NA 15.33 ± 7.98 NA 0.645
Age (years) 47.96 ± 12.97 NA 55.97 ± 9.30 NA <0.0001
Weight (Kg) 94.21 ± 18.60 77.94 ± 15.67 <0.0001 89.92 ± 17.84 75.28 ± 13.95 <0.0001 0.510
BMI (kg/m2) 34.46 ± 5.69 28.67 ± 5.04 <0.0001 33.01 ± 6.01 27.56 ± 4.68 <0.0001 0.334
Fat Mass (Kg) 37.51 ± 9.57 25.93 ± 9.45 <0.0001 33.05 ± 9.96 22.31 ± 7.99 <0.0001 0.742
Fat Free Mass (Kg) 56.69 ± 14.10 52.01 ± 11.10 <0.0001 56.88 ± 12.55 52.97 ± 10.72 <0.0001 0.521
Skeletal Muscle Mass (Kg) 37.08 ± 10.05 33.58 ± 8.14 <0.0001 37.54 ± 9.00 34.40 ± 8.13 <0.0001 0.542
TBW (Lt) 42.04 ± 10.00 38.60 ± 7.86 <0.0001 42.98 ± 9.76 40.03 ± 8.47 <0.0001 0.494
ECW (Lt) 19.53 ± 4.49 18.42 ± 4.04 <0.0001 19.74 ± 4.50 18.78 ± 3.90 <0.0001 0.238
ICW (Lt) 22.51 ± 5.91 20.37 ± 4.78 <0.0001 23.20 ± 5.67 21.23 ± 5.19 <0.0001 0.561
SBP (mmHg) 136.6 ± 14.5 130.9 ± 10.8 <0.0001 137.6 ± 12.2 132.2 ± 9.2 <0.0001 0.998
DBP (mmHg) 82.6 ± 7.2 77.3 ± 5.2 <0.0001 81.4 ± 6.7 77.4 ± 4.6 <0.0001 0.841
Creatinine (mg/dL) 0.70 ± 0.11 0.71 ± 0.12 0.43 0.93 ± 0.16 0.88 ± 0.17 0.002 0.414
eGFR (ml/min/1.73m2) 107.22 ± 11.20 105.28 ± 14.32 0.263 76.32 ± 10.44 82.21 ± 15.14 0.002 0.901
BUN (g/L) 0.34 ± 0.08 0.38 ± 0.07 <0.0001 0.39 ± 0.11 0.41 ± 0.11 0.052 0.822
Glucose (mg/dL) 96.7 ± 14.5 91.73 ± 15.23 0.002 91.59 ± 11.0 86.60 ± 10.02 0.005 0.053
HbA1c (\%) 5.68 ± 0.96 5.30 ± 0.38 <0.0001 5.60 ± 0.46 5.39 ± 0.40 0.007 0.470
AST (mg/dL) 28.33 ± 19.49 22.08 ± 6.71 0.218 20.44 ± 6.17 19.36 ± 5.26 0.732 0.724
ALT (mg/dL) 38.07 ± 31.45 26.23 ± 15.35 0.15 22.11 ± 9.85 20.42 ± 8.36 0.759 0.766
Total cholesterol (mg/dL) 206.65 ± 48.24 182.61 ± 35.54 <0.0001 207.27 ± 43.62 187.13 ± 49.28 0.148 0.279
LDL (mg/dL) 122.74 ± 44.53 121.71 ± 30.31 0.233 117.04 ± 42.32 113.04 ± 46.14 0.958 0.309
HDL (mg/dL) 53.59 ± 9.59 51.21 ± 8.61 0.491 58.67 ± 25.13 52.25 ± 14.27 0.199 0.550
Triglycerides (mg/dL) 172.37 ± 101.64 99.33 ± 30.48 <0.0001 136.27 ± 73.48 106.94 ± 42.28 0.061 0.249
Total Protein (g/dL) 7.11 ± 0.41 7.11 ± 0.40 0.48 7.02 ± 0.34 7.00 ± 0.38 0.352 0.185
Albumin (g/dL) 4.08 ± 0.26 4.17 ± 0.27 <0.0001 4.21 ± 0.21 4.20 ± 0.30 0.968 0.226
Uric Acid (mg/dL) 5.06 ± 0.98 4.75 ± 1.21 <0.0002 5.49 ± 1.10 4.96 ± 0.94 0.008 0.561
Calcium (mg/dL) 9.41 ± 0.46 9.56 ± 0.44 <0.0003 9.42 ± 0.39 9.59 ± 0.44 0.002 0.401
Phosphorus (mg/dL) 3.46 ± 0.54 3.67 ± 0.49 <0.0004 3.62 ± 0.45 3.75 ± 0.39 0.168 0.864
Sodium (mmol/L) 140.60 ± 2.30 141.16 ± 2.02 0.113 141.89 ± 2.49 142.00 ± 2.06 0.865 0.883
Potassium (mmol/L) 4.48 ± 0.43 4.51 ± 0.42 0.345 4.59 ± 0.35 4.62 ± 0.36 0.505 0.920
PTHi (ng/L) 39.04 ± 11.89 36.66 ± 14.54 0.627 32.33 ± 8.16 29.67 ± 7.42 0.33 0.528
Ferritin (ug/L) 121.56 ± 146.19 90.44 ± 67.44 0.238 95.21 ± 74.22 116.14 ± 67.74 0.036 0.449
Urinary protein (mg/dL) 13.54 ± 10.74 11.34 ± 7.30 0.157 10.74 ± 7.16 10.56 ± 6.98 0.882 0.577
Data shown as means ± standard deviation (SD) of the mean. The p value is from a dependent sample t-test for the within group analysis. The p * value shown is from a general linear
mixed model analysis of end of diet variables of the NF and MCKD groups for the between groups analysis. The variables of group, baseline values, age and gender were included in the
model as fixed effects. For BP, HTN treatment was taken into account. NA, not applicable; NKF, Normal Kidney Function; MCKD, Mild Chronic Kidney Disease; HTN, hypertension; BMI,
Body Mass Index; TBW, Total Body Water; ECW Extra Cellular Water; ICW Intra Cellular Water; SBP, Systolic Blood Pressure; DBP, Diastolic Blood Pressure; eGFR, estimated Glomerular
Filtration Rate; BUN, Blood Urea Nitrogen; LDL, Low Density Lipoprotein; HDL, High Density Lipoprotein. Significant p values are highlighted in bold.
Nutrients 2020, 12, 333 7 of 10
4. Discussion
Management of obesity is of constantly increasing concern nowadays, and chronic kidney disease
is a possible complication that may require extra care. Among the available strategies for weight
loss and maintenance, VLCKDs are an effective tool, but some concern is present with regard to the
treatment of patients with renal failure due to the relative dietary protein excess.
In our real-life observational study, we first assessed the entire enrolled population without taking
renal function into account. We report a significant overall weight reduction as expected, with a mean
body weight decrease of nearly 20\%, and a significant reduction in fat mass (80\% of total weight
loss) in less than 3 months of dietary treatment, consistent with previous studies [15]. A statistically
significant reduction in MM, of little clinical relevance, was observed, and this decrease was paralleled
by a reduction in TBW as previously described [21]. The increase diuresis known to happen during
a ketogenic diet might explain the TBW finding, that could, in turn, play a role in the BIA assessed
MM reduction known to be influenced by body hydration [21]. Both systolic and diastolic blood
pressure were reduced as expected. Lipid and glucose metabolism significantly improved, and no
safety concern arose.
In fact, hepatic enzymes AST and ALT showed a tendency to decrease, despite not reaching
significance, and triglycerides were profoundly decreased, all of which is consistent with reduced
intrahepatic triglyceride content and liver size reduction, as previously described in patients with
obesity undergoing a VLCKD before bariatric surgery [19,20]. No changes were detected in sodium or
potassium levels, suggesting that a VLCKD does not impair the hydroelectrolytic balance. Uric acid was
finally significantly decreased, excluding a correlation between VLCKD and hyperuricemia. A recent
metanalysis reported an overall neutral effect on uric acid by VLCKDs [22], and a previous study
reported similar effect to ours, where a decrease in urate was observed after a VLCKD but not after an
LCD [23]. These controversial results might find their explanation in timing and weight loss amplitude,
as food groups that typically increase serum uric acid levels are widely consumed in ketogenic diets
and might lead to such an effect in the short term [24]. However, it is acknowledged that weight loss
is associated with a significant reduction in urate levels [25]. As our patients experienced a mean
weight loss of nearly 20\% of baseline values, it seems reasonable to conclude that this aspect might
have played a predominant role in modulating uric acid levels.
We also observed a significant but slight increase in calcium and phosphorus levels (though
remaining in the normal range), that might be attributable to two possible reasons: First,
mild dehydration, as observed by TBW reduction, and expected as a result of the significant diuretic
effects of VLCKDs, might be responsible for the relative increase in calcium and phosphorus levels due
to simple hemoconcentration. The observed elevation in albumin levels point in the same direction.
Second, calcium and phosphorus levels could also be increased following bone loss, as it was previously
observed in patients on severely calorie-restricted regimens with profound weight reduction [26].
Of note, PTH levels were not altered by the intervention, suggesting that bone metabolism was
unaffected, and adequate protein intake and electrolytes supplementation were provided throughout
the study, making the latter option less plausible. That being said, as no evidence is currently available
regarding change in bone density and quality in patients undergoing a VLCKD regimens, further
studies are warranted to look into this safety outcome.
Ferritin levels were also marginally modified by dietary intervention, with a significant reduction
over time. As ferritin has been shown to be a marker of inflammation rather than iron deficiency
in subjects with obesity [27], and ketosis has been widely proven to have an anti-inflammatory
effect [28], we believe that this reduction parallels reduced systemic inflammation in our patients.
However, no other inflammatory markers were assessed in this study, and we therefore cannot confirm
this hypothesis.
Subjects included in this study were then stratified in two groups based on renal function.
No differences between groups were shown in anthropometric parameter changes (body weight, BMI,
BIA data) or metabolic profile improvement. Interestingly, a significant proportion of MCKD patients
Nutrients 2020, 12, 333 8 of 10
reported a full recovery (eGFR ≥ 90 mL/min/1.73m2) of kidney function at the end of the dietary
intervention, suggesting that not only is VLCKD an effective and safe weight loss tool in MCKD
patients with obesity, but that it also could help ameliorate renal function.
Relative protein excess typical of VLCKDs has been of major concern among clinicians for its
kidney-damaging potential, preventing many to propose this intervention to patients with CKD
in need of weight loss. In order to assess this safety outcome, creatinine, BUN, eGFR and urinary
proteins were evaluated. Creatinine and eGFR were not affected by the dietary intervention and no
differences were observed in the between group analysis. Conversely, BUN was slightly increased,
most likely as a consequence of increased protein metabolism as previously described [29], with again
no difference between the two groups. Current guidelines are inconclusive regarding recommended
dietary protein intake in patients with early stages of CKD, with some suggesting .8 g/kg body weight
as the optimum [30], and others recommending up to 1.4 g/kg body weight [31]. Recent evidence
suggests that the impact of dietary protein on renal function may depend on the protein source,
with red meat intake being harmful in a dose dependent manner, and other protein sources such as
poultry, fish, egg and dairies not showing such a deleterious effect [32]. Moreover, studies assessing
plant-based protein sources (soy and vegetable derived) seem …
International Journal of Chemistry; Vol. 12, No. 2; 2020
ISSN 1916-9698 E-ISSN 1916-9701
Published by Canadian Center of Science and Education
6
Influence of Low Carbohydrate High Fat Ketogenic Diets on Renal and
Liver Parameters
B. O. Eiya1, R. O. Aikpitanyi-iduitua1
1Department of Physiology, School of Basic Medical Sciences, College of Medical Sciences, University of Benin,
Benin
Correspondence: B. O. Eiya, Department of Physiology, School of Basic Medical Sciences, College of Medical
Sciences, University of Benin, Benin.
Received: March 30, 2020 Accepted: May 28, 2020 Online Published: July 2, 2020
doi:10.5539/ijc.v12n2p6 URL: https://doi.org/10.5539/ijc.v12n2p6
Abstract
In recent times the use of high fat ketogenic diet as a treatment strategy in some diseases and weight control has been on
the increase. This study aims to elucidate the effect of high fat ketogenic diet on some renal and liver parameters. Forty
albino rats were used and divided into four groups. Group A was control; B, C, and D were fed with diets including
butter, coconut oil and olive oil respectively for eight weeks. Urine and serum samples were assayed
spectrophotometrically. There was a significant difference in urinary albumin (0.13±0.01g/dl) of group D when
compared with control (0.22 ± 0.03g/dl). Urinary creatinine concentrations of group D (4.32±0.70mg/dl) was higher
than group C (1.75±0.46 mg/dl). Urea of group B (39.40±4.70 mg/dl), group C (29.90±1.46 mg/dl) and group D
(40.20±2.62mg/dl) were lower than control group (64.20±3.41mg/dl). Serum creatinine concentrations of group B
(1.05±0.09mg/dl), group C (0.85±0.07lmg/dl) and group D (1.03±0.07 mg/dl) were reduced significantly. Albumin:
creatinine ratio of group A (120.6±32.04) was higher than that of group D (41.31±8.28). AST (260.1±17.80) was higher
in group C compared with A (160.1± 9.510). ALT for D (91.20±18.70), group A (36.00±3.84), serum albumin
concentrations of group D (3.590±0.1286), group C (3.590±0.1286) and group A (4.100±0.1814). Total protein
concentration of group C (5.390±0.2105), D (5.280± 0.1104) and group A (6.190±0.2496g). Body weight of
experimental groups reduced while the control groups increased. This study has confirmed that high fat ketogenic diet
can be used for weight management however it could be harmful to the liver but did not show any harmful effects on
the kidneys.
Keywords: high fat, ketogenic diet, kidney and liver
1. Introduction
The use of LCHF ketogenic diet dates back to over 90 years, when the diet was used in the treatment of refractory
epilepsy (Wilder, 1921). The name ketogenic diet arose from the fact that intake of this diet resulted in the production of
ketone bodies which serves as an alternative source of energy to the brain. In recent time, ketogenic diet have also been
employed in weight management, this is based on the hypothesis that energy from the diet when used up for energy
would result in calories waste, thus supporting weight loss (Feinman & Fine,2007, Halton & Hu 2004). Outside its use
in weight control, the use of high fat ketogenic diet have been shown to be effective in type 11 diabetes mellitus
management as well as in the management of renal impairment (Michal et al., 2011). Tonna et al., (2010) reported the
ability of high fat ketogenic diet to reverse diabetic nephropathy. One hypothesis is that the use of energy from proteins in
ketogenic diet is an expensive process for the body and so leads to a waste of calories, therefore increasing weight loss
(Feinman and Fine,2007;Fine and Feinman,2004;Halton and Hu,2004) and it’s a remedy for obesity, a major problem
affecting up to 30\% of the adult population (Kramer and Luke, 2007), with complications including type 2 diabetes
mellitus and renal function impairment (Michal et al., 2011). Stereotypical results have proven that the numerical density
of glomerulus and total number of glomeruli in rats fed the high fat ketogenic diet for a prolonged period of time was
significantly decreased, indicating glomerular atrophy (Altakunak et al., 2008;). In another study, the ketogenic diet was
proven to reverse diabetic nephropathy (Tonna et al., 2010) by producing prolonged exposure to the ketone
3-beta-hydroxybutyric acid (3-OHB), which blocks the inhibition of agouti-related peptide by glucose (Poplawski et al.,
2011; Bailey et al., 2005). The diet reversed blood glucose to normal in akita mice (Susztak et al., 2006) as indicated by
urinary albumin/creatinine ratios, and in patients with type 2 diabetes mellitus it greatly improved fasting glucose levels
(Gannon and Nuttall, 2004) and increased insulin sensitivity (Samaha et al., 2003), thereby alleviating glycosuria and
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reversing kidney damage due to excessive glucose excretion. The ketogenic diet halted the progression of renal
insufficiency in patients with chronic renal failure, as accessed by serial determination of creatinine levels.
Liver disease have in the past being linked to high consumption of alcohol resulting in alcoholic liver disease, in recent
times, a new type of liver disease known as the nonalcoholic fatty liver disease (NAFLD) has been associated to unhealthy
diet, resulting in gradual accumulation of fat in the liver. In fact; some school of thoughts feels that high carb diets and not
high fat diets results in NAFLD. They attributed it to the synthesis of fat from de novo lipogenesis, (ie the liver creating fat
from excess carbohydrate and protein).
There are controversies as to the impact of high fat ketogenic diet on liver functions, while some studies have reported that
this diet can impact negatively on the kidney, others have reported otherwise. Arslan et al., (2016) observed high levels of
Aspartate Transaminases (AST) & Alanine Transaminases (ALT) after two months intake of the diet. Hepatic steatosis in
both short- and long- term intake of the diet was observed in mice fed with this diet (Ellenbrok, 2014). In a meta- analysis
study carried out by Fahimeh et al., (2014), they concluded that intake of LCD improves liver fat contents but not serum
enzymes.
Information on the relationship between ketogenic diets and the type of fat used are limited, in ketogenic diet the
consumption of both saturated and unsaturated fats is encouraged. The consumption of saturated fat is generally
considered a risk factor for dyslipidemia, which in turn is a risk factor for some types of cardiovascular disease (Canon et
al., 2006). Abnormal blood lipid levels, that is high in total cholesterol, high levels of triglycerides, high levels
of low-density lipoprotein (bad cholesterol) or low levels of high density lipoprotein (Good cholesterol), are all
associated with increased risk of heart disease and stroke. Many health authorities such as the America Diabetic
Association (Kris-Etherton and Innis, 2007), the British Diabetic Association, America Heart Association (Sacks et al.,
2017), the World Heart Federation, the British National Health Service, among others, advise that saturated fat is a risk
factor for cardiovascular diseases. The World Health Organization in May 2015 recommends switching from saturated to
unsaturated fats. A limited number of systematic reviews have examined the relationship between saturated fat and
cardiovascular diseases and have come to different conclusions. A 2015 systematic review found no association between
saturated fat consumption and risk of heart disease, stroke, diabetes, or death. There are no enough literatures on the effect
of the type of fat used in the preparation of ketogenic diets on biochemical parameters, most of the available literatures
emphasis more on the effects of the diet on biochemical parameters without looking at the nature of fat used in preparing
the diet, it is therefore the aim of this study to look at the effect of three different fats type ketogenic diets and ascertain if
the type of fats used in the preparation of the diet has any significant effect on liver and renal function of Wistar albino
rats.
2. Methods
2.1 Experimental Design
Forty (40) albino Wistar rats of either sex were used for this study. They were weighed on arrival and acclimatized for two
weeks. At the end of acclimatization, the rats were divided into four groups of 10 each; Group A (10) was the control
group fed normal rat chow, while Group B (10), Group C (10), Group D (10) were experimental groups fed with 65\%
ketogenic diet made with butter, coconut oil and olive oil respectively. They were fed with these diets for a period of eight
weeks (2 months) and were allowed free access to feeds and water during the duration of the experiment.
2.2 Diet Formulation
The experimental groups were fed 65\% ketogenic diets made of butter, coconut oil and olive oil along with the other
constituents of the diets for a period of eight (8) weeks, Percentage Composition of Experimental Diet
CONSTITUENTS CONTROL
(A)
EXPERIMENTAL
GROUP (B)
EXPERIMENTAL
GROUP (C)
EXPERIMENTAL
GROUP (D)
COCONUT FLOUR - 15g 15g 15g
MAIZE 70g - - -
PALM OIL 10g - - -
BUTTER - 65g - -
COCONUT OIL - - 65g -
OLIVE OIL - - - 65g
FISH MEAL 15g 15g 15g 15g
BONE MEAL 2g 2g 2g 2g
LIMESTONE 1g 1g 1g 1g
SALT 0.5g 0.5g 0.5g 0.5g
VIT-MIN PREMIX 0.5g 0.5g 0.5g 0,5g
LYSINE 0.3g 0.3g 0.3g 0.3g
MET & CYS 0.3g 0.3g 0.3g 0.3g
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2.3 Collection of Samples
After two weeks of feeding the rats with the diets, 24-hour urine samples were collected using metabolic cages and the rats
were ascertained to be in ketosis using a urinalysis test strip. At the end of two months they were weighed and put in
metabolic cages, 24-hour urine samples were again collected into universal containers. The rats were put to sleep using
chloroform in a closed chamber and fasted blood samples were collected from the abdominal aorta and then the heart into
plain test tubes. The blood was allowed to clot and the clot was dislodged, spun on a centrifuge to get the serum which was
used for the analysis.
2.4 Determination of Biochemical Parameters
Creatinine: This was assayed by two-point’s kinetic-using the modified Jaffe increasing reaction proposed by Bartels and
Bolumer (1972). Serum urea was determined by Berthelot method (Newman and Price, 1999). Urinary microalbumin was
determined by method described by feldi-Rasmussen et al., (1985). Aspartate amino transferase and Alanine amino
transferase were estimated using the Colorimetric Method of Reithman and Frankel (1975).
2.5 Statistical Analysis
Data is presented in mean ± standard error of mean (SEM). Analysis of variance (ANOVA) was used to compare in
between groups, while turkey test was considered the post-hoc test. P≤ 0.05 was considered as the statistical significance.
Figure 1. Effect of different ketogenic diet made of butter, coconut oil, and olive oil on the rat body weight
3. Results
Table 1. mean values of urine and blood renal parameters in rats fed with 65\% high fats ketogenic diets
Parameters Control Butter Coconut oil Olive oil
Urine albumin (g/dl) 0.22 ± 0.03 0.19 ± 0.02 0.15 ± 0.02 0.13 ± 0.01*ᵃ
Urine creatinine (mg/dl) 3.84 ± 1.02 3.84 ± 0.90 1.75 ± 0.46 4.31 ± 0.70ᵇ
Serum urea (g/dl) 64.20 ± 3.41 39.40 ± 4.70* 29.90 ± 1.46* 40.20 ± 2.62*ᵇ
Serum creatinine (mg/dl) 1.42 ± 0.04 1.05 ± 0.09* 0.85 ± 0.07* 1.03 ± 0.07*
Albumin-creatinine ratio 120.6 ± 32.04 94.64 ± 23.03 174.7 ± 61.82 41.31 ± 8.28*ᵃᵇ
*P ≤ 0.05 indicates significant difference at the different diets compared with the control.
ᵃP ≤ 0.05 indicates significant difference when coconut oil or olive oil ketogenic fed rats are compared with butter
ketogenic fed rats.
ᵇP ≤ 0.05 indicates significant difference when coconut oil ketogenic fed rats are compared with olive oil ketogenic fed
rats
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Table 2. Mean values of some liver functions markers of rats fed with 65\% high fats ketogenic diets
Parameters Control Butter Olive Oil Coconut Oil
AST (U/I) 160.1 ± 9.510 143.4 ± 8.218 196.1 ± 3.926 260.1 ± 17.80*
ALP (U/I) 9.900 ± 0.7371 24.90 ± 2.536* 23.00 ± 2.591* 21.80 ± 3.422*
ALT (U/I) 36.00 ± 3.884 40.10 ± 2.173 50.30 ± 2.996 91.20 ± 18.70*
ALBUMIN (g/dl) 4.100 ± 0.1814 3.590 ±0.1286* 3.300 ± 0.1085* 3.390 ± 0.0862*
GLOBULIN(g/dl) 2.160 ± 0.1097 1.870 ± 0.1193 2.180 ± 0.08138 1.960 ± 0.1352
TOTAL PROTEIN
(g/dl)
6.190 ± 0.2496 5.320 ± 0.12818* 5.390 ± 0.2105* 5.280 ± 0.1104*
*P< 0.05 indicates significant difference between groups
4. Discussion
In this current study, we looked at the effect of three fat (coconut oil, olive oil and butter) types’ ketogenic diets on liver
and renal parameters in wistar rats for a period of two months. We observed a significant increase in AST and ALT
levels only in coconut oil based diet when compared with control, while there was a significant increase in ALP levels
of all three ketogenic diets (olive oil, butter and coconut oil), when compared with control. Albumin levels were also
significantly reduced in the ketogenic fed groups when compared with control, there was however no significant change
in globulin levels, total protein levels reduced significantly only in the olive oil and coconut oil based diets. Previous
studies have shown that intake of ketogenic diet have long and short term effects, dyslipidemia, kidney stones,
carnithine deficiency are some of the long term effects (Cervenka et al., 2016). Kang et al., (2004) and Arslan et.al
(2016), have reported an alteration in serum levels of liver enzymes following intake of ketogenic diet, Arslan et al., in
their study observed an increase in ALT and AST in 2 out of every 3 rats after intake of ketogenic diet, their findings is
similar to our observation in this current study as AST and ALT levels of rats in this study increased significantly when
compared with control. In another study by Henrietta and Olumese (2010), they reported increased levels of AST and
ALT in rats fed ketogenic diet, this is also in agreement with our findings, where we observed significant increase in
ALP in all the ketogenic fed groups while AST and ALT increased only in rats fed the coconut based diet. ALP is a
sensitive detector for early intra hepatic and extra hepatic bile obstruction, the presence of infiltrative disease of the
liver and all bone diseases associated with osteoblastic activities, (Friedman et al., 2003). Albumin levels significantly
decreased in ketogenic groups compared with control, the liver is the only site for albumin synthesis hence anything that
affect the liver will affect its synthesis (Marjolain et al., 2008). This finding also is in agreement with that of Henrietta
and Olumese (2010), but disagree with the observations of Imafidon and Okunrobo, (2012), who reported a significant
increase in levels of albumin and total protein after intake of ketogenic diet. The varying effects the different diets have
on the various parameters can be attributed to the fact that different types of fats were used to constitute the diets and
since they contain different fatty acids making some more ketogenic than others. Coconut oil is a medium chain fatty
acid which contains 50\% of Lauric acid which easily enters the mitochondria independently of carnithine transport
system and therefore easily transfers into the mitochondria. One mechanism that has been attributed to liver disease is
depletion of carnitine, an amino acid derivative that is responsible for the transport of long chain fatty acids (LCFA) to
the mitochondria (Lheureux, & Hantson, 2009). The fact that coconut oil bypasses the carnitine pathway thus getting to
the mitochondria faster than the other fatty acids could be responsible for its fast effects observed in this study.
Findings from this study did not show any significant increase in renal parameters in rats fed with ketogenic diets,
rather the diets were observed to improve renal parameters (serum creatinine, urinary albumin and urea) when
compared with control. However, when the different ketogenic diets (KD) (butter, coconut oil and olive oil) were
compared between each other, there were significant differences between groups but the values were within the control
values. These results have further buttressed the fact that high fat ketogenic diet does not affect the kidney as seen in the
creatinine and urinary albumin values which were lower in the ketogenic groups when compared with control (Allen
2002). This is in agreement with reports of researchers from Mount Sinai School of Medicine who observed in their
study that intake of high fat ketogenic diet could reverse impaired kidney function in type 1& 2 diabetic subjects. In
their study they reported the reversed expression of genes associated with diabetic related kidney failure after 8 weeks
intake of KD in mice, this is similar to findings in our study as rats fed KD diet were seen to have improved renal
parameters when compared with control. Chikako et al, (2016), further confirmed this in their study where they looked
at the effect of LCD on renal parameters in over weight obese individuals without chronic kidney disease and their
result showed that eGFR was greater in LCD fed individuals compared with control diet. The various ketogenic diet
also resulted in weight loss when initial weight of the rats were compared with the final weight after eight weeks intake
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of the diet as compared to the control rats that gained weight. This is in agreement with our previous studies, (Eiya &
Osakue, 2019, 2018), it also agrees with the findings of Feinman and Fine (2004). The reduction in weight can be
attributed to reduced caloric intake and the rats burning fat as an alternative source of fuel to glucose
(Westerterp-Platenga et al., 2009; Seyfried & Murkherjee, 2005). When compared in between groups, there was
significant reduction in weight in groups fed olive oil and coconut oil, compared with the group fed butter.
5. Conclusion
This study has again confirmed that high fat ketogenic diet can be used for weight management however intake of
LCHF diet could be harmful to the liver due to its effect on transaminases and serum albumin, it has also shown that the
type of fat used in constituting the diet can contribute to the level of damage the diet can cause to the liver as observed
in rats fed with the coconut oil based diet. The diet did not have any harmful effect on the kidney rather it was observed
to improve renal function as was shown by the reduction of serum creatinine, urea and urinary albumin levels. The need
for further studies to ascertain the best fat type that will be used in the preparation of HFLC diet is very important; this
will no doubt ensure the benefit of this diet is optimized.
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https://doi.org/10.2337/diabetes.55.01.06.db05-0894
https://doi.org/10.1038/nrneph.2010.55
https://doi.org/10.1146/annurev-nutr-080508-141056
2274
Abstract. – OBJECTIVE: To verify safety re-
spect to weight loss, cardiometabolic diseases
of short-term Very low-calorie ketogenic diets
(VLCKDs, <800 kcal day-1).
PATIENTS AND METHODS: Randomized
cross-over trial with placebo. The study had no. 2
dietary treatment (DT), conducted in two arms: (1)
VLCKD1 in which 50\% of protein intake is replaced
with synthetic amino acids; (2) VLCKD2 with pla-
cebo. The VLCKDs (<800 kcal day-1) were different
in term of protein content and quality each arm
lasted three weeks (wks). Between the two arms
a 3-wks washout period was performed to avoid
additive effects on DT to follow. At the baseline,
at start and end of each arm, all the subjects were
evaluated for their health and nutritional status, by
anthropometric analysis, body composition (Dual
X-ray Absorptiometry (DXA), Bioimpedentiometry,
biochemical evaluation, and Peroxisome Prolifer-
ator-Activated Receptor γ (PPAR) γ expression by
transcriptomic analysis.
RESULTS: After VLCKD1 were reduced: Body
Mass Index (BMI) (Δ\%=-11.1\%, p=0.00), Total
Body Water (TBW) (p<0.05); Android Fat Percent-
age (AFP) (Δ\%=-1.8\%, p=0.02); Android Fat Mass
(AFM) (Δ\%=-12.7\%, p=0.00); Gynoid Fat Mass
(GFM) (Δ\%=-6.3\%, p=0.01); Intermuscular Adi-
pose Tissue (IMAT) (Δ\%= -11.1\%, p=0.00); Homeo-
stasis Model Assessment of Insulin Re-sistance
(HOMA-IR) (Δ\%=-62.1\%, p=0.01). After VLCKD1 a
significant increase of uricemia, cre-atinine and
aspartate aminotransferase (AST) (respectively
Δ\%=35\%, p=0.01; Δ\%=5.9\%, p=0.02; Δ\%=25.5\%,
p=0.03). After VLCKD2 were reduced: BMI (Δ\%=-
11.2\%, p=0.00); AFM (Δ\%=-14.3\%, p=0.00); GFM
(Δ\%=-6.3\%, p=0.00); Appendicular Skeletal Mus-
cle Mass Index (ASMMI) (Δ\%=-17.5\%, p=0.00);
HOMA-IR (Δ\%=-59,4\%, p=0.02). After VLCKD2,
uricemia (Δ\%=63.1\%, p=0.03), and Vitamin D
levels (Δ\%=25.7\%, p=0.02) were increased. No
significant changes of car-diovascular disease
(CVD) indexes were observed after DTs. No sig-
nificant changes of PPARγ lev-el in any DTs.
CONCLUSIONS: 21-days VLCKDs not impair
nutritional state; not cause negative changes in
global measurements of nutritional state includ-
ing sarcopenia, bone mineral content, hepatic,
renal and lipid profile.
Key Words
Very-low-calorie, Ketogenic Diet, Randomized
crossover clinical trial, Obesity, Body Composition, Vi-
tamin D, PPARγ.
Introduction
In recent years we are observing a rapid
growth in the prevalence of chronic non-com-
European Review for Medical and Pharmacological Sciences 2017; 21: 2274-2289
C. COLICA1, G. MERRA2, A. GASBARRINI3, A. DE LORENZO4,
G. CIOCCOLONI5, P. GUALTIERI5, M.A. PERRONE6,7, S. BERNARDINI6,
V. BERNARDO8, L. DI RENZO4, M. MARCHETTI9,10
1CNR, IBFM UOS of Germaneto, University “Magna Graecia” of Catanzaro, Campus “Salvatore
Venuta”, Catanzaro, Italy
2Department of Emergency Medicine, Catholic University of the Sacred Heart, “Polyclinic Gemelli
Foundation, Rome, Italy
3Division of Internal Medicine and Gastroenterology, Catholic University of the Sacred Heart,
Polyclinic Gemelli Foundation, Rome, Italy
4Department of Biomedicine and Prevention, Section of Clinical Nutrition and Nutrigenomic,
University of Rome “Tor Vergata”, Rome, Italy
5School of Medical-Surgical Applied Sciences, University of Rome “Tor Vergata”, Rome, Italy
6Division of Clinical Biochemisty and Clinical Molecular Biology, University of Rome “Tor Vergata”,
Rome, Italy
7Division of Cardiology, University of Rome “Tor Vergata”, Rome, Italy
8“San Camillo de Lellis” Hospital, Rieti, Italy
9Department of Surgical Sciences, Policlinico “Umberto I”, “Sapienza” University of Rome, Rome, Italy
10USL 1 Umbria, Castiglione del Lago, Perugia, Italy
C. Colica and G. Merra contributed equally
Corresponding Author: Giuseppe Merra, MD; e-mail: [email protected]
Efficacy and safety of very-low-calorie
ketogenic diet: a double blind randomized
crossover study
Safety of very-low-calorie ketogenic diet
2275
municable diseases (CNCDs)1. The effects of diet
compounds on metabolic pathways related to dia-
betes mellitus, cardiovascular diseases, and other
CNCD is currently under investigation and it is
leading the traditional nutritional counseling to
a more complex approach. The primary deter-
minant of weight loss is energy deficit. Low-fat,
low-carbohydrate or high-protein, low glycemic
index, and balanced deficit diets have been com-
pared in many studies to verify the difference in
weight loss2. However, it does not seem that there
is a better diet of another. The most commonly
used diet therapy is based on relatively high levels
of carbohydrates and low in fat, but these diets
often result in modest weight loss3, and adherence
to diet is quite low in the long term, because
obese individuals tend to have preference for
foods with a high fat content4. Furthermore, as a
result questionable effectiveness for weight loss
of these types of diet, there was a growing inter-
est in low-carbohydrate ketogenic diets (LCDs),
very low-calorie ketogenic diets (VLCKDs, <800
kcal day-1), or simply ketogenic diets (KDs)5.
They can lead to a state of ketosis, in which
the concentration of blood ketones (acetoacetate,
3-β-hydroxybutyrate, and acetone) increases due
to increased fatty acid breakdown and activity of
ketogenic enzymes. These diets are used as part
of a comprehensive intervention that includes
medical monitoring and a program of lifestyle
modification, and they are considered safe and
effective when used by appropriately selected
individuals under careful medical supervision6.
VLCKDs and low energy consumption provid-
ing a daily energy intake lower than the basic
metabolism, could be a choice for a rapid loss
of body fat and weight in obese individuals at
risk of metabolic complications7. In fact, VLCDs
and VLCKDs have undoubtedly proven to be
effective not only for weight loss, at least in the
short and medium term, but also against hyper-
lipidemia and some cardiovascular risk factors8,9.
KD seems to have a role in the management of
hepatic steatosis in obese subjects. As a matter of
fact, Pérez-Guisado et al10 demonstrated that KD
improved aspartate aminotransferase (AST), ala-
nine aminotransferase (ALT) levels, and reduced
steatosis degree in 93\% of obese patients, under-
lining that KD could be a safe and effective treat-
ment for NAFLD. However, it is widely thought
that a diet low in carbohydrates, and high in pro-
tein and fat content is not safe, since it can cause
an increase in LDL cholesterol, triglycerides, glo-
merular pressure and hyperfiltration11-13. Possible
adverse renal effects represent additional safety
assessment of KD. In fact, high levels of nitrogen
excretion during protein metabolism caused an
increase in glomerular pressure and hyperfiltra-
tion14. After six months of KD, often creatinine
ratios, acid urine and hypercalciuria increased,
while urinary citrate excretion decreased and uric
acid excretion remained normal. This conditions
in conjunction with low fluid intake increased
the risk for calcium stone formation15. KD are
previously investigated about their impact on
bone mineral content, osteopenia and osteoporo-
sis, as well as common consequences related to
this dietary treatment, like hypercalciuria, urine
acidification and hypocytraturia16. Given the role
of the crosstalk between adipose tissue and bone,
it must also evaluate the effect of KD on bone
metabolism. A reduction of serum 25-(OH)2-Vi-
tamin D3 (25(OH)D3) levels and calcium con-
centration in epileptic subjects who were treated
with ketogenic diets were noticed. However, bone
mineral content (BMC) loss during ketogenic diets
could be a common consequence downside of an-
tiepileptic drugs used during the therapy, alone or
in combination with ketogenic diets17-20. 25(OH)D3
is also able to reduce the expression of Peroxisome
Proliferator-Activated Receptor (PPAR)γ and oth-
ers genes involved in to adipogenic transcription,
as well as some adipocyte markers like fatty
acid synthase, lipoprotein lipase and adipocyte
lipid-binding protein21, inhibiting adipogenesis in
a dose dependent manner. PPARγ belongs to the
nuclear hormone receptor superfamily, and has
anti-inflammatory effects22. Some splice variants
in the transcription of insulin-sensitizing nuclear
receptor PPARγ factor show different lipogenic ac-
tivities in different contexts23; for example, PPARγ
2 loss worsens lipotoxicity and insulin resistance24.
Moreover, the activation of PPARγ may ameliorate
hepatic stress of endoplasmic reticulum (ER)25.
The effect of the KD and VLCKD on glucose liver
and the mechanisms through which it can promote
weight loss remains controversial26. According to
Ellenbroek et al27, KD lead to glucose intolerance
and insulin resistance, without weight loss after
long-term treatment. The purpose of this work is
to identify the criteria of effectiveness and safety
in the short-term VLCKD. We assume a possible
relationship between cardiovascular disease risk
(CVD) indexes, AST, ALT, creatinine, Blood
Urea Nitrogen (BUN), uric acid, 25-(OH)2-Vita-
min D (25(OH)D), PPAR-γ gene expression and
body composition parameters after VLCKDs.
We conducted a randomized controlled trial with
C. Colica, G. Merra, A. Gasbarrini, A. De Lorenzo, G. Cioccoloni, P. Gualtieri, et al.
2276
placebo, and we comprehensively analyzed nutri-
tional status by anthropometric parameters, body
fat and lean mass, body water compartments,
serum metabolites, and gene expression.
Patients and Methods
Study Design
The clinical trial was conducted with a ran-
domized crossover design (Figure1) between Oc-
tober 2015 and April 2016.
The study had no. 2 dietary treatment (DT)
conducted in two arms: 1) a VLCKD1 in which
50\% of protein intake is replaced with synthetic
amino acids; 2) a VLCKD2 with placebo.
Each arm lasted three weeks (wks). Between
the two arms a 3-wks washout period was per-
formed to avoid additive effects on DT to follow.
At arm no.1 the intervention group (IG) received
the VLCKD1, and the control group (CG) received the
VLCKD2. At arm no. 2 each groups were reversed.
Analysis was performed at the Section of
Clinical Nutrition and Nutrigenomic, Department
of Biomedicine and Prevention of the University
of Rome “Tor Vergata”.
The study was reviewed and approved by the
Ethics Committee “Centro, Regione Calabria”
30.11.02.2016. The study has been registered by
ClinicalTrials.gov Id: NCT01890070.
Endpoints
The primary endpoint was the evaluation of
body composition changes after DTs, by anthro-
pometry Dual X-ray Absorptiometry (DXA),
and bioimpedentiometry. The secondary end-
point was the evaluation of metabolic profile
by blood analysis. The third endpoint was the
evaluation of PPARγ expression by transcrip-
tomic analysis.
Figure 1. Flowchart of clinical study design.
Safety of very-low-calorie ketogenic diet
2277
Patients
Inclusion criteria: patients who were between
18 and 65 years old, body mass index, BMI ≥25
kg/m2, percentage of body fat (PBF) ≥ 25\% for
male, and ≥ 30\% for female.
Exclusion criteria: pregnancy, breast-feeding,
type 1 diabetes, heart failure, endocrine disorders,
liver dysfunction, liver, kidney, autoimmune, viral
chronic (Hepatitis C, B, HIV), and neoplastic
diseases; corticosteroid and chronic inflammatory
therapy; participating in another diet trial.
Study Methods
Subjects were recruited sequentially, within
a program of routine medical check-up at the
Section of Clinical Nutrition and Nutrigenomic,
University of Rome “Tor Vergata”, Italy.
Eligible patients were randomly (R) divided
into IG and CG in a 1:1 ration.
The randomization was determined by an
external contract research organization and co-
ordinated with the Section of Clinical Nutrition
and Nutrigenomic, at the University of Rome
“Tor Vergata”, Italy, independently of the investi-
gators. The study was conducted in double-blind.
All participants were instructed to maintain
their pre-trial lifestyle habits and physical activ-
ity habits. Any adverse effect has been properly
signed.
At the Baseline (T0), at start and end of each
arm (T1-T2), all the subjects were evaluated for
their health and nutritional status, by anthropo-
metric analysis, body composition, biochemical
evaluation, and genomic profile.
All subjects provided informed written provid-
ed informed written at study enrollment, accord-
ing to principles of the Declaration of Helsinki.
All procedures followed were in accordance with
the ethical standards of the responsible Commit-
tee on Human Experimentation. The participants
received no financial compensation or gifts.
Sample Size
The minimum sample size was calculated on a
two-tailed one-sample Student’s t-test, consider-
ing as (i) insulin level to be detected between the
two DTs |δ| ≥15 µU/mL - 1, (ii) SD of the paired
differences SD=15 µU/mL - 1, (iii) type I error
probability α=0.05 and power 1 - β=0.90. The re-
sult was a minimum sample size of 10 per group.
Dietary Treatment
The average macronutrients distribution of
VLCKD1 was:
a) 450-500 kcal per day for female, with 35-45\%
of calories from proteins (corresponding to 1,2
g/kg of ideal body weight), 45-50\% from fat
(<10\% of calories from saturated fat), and 15\%
from carbohydrates (< 20 g).
b) 650-700 kcal per day for male, with 50-55\%
of calories from proteins (corresponding to 1,5
g/kg of ideal body weight), 35-40\% from fat
(<10\% of calories from saturated fat), and 10\%
of calories from carbohydrates (< 20 g).
The half of the amount of daily protein was
reached using synthetic aminoacid supplemen-
tation (SAS), contained: whey protein (13.42/
bag), carbohydrate (0.03/bag), fat (0.15/bag), iso-
leucine (0.31/bag), ornithine alpha-ketoglutarate
(0.25/bag), L-citrulline (0.25/bag), taurine, (0.25/
bag), L-tryptophan (0.05/bag), potassium citrate
(0.45/bag), for a total of 64 kCal (268 Kj) (Amin
21K, Italfarmacia, Rome, Italy). The powder of
aminoacid was dissolved in water and drunk at
breakfast and lunch or dinner.
The average macronutrients distribution of
VLCKD2 was:
a) 450-500 kcal for female with 25-35\% of calo-
ries from proteins (corresponding to 0,9 g/kg
of ideal body weight), 45-50\% from fat (<10\%
of calories from saturated fat) and 20-25\% of
calories from carbohydrates (< 30 g; >35\%
from complex sugars).
b) 650-700 kcal per day for male with 45-50\% of
calories from proteins (corresponding to 1,1
g/kg of ideal body weight), 35-40\% fat (<10\%
of calories from saturated fat) and 15-20\%
of calories from carbohydrates (<30 g; >35\%
from complex sugars).
The CG1 received VLCKD2 supplemented
with the placebo, represented by inert material
(flour type 00). The powder of placebo was dis-
solved in water and drunk at breakfast and lunch
or dinner. All DTs provided an intake of 20 mg
of fiber per day. IG and CG received a capsule of
multivitamin, multimineral salts and an alkaliz-
ing product. The correct administration of diet
was evaluated by urinary keto-stick.
Anthropometric Evaluation
Height, weight and waist circumference were
measured according to standard method28,29. Body
weight (kg) was measured to the nearest 0.1 kg,
using a balance scale (Invernizzi, Rome, Italy).
Height (m) was measured using a stadiometer to
the nearest 0.1 cm (Invernizzi, Rome, Italy). BMI
was calculated using the formula: BMI = body
weight /height2 (kg/m2).
C. Colica, G. Merra, A. Gasbarrini, A. De Lorenzo, G. Cioccoloni, P. Gualtieri, et al.
2278
Bioelectrical Impedance Analysis (BIA)
Resistance, reactance, impedance and phase
angle at 50 kHz frequencies were measured using
a BIA phase sensitive system (BIA 101S, Ak-
ern/RJL Systems-Florence, Italy). Measurements
were taken according to Di Renzo et al30. Total
body water (TBW), extracellular water (ECW),
intracellular water (ICW), Na/K ratio, phase an-
gle (PA), body cell mass (BCM), and body cell
mass index (BCMI) were calculated from bio-
electrical measurements and anthropometric data
by applying the software provided by the manu-
facturer, which incorporated validated predictive
equations31,32.
Dual X-ray Absorptiometry (DXA)
Bone Mineral Density (BMD), Bone Mineral
Content (BMC), Total body fat mass (TBFat) and
total body lean mass (TBLean) were assessed
using a dual-energy X-ray absorptiometry (DXA)
(i-DXA, GE Medical Systems, Milwaukee, WI,
USA).
TBFat, TBLean, android fat (AF), and gynoid
fat (GF) were expressed in kilogram (kg) and as
a percentage (\%) of the total body mass. BMC
was expressed in grams (g), and (BMD) in g/
cm2. TBFat, TBLean, android fat mass (AFM),
and gynoid fat mass (GFM), android lean mass
(ALM) and gynoid lean mass (GLM) were ex-
pressed in kilogram (kg) and as a percentage (P,
\%) respect to the total body weight of the total
body mass. AF to GF ratio (A/G) and TBF to
TBL ratio (TBF/TBL) were calculated.
Android region was considered to extend from
pubis cut up to the fifth bottom of an ideal line
extending from the pubis to the jugulum. The
gynoid region was considered delimited upper
by the upper greater trochanters, and by a lower
boundary defined at a distance up to twice the
height of the android region. Both AF and GF
were expressed in kilogram (kg) and as a per-
centage of the TBFat.
Total body fat percentage (PBF) = (TBFat +
TBLean + TBBone) x 100.
TBBone is total body bone mass
Region (\%) = TBFat (kg) / (TBFat (kg)+
TBLean (kg) + BCM (kg) x 100
Appendicular Skeletal Muscle Mass Index
(ASMMI) = (Legs Muscle Mass (kg) + Arms
Muscle Mass (kg)/Height (m2); (Men <7.59 kg/
m2, Women <5.47 kg/m2).
Intermuscular Adipose Tissue (IMAT) was
calculated according to Bauer et al14 with the
following formulas: Log (IMAT) = -2.21 + (0.12
x fat) + (-0.0013 x fat2) for women, Log (IMAT)
= -2.05 + (0.12 x fat) + (-0.0013 x fat2) for men.
Resting metabolic rate (RMR)= (3.94 x VO2)
+ (1.106 x VCO2) x 1.44 VO2, VO2 is the volume
of oxygen uptake (mL/min), estimated with the
following formulas: VO2 Woman = TBLean DXA
x 4.5; VO2 Man = TBLean DXA x 5.3; VCO2 is
the volume of carbon dioxide output (mL/min),
estimated with the following formulas: VCO2 =
VO2 x 0.85.
Analysis of Blood Samples
Blood tests were performed at each time, after
a 12 h overnight fast. All materials were imme-
diately placed on ice and plasma was separated
by centrifugation at 1600 x g for 10 min at 4°C.
Laboratory test included Total cholesterol
(TC), HDL-cholesterol (HDL-C), LDL-choles-
terol (LDL-C), triglycerides (Tg), AST, ALT,
creatinine, uric acid, BUN, and 25(OH)D total
levels were recorded at baseline, and at the end of
each arms. Plasma glucose concentrations were
measured using the glucose oxidase method with
an automated glucose analyzer (COBAS INTEG-
RA 400, Roche Diagnostics, Indianapolis, IN,
USA). Creatinine and BUN measurements were
performed using a chemiluminescent enzyme
immunoassay in homogeneous phase (Dimen-
sion VISTA 1500, Siemens, Munich, Germany).
Plasma 25(OH)D total levels were analysed using
a quantitative chemiluminescence (CLIA) test,
LIAISON® 25 OH Vitamin D TOTAL Assay –
DiaSorin (REF 310600, Vercelli, Italy)33. During
the first incubation, 25(OH)D is separated from
its binding protein and the specific antibody binds
to the solid phase. After 10 min is added as a trac-
er vitamin D, linked to a derivative isoluminol.
After a second 10 min incubation, the unbound
material is removed by a washing cycle. Subse-
quently, the starter reagents that induce a reaction
of the chemiluminescent flash type are added.
The light signal is measured by a photomultipli-
er as relative light units (RLU) and is inversely
proportional to the concentration of vitamin D
25(OH) present in calibrators, controls or sam-
ples. Reference values for this test are 4.0-150 ng/
ml (10-375 nmol/L) (DiaSorin LIAISON® 25 OH
Vitamin D TOTAL Assay, DiaSorin, Stillwater,
MN, USA).
Plasma lipid profile components were deter-
mined by standard enzymatic colorimetric tech-
niques (Roche143 Modular P800, Roche Diag-
nostics, Indianapolis, IN, USA).
To derive a surrogate for whole body insulin
Safety of very-low-calorie ketogenic diet
2279
sensitivity, Quantitative Insulin Sensitivity Check
Index (QUICKI) was calculated as QUICKI = 1/
log(I0) + log(G0), where I0 is fasting insulin (µU/
ml) and G0 is fasting glucose (mg/dl).
To assess the insulin-resistance, Homeostasis
Model Assessment of Insulin Resistance (HO-
MA-IR) was estimated with the following formula:
HOMA-IR = (Fasting glucose (mg/dL) x Fast-
ing insulin (µU/ml)) / 405.
Cardiovascular disease (CVD) risk indexes
were determined with the following ratios:
• CVD risk 1: Total Cholesterol (mg/dL)/ HDL–
Cholesterol ((mg/dL);
• CVD risk 2: LDL-Cholesterol (mg/dL)/HDL–
Cholesterol (mg/dL);
• CVD risk 3: Triglycerides (mg/dL)/HDL–
Cholesterol (mg/dL).
Visceral Adiposity Index (VAI) was calculat-
ed according to Amato et al34, with the following
formula:
• WC/39.68+(1.88 x BMI) x Tg/1.03 x 1.31/HDL
for man;
• WC/36.58+(1.89 x BMI) x Tg/0.81 x 1.52/HDL
for woman.
Analyses were carried out at the accredited
Clinical Chemical Laboratories of the “Polyclinic
Tor Vergata (PTV)” of Rome, Italy.
Sample Collection, RNA Extraction
and Reverse Transcription
Blood sample was collected and stabilized in
Tempus Blood RNA Tubes (Applied Biosystems,
Foster City, CA, USA), and stored at -20°C until
RNA extraction. The total RNA of each collected
sample was purified using the Stabilized Blood to
Ct Nucleic Acid Preparation Kit for qPCR (Life
Technologies, Carlsbad, CA, USA). Aliquots of
total RNA were quantified and assessed for qual-
ity by spectrophotometry (Nanodrop, Wilming-
ton, DE, USA). Reverse transcription of each
sample of RNA was performed with High Ca-
pacity RNA-to-cDNA Kit (Applied Biosystems,
Foster City, CA, USA).
Quantitative Real Time PCR
and Data Analysis
Real-time PCR was performed using Taqman
Gene Expression Assay primer-probe sets (Ap-
plied Biosystems, Foster City, CA, USA) for Per-
oxisome proliferator activated receptor-γ (PPAR-γ)
(Hs00234592_m1). qRT-PCR experiment was per-
formed in triplicate and repeated at least twice,
according to manufacturer’s instruction.
Comparative threshold (Ct) cycle was used
to determine gene expression level about the
calibrator from controls. The Ct value for the
gene was normalized using the formula Δ Ct =
Ct (gene) – Ct (Housekeeping Gene). The house-
keeping gene used for this analysis was Actin-β
(Hs01060665_g1) (Applied Biosystems, Foster
City, CA, USA).
Statistical Analysis
A paired t-test or a non-parametric Wilcoxon
test was performed to evaluate differences at
baseline and after nutritional intervention.
The differences between parameter at baseline
and after diet were calculated as the follow: Δ\%
= (Z-W)/W x100, where Δ\% is the percentage
variation of each parameter, calculated as ratio
of absolute variation to the base value.
Pearson correlation was performed to evaluate
a linear correlation between variables before and
after nutritional intervention. The null hypothesis
was rejected at the 0.05 level of probability.
Results
Patients Flow
Of the forty-five subjects enrolled, three of
them did not meet the inclusion criteria, there-
fore, forty-two participants resulted eligible for
the study, and were randomized into IG and CG
(Figure 1). Two subjects declined to participate
after one week. Twenty patients completed the
study (Figure 1).
All baseline characteristics were similar for
the enrolled subjects, on demographics, anthro-
pometrics and body composition, blood tests.
Furthermore, no difference in dietary intake at
baseline was observed (data not shown).
As shown in Table I, at baseline (T0), accord-
ing to BMI the 50\% of the population was obese.
All the subjects were obese according to TBFat
percentage estimated by DXA. No sarcopenic
subjects were highlighted by BCMI or ASMMI.
The frequency of insulin resistant subjects ac-
cording to HOMA-IR>2.5 were 70\%.
Clinical Outcomes During DTs
The characteristics of the participants after 3
weeks of each DTs are shown in Table II and III.
Both groups had a significant decreased in
BMI: after VLCKD1 the Δ\% of BMI was -11.1\%,
( p=0.00); after VLCKD2 the Δ\% of BMI was
=-11.2\%, ( p=0.00).
Both groups lost weight, but the reduction
C. Colica, G. Merra, A. Gasbarrini, A. De Lorenzo, G. Cioccoloni, P. Gualtieri, et al.
2280
was greater in the VLCKD2 (Δ\%=-7.92\% p=0.00)
compared to VLCKD1 (Δ\%=-5.61\%; p=0.00). Af-
ter VLCKD1, it was noticed a significant reduc-
tion of TBW, ( p<0.05) after VLCKD1.
After VLCKD1 treatment, a significant de-
crease for region AFP, (Δ\%=-1,8\%, p=0.02), and
AFM (kg) (Δ\%=-12.7\%, p=0.00) was observed.
Furthermore, GFM (kg) (Δ\%=-6.3\%, p=0.01) was
significantly reduced after VLCKD1.
VLCKD2 determined a significant decrease
of AFM (kg) (Δ\%=-14.3\%, p=0.00), GFM (kg)
(Δ\%=-6.3\%, p=0.00). Left (sx) femur BMC was
significantly increased after VLCKD1 (Δ\%=1.5\%,
p=0.04). No other significant changes in BMC or
BMD were observed after DTs.
It was observed a significant reduction of
ALM (kg) (Δ\%=-6.3\%, p=0.01) and GLM (kg)
(Δ\%=-4.8\%, p=0.01) as a result of VLCKD1 treat-
ment. At the same time, after VLCKD2 treatment
there was a significant lowering of ALM (kg)
(Δ\%=-10.8\%, p=0.01) and GLM (kg) (Δ\%=-6.1\%,
p=0.01). Pearson’s r-value was significant posi-
tive between creatine and ALM (kg) ( p=0.01) in
VLCKD1.
RMR decreased significantly after both DTs
(VLCKD1 Δ\%=-4.8\%, p=0.00; VLCKD2 Δ\%=-
7.8\%, p=0.00).
IMAT value decreased in all diet treatments,
but only in VLCKD1 a significant reduction was
observed (Δ\%=-11.1\%, p=0.00). Pearson’s r-value
was significant negative between serum 25(OH)
D and IMAT ( p=0.04) in VLCKD2.
VLCKD2 determined a significant decrease of
ASMMI (Δ\%=-17.5\%, p=0.00). Pearson’s r-value
was significant positive between ASMMI and
creatinine ( p=0.02), and ALM (kg) ( p=0.01) in
VLCKD1.
After VLCKD1, blood tests underlined a
significant increase of uricemia, creatinine and
AST (respectively Δ\%=35\%, p=0.01; Δ\%=5.9\%,
p=0.02; Δ\%=25.5\%, p=0.03). No significant
changes were observed for ALT and BUN values
in any dietary treatment. After VLCKD2, urice-
mia was significantly increased (Δ\%= 63.1\%, p=
Table 1. Baseline characteristics of anthropometric mea-
surements, body composition parameters and blood tests of
the study population.
Parameters Mean ± SD (Min – Max)
Age 45.40 ± 14.20 (22.00 – 64.00)
Weight (kg) 85.50 ± 12.38 (69.00 – 105.00)
BMI 30.45 ± 2.64 (23.76 – 32.78)
R (Ohm) 498.18 ± 77.86 (341.00 – 607.80)
Xc (Ohm) 55.67 ± 6.71 (43.00 – 65.00)
PA 6.58 ± 1.05 (5.10 – 8.70)
BCM (kg) 31.85 ± 9.27 (21.70 – 48.40)
BCMI 11.56 ± 2.71 (8.50 – 18.22)
TBW (L) 41.25 ± 10.02 (32.80 – 59.80)
ECW (L) 17.94 ± 3.75 (14.60 – 27.10)
ICW (L) 23.32 ± 6.84 (16.30 – 35.20)
AFP (\%) region 0.47 ± 0.06 (0.38 – 0.59)
AFM (kg) 3.06 ± 0.72 (2.24 – 4.73)
ALM (kg) 3.38 ± 0.91 (2.51 – 5.12)
GFP (\%) region 0.44 ± 0.07 (0.29 – 0.51)
GFM (kg) 5.93 ± 0.93 (4.43 – 7.23)
GLM (kg) 7.61 ± 2.07 (5.56 – 10.89)
TBFat (\%) region 39.50 ± 1.08 (30.00 – 48.00)
TBFat/TBLean 0.75 ± 0.20 (0.47 – 1.03)
ASMMI 8.15 ± 1.48 (6.42 – 11.34)
IMAT 1.49 ± 0.19 (1.08 – 1.73)
Total T-score 1.15 ± 1.14 (–0.70 – 2.50)
Total BMD (g/cm2) 1.23 ± 0.12 (1.06 – 1.38)
Total BMC (g) 2600.50 ± 475.68 (2150.00 – 3543.00)
Dx T-score 0.37 ± 1.16 (-1.30 – 2.40)
Dx BMD (g/cm2) 1.07 ± 0.13 (0.91 – 1.29)
Dx BMC (g) 36.22 ± 5.76 (29.10 – 45.59)
Sx T-score 0.44 ± 1.08 (-1.20 – 2.30)
Sx BMD (g/cm2) 1.08 ± 0.13 (0.93 – 1.28)
Sx BMC (g) 36.27 ± 5.35 (31.05 – 46.00)
L1L4 T-score -0.21 ± 1.27 (-1.80 – 1.70)
L1L4 BMD (g/cm2) 1.17 ± 0.15 (0.96 – 1.38)
L1L4 BMC (g) 64.23 ± 7.56 (50.93 – 73.26)
Uric acid (mg/dL) 3.88 ± 1.24 (2.30 – 6.50)
BUN (mg/dL) 30.71 ± 7.68 (21.00 – 43.00)
Creatinine (mg/dL) 0.69 ± 0.14 (0.51 – 0.94)
Vitamin D (ng/mL) 21.74 ± 2.38 (18.7 – 24.9)
AST (μL) 16.90 ± 7.00 (2.99 – 27.00)
ALT (μL) 28.80 ± 8.35 (13.00 – 46.00)
Glycemia (mmol/L) 4.93 ± 0.58 (4.28 – 6.11)
Insulin (μU/mL) 17.41 ± 9.90 (5.47 – 41.11)
HOMA-IR (ng/mL) 4.01 ± 2.85 (1.08 – 11.17)
QUICKI 0.32 ± 0.03 (0.27 – 0.38)
TC/HDL-C 3.45 ± 0.98 (1.91 – 5.33)
LDL-C/HDL-C 2.19 ± 0.81 (0.76 – 3.67)
Tg/HDL-C 1.90 ± 1.11 (0.62 – 4.46)
VAI …
Received 10/01/2019
Review began 11/11/2019
Review ended 01/03/2020
Published 01/08/2020
© Copyright 2020
Anekwe et al. This is an open access
article distributed under the terms of
the Creative Commons Attribution
License CC-BY 3.0., which permits
unrestricted use, distribution, and
reproduction in any medium, provided
the original author and source are
credited.
Ketogenic Diet-induced Elevated
Cholesterol, Elevated Liver Enzymes and
Potential Non-alcoholic Fatty Liver Disease
Chika V. Anekwe , Poongodi Chandrasekaran , Fatima C. Stanford
1. Weight Center, Massachusetts General Hospital, Boston, USA 2. Internal Medicine, North Shore
Physicians Group, Salem, USA 3. Endocrinology and Pediatric Endocrinology, Massachusetts General
Hospital, Boston, USA
Corresponding author: Chika V. Anekwe, [email protected]
Abstract
A 57-year-old woman with class I obesity (BMI = 31.42 kg/m 2) and a medical history significant
for binge-eating disorder with emotionally-triggered eating, post-traumatic stress disorder, and
untreated depression and anxiety, presented for follow-up of weight management with
laboratory values revealing acutely-worsened hyperlipidemia and elevated liver enzymes.
Abdominal ultrasound showed a mildly heterogenous and echogenic liver, without focal
lesions, suggestive of non-alcoholic fatty liver disease. The only significant change from
previous consultation four months prior was introduction of a ketogenic diet consisting of
eggs, cheese, butter, oil, nuts, leafy green vegetables and milk (almond and coconut). The
patient reported a reduction in hunger on this diet. Immediate discontinuation of the diet
resulted in modest reduction of low-density lipoprotein cholesterol (LDL-C) and liver enzymes
two weeks later. Resolution of liver enzymes was seen within eight months and LDL-C levels
normalized one year later. This case report discusses the rationale, benefits and risks of a
ketogenic diet and encourages increased vigilance and monitoring of patients on such a diet.
Categories: Gastroenterology, Endocrinology/Diabetes/Metabolism, Preventive Medicine
Keywords: ketogenic diet, fatty liver, transaminitis, elevated liver enzymes, obesity, nutrition
Introduction
The ketogenic diet was originally developed for implementation under medical supervision to
treat refractory epilepsy in infants and children [1]. It is a high-fat, low-carb, moderate-protein
diet that produces metabolic changes similar to those seen in a state of starvation. These
changes include increased levels of free fatty acids and serum ketones (acetoacetate, acetone
and beta-hydroxybutyrate) and decreased levels of insulin, glucose and glucagon [2]. The
theory is that ketone bodies are anti-convulsant when they cross the blood-brain barrier [3].
There are four types of ketogenic diets used for treating epilepsy - the classic ketogenic diet,
the medium chain triglyceride diet, the modified Atkins diet and the low glycemic index
treatment, each of which has respectively less restrictive requirements for fluid, protein and fat
intake [4]. In recent years, however, the ketogenic diet has transitioned from a medically-
monitored tool for treating epilepsy to become a mainstream interpretation of the low-
carbohydrate dietary plan used to induce weight loss [2]. Individuals on ketogenic diets have
been shown to lose and keep off more weight than those on low-fat diets. They also tend to
report decreased hunger and maintain higher metabolism rates than low-fat dieters [5]. The
ability to achieve and maintain weight reduction for individuals with overweight or obesity
reduces cardiometabolic risk. Despite these benefits of the ketogenic diet, it is not completely
without risk. In particular, it has the potential to increase blood cholesterol levels and induce
1 2 3
Open Access Case
Report DOI: 10.7759/cureus.6605
How to cite this article
Anekwe C V, Chandrasekaran P, Stanford F C (January 08, 2020) Ketogenic Diet-induced Elevated
Cholesterol, Elevated Liver Enzymes and Potential Non-alcoholic Fatty Liver Disease. Cureus 12(1):
e6605. DOI 10.7759/cureus.6605
https://www.cureus.com/users/134711-chika-v-anekwe
https://www.cureus.com/users/134720-poongodi-chandrasekaran
https://www.cureus.com/users/134719-fatima-c-stanford
elevations in liver enzymes. This case report illustrates the risks and benefits of the ketogenic
diet.
Case Presentation
A 57-year-old woman with class I obesity, binge eating disorder, emotionally-triggered eating,
post-traumatic stress disorder, depression and anxiety presented in 2012 with a BMI of 31.6
kg/m2 for treatment of her obesity. At initial evaluation, she reported no weight problems up
until 2003, when she started noticing weight gain. At that point she was living in Iraq during the
Iraq war, was very sedentary, stayed indoors most of the time and consumed a high-calorie diet.
In 2008, she immigrated to the USA with her family. She continued to lead a sedentary lifestyle
with rare formal exercise. She worked as the director of a social refugee agency and had many
responsibilities caring for her household and family. She suffered from sleep disturbance and
was taking clonazepam daily for sleep, which she obtained from her husband. She reported high
stress levels, a strong desire to lose weight, and a lack of support in her daily life.
At initial presentation, she had symptoms consistent with dysthymia and was recommended to
undergo treatment for mood stabilization with psychotherapy and/or psychopharmacology. She
was also prescribed a low dose of topiramate, given off-label for appetite reduction. She
suffered an adverse reaction to topiramate with an episode of significant anxiety and emotional
outburst, resulting in a visit to the emergency department. Topiramate was discontinued and
she began treatment with metformin for both obesity and metabolic syndrome; she was also
instructed to introduce structured lifestyle changes including keeping records of dietary intake,
exercise and sleep routine.
Metformin was not effective for weight reduction, and she in fact gained approximately 2.8 lbs
during the four-month period during which the dose was titrated to 1000 mg twice daily.
Although she was continued on metformin, she was recommended to discontinue using
clonazepam for insomnia and instead start melatonin 3 mg and zonisamide 100 mg daily, both
at bedtime. Zonisamide was titrated up to 200 mg at bedtime. Similar to topiramate, zonisamide
is an anti-epileptic medication used off-label for appetite reduction in the treatment of obesity.
As it can cause drowsiness, it is often dosed at bedtime. She lost 5.4 lbs (3\% total body weight)
within two months on this medication regimen, however was subsequently lost to follow-up,
with her last visit on 5/21/13.
She was treated at an outside clinic from 2015 to 2018 with a variety of anti-obesity agents
including naltrexone/bupropion, phentermine/topiramate ER and lorcaserin. Labs obtained on
2/24/16 showed hypercholesterolemia with total cholesterol (TC) = 271 mg/dL, low-density
lipoprotein cholesterol (LDL-C) = 156 mg/dL and normal high-density lipoprotein cholesterol
(HDL-C) = 102 mg/dL (see Table 1). Lipid values improved slightly with dietary modification
and simvastatin, although she did not take simvastatin consistently. At her nadir weight in
October 2015, she was 151 lbs (BMI = 28.5 kg/m2), and had achieved 17\% reduction in total
body weight from her heaviest weight of 182.8 lbs in March 2013.
2020 Anekwe et al. Cureus 12(1): e6605. DOI 10.7759/cureus.6605 2 of 7
Laboratory reference
ranges
Prior to
KD
During
KD
Two weeks after
KD
Eight months after
KD
One year after
KD
AST (15-41 U/L) 21 U/L 67 U/L 55 U/L 27 U/L -
ALT (10-35 U/L) 18 U/L 119 U/L 80 U/L 25 U/L -
LDL-C (40-130 mg/dL) 156 mg/dL
216
mg/dL
209 mg/dL 157 mg/dL 80 mg/dL
Tot-C (0-200 mg/dL) 271 mg/dL
323
mg/dL
- 268 mg/dL -
HDL-C (>39 mg/dL) 102 mg/dL 98 mg/dL - 84 mg/dL -
TG (0-150 mg/dL) 66 mg/dL 45 mg/dL - 133 mg/dL -
TABLE 1: Patients laboratory values before, during and after ketogenic diet
KD: Ketogenic diet; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LDL-C: Low-density lipoprotein cholesterol; Tot-
C: Total cholesterol; HDL-C: High-density lipoprotein cholesterol; TG: Triglycerides.
The patient returned for follow-up of obesity management in April 2018. At this point she was
off all anti-obesity medications and was in fact on the weight-promoting medication seroquel;
at 170.5 lbs, she had regained a significant portion of her lost weight. She was restarted on
bupropion and zonisamide. In September 2018, the patient self-initiated a ketogenic diet,
consuming predominantly eggs, cheese, butter, oil, nuts, leafy green vegetables and
almond/coconut milk. This resulted in a modest weight loss of about 6 lbs over two months.
However, she also suffered a marked increase in liver enzymes and total and LDL cholesterol
levels.
Laboratory testing on 12/21/18 revealed aspartate aminotransferase (AST) = 67 U/L and alanine
aminotransferase (ALT) = 119 U/L (alkaline phosphatase was normal at 77 U/L). TC = 323 mg/dL
and LDL-C = 216 mg/dL (triglycerides, TG, were normal at 45 mg/dL). Also of note was an
elevated Vitamin B12 level of 1,156 pg/mL, despite the patient not taking any B12
supplementation. In addition, 25-hydroxy Vitamin D levels were insufficient, at 22 ng/mL, and
ferritin levels were elevated at 155 ug/L. Previous TC level obtained by her primary physician on
3/15/18 was 267 mg/dL; LDL-C and TG values were not obtained. Previous liver chemistries on
4/10/18 were within normal limits, with AST = 21 U/L and ALT = 18 U/L (see Table
1). Abdominal ultrasound obtained on 1/10/19 revealed a mildly heterogenous and echogenic
liver, with no focal lesions visualized and no significant biliary ductal dilation (see Figure 1 and
Figure 2). These findings are highly suggestive of hepatic steatosis, or fatty liver disease.
2020 Anekwe et al. Cureus 12(1): e6605. DOI 10.7759/cureus.6605 3 of 7
FIGURE 1: Left transverse view of liver
2020 Anekwe et al. Cureus 12(1): e6605. DOI 10.7759/cureus.6605 4 of 7
https://assets.cureus.com/uploads/figure/file/86483/lightbox_7b7cd780011d11ea9e769db4adc452da-Webp.net-resizeimage-1-.png
https://assets.cureus.com/uploads/figure/file/86485/lightbox_135e5f60011e11ea93b73f40eb27bf14-Webp.net-resizeimage.png
FIGURE 2: Right sagittal view of liver
The patient agreed to discontinue the ketogenic diet and follow up with a registered dietician.
She continued bupropion 150 mg twice daily and zonisamide 200 mg in the evening. She also
continued cholecalciferol 2000 IU daily for hypovitaminosis D. She was encouraged to consume
a high-quality diet and engage in regular physical activity. In addition, due to her LDL-C value
of 216, she was prescribed atorvastatin 20 mg daily. The National Cholesterol Education
Program Adult Treatment Panel III recommends statin therapy for low-risk individuals (one or
no risk factors) who have an LDL-C > 190 mg/dL, with lower LDL-C cut-off values for higher risk
populations [6]. The patient has no reported history of premature cardiovascular events in
first-degree relatives; LDL-C levels of her first-degree relatives were not accessible.
The patient followed up with her primary physician four days after her weight management
visit and reported having stopped the ketogenic diet, while affirming adherence to a high-
quality diet and regular exercise. She also reported taking an omega-3 DHA/EPA 1000 mg (120
mg/180 mg) fish oil capsule daily. Her weight was 164 lbs. She received counseling to follow a
low-fat and low-carbohydrate diet rich in fruits and vegetables. She was counseled to engage in
routine aerobic exercise at least three times per week and advised against implementing any
diet that promotes rapid weight loss. Repeat laboratory testing 10 days after visit to the
primary physician revealed improved liver enzymes (AST = 55 U/L, ALT = 80 U/L), and a slightly
decreased direct LDL-C of 209 mg/dL. Liver enzymes resolved completely within eight months,
while LDL-C levels resolved by one year (see Table 1). She was advised to continue follow-up for
monitoring of weight and laboratory values as well as continued lifestyle counseling.
Discussion
Individuals with obesity or overweight often implement what they hope to be the next “quick
fix” for reversing their increased fat mass. Often these self-initiated diets are implemented
without the guidance of a licensed health care provider. The ketogenic diet is one example of a
dietary pattern that has gained popularity, with mainstream use as an effective strategy for
weight loss.
The ketogenic diet was originated in the 1920s and 1930s as an alternative to fasting for the
reduction of seizure frequency in children with epilepsy [1]. Individuals in ketosis release
ketone bodies from the breakdown of body fat, and these ketones are used, instead of glucose,
as the primary source of energy [2]. This ketotic state has been shown to alter genes involved in
energy metabolism in the brain, which helps stabilize the function of neurons susceptible to
epileptic seizures [3].
The ketogenic diet is very low in carbohydrates and very high in fat. Clinical ketogenic diets
limit carbs to 20 to 50 g per day, primarily from non-starchy vegetables, with very low carb
ketogenic diets restricting carbs to less than 20 g per day [5]. Protein is kept high enough to
maintain lean body mass, but low enough to preserve ketosis. The amino acids alanine and
glutamine can be converted to glucose through gluconeogenesis, thus removing the body from
a ketotic state [7].
The diet works, simply, by altering energy metabolism. After three to four days of fasting or
following a very low-carbohydrate diet, the body becomes deprived of dietary sugar and starch,
and reacts by reducing insulin secretion and switching to primarily burning fat for fuel. The
resulting overproduction of acetyl-CoA leads to formation of ketones (acetoacetate, acetone
and beta-hydroxybutyric acid) in a process known as ketogenesis [8]. While the brain is unable
to use fatty acids for fuel, ketones can cross the blood-brain barrier, thereby providing fuel to
2020 Anekwe et al. Cureus 12(1): e6605. DOI 10.7759/cureus.6605 5 of 7
the typically glucose-dependent brain. The full transition to physiological, or nutritional,
ketosis usually takes a week [8]. The true ketogenic diet contains 75\% to 90\% calories from fat,
10\% from protein, and 5\% from carbs. Careful monitoring of dietary intake and blood (not
urinary) ketone levels is required in order to ensure an adequate state of ketosis. Protein intake
may need to be increased for individuals doing resistance training, in order to prevent muscle
degradation [2].
The ketogenic diet has both benefits and risks. Advantages of the diet include weight loss,
reduction in cravings and appetite (likely due to the satiating effects of fat and protein as well
as the stabilizing effect on blood sugar levels), and a more stable flow of energy to organs and
tissues, due to the reliance of fat catabolism rather than dietary intake for energy [2, 7]. The
weight loss occurs partly due to the diuretic effect of glycogen utilization and the likely calorie
reduction resulting from the restricted dietary variety, but primarily because the reduction in
blood glucose and insulin leads to less fat storage, as insulin is known to promote the
conversion of excess glucose to fat [5]. Research also suggests that the ketogenic diet improves
insulin sensitivity and glycemic control, although the mechanisms are unclear [8].
One potential risk of the ketogenic diet is an increase in LDL-C, TC and liver enzymes. Notably,
in rodents, development of nonalcoholic fatty liver disease (NAFLD) and insulin resistance
have been described [9]. Despite this risk, some studies show that the higher-risk small dense
LDL particles were decreased in individuals on a ketogenic diet, while HDL cholesterol and
triglycerides tend to improve [9, 10]. It should be noted, however, that the reduction in small
dense LDL particles is observed only in individuals with certain variants of the apolipoprotein
gene which is known to play a key role in lipid metabolism [11]. Depending on an individual’s
response to the diet, benefits of improved glycemic control may outweigh potential risks of an
elevated LDL. One way to mitigate the negative effects of the diet on LDL cholesterol is to
replace saturated fats from animal sources with polyunsaturated fats found in avocados, nuts,
seeds, coconut and olive oil.
Another side effect of the ketogenic diet is a constellation of symptoms known as “keto flu,”
which includes lightheadedness, fatigue, headaches, nausea, and constipation. These
symptoms are a result of the body’s rapid excretion of sodium and fluids as carbohydrate intake
is restricted and glycogen stores are depleted. Increasing sodium by 1-2 g per day may restore
electrolyte balance [2].
Finally, the extreme limitation of carbohydrates in a ketogenic diet poses concern regarding the
potential impact on micronutrient intake and gut health. Ketogenic diets eliminate not only
sugar and refined carbohydrates but also pulses, whole grains, fruits and starchy vegetables, all
of which contain vitamins, minerals, antioxidants, phytochemicals and fiber, including healthy
gut microbiota-promoting prebiotic fiber. Although this alteration in the gut microbiome may
be beneficial for individuals with epilepsy, research is lacking on the impact on populations
using the diet for weight loss or diabetes management [8].
Conclusions
The ketogenic diet is a high-fat, moderate-protein, low-carbohydrate diet that can induce
weight loss and improvement in glycemic control, but poses a risk of inducing hyperlipidemia,
elevation of liver enzymes and onset of fatty liver disease. Like any other restrictive dietary
plan, the ketogenic diet is often difficult to maintain long-term. Cycling in and out of ketosis
reduces its metabolic effects. Patients on a ketogenic diet should be monitored with frequent
laboratory testing of blood ketones, lipids, and liver enzymes as well as frequent assessment of
cognitive function and energy levels.
2020 Anekwe et al. Cureus 12(1): e6605. DOI 10.7759/cureus.6605 6 of 7
Additional Information
Disclosures
Human subjects: Consent was obtained by all participants in this study. Conf licts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following:
Payment/services info: All authors have declared that no financial support was received from
any organization for the submitted work. Financial relationships: All authors have declared
that they have no financial relationships at present or within the previous three years with any
organizations that might have an interest in the submitted work. Other relationships: All
authors have declared that there are no other relationships or activities that could appear to
have influenced the submitted work.
References
1. Kossoff EH: Ketogenic dietary therapies for the treatment of epilepsy . UpToDate. 2019,
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energy production, increasing neuron stability. Science Daily. 2005,
4. Sampaio LP: Ketogenic diet for epilepsy treatment. Arq Neuropsiquiatr. 2016, 74:842-848.
10.1590/0004-282X20160116
5. Abbasi J: Interest in the ketogenic diet grows for weight loss and type 2 diabetes . JAMA. 2018,
319:215-217. 10.1001/jama.2017.20639
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recommendable?. Eur J Nutr. 2018, 57:1301-1312. 10.1007/s00394-018-1636-y
8. Paoli A: Ketogenic diet for obesity: friend or foe? . Int J Environ Res Public Health. 2014,
11:2092-2107. 10.3390/ijerph110202092
9. Kosinski C, Jornayvaz FR: Effects of ketogenic diets on cardiovascular risk factors: evidence
from animal and human studies. Nutrients. 2017, 9:517. 10.3390/nu9050517
10. Volek JS, Sharman MJ, Forsythe CE: Modification of lipoproteins by very low-carbohydrate
diets. J Nutr. 2005, 135:1339-1342. 10.1093/jn/135.6.1339
11. Ordovas JM: The genetics of serum lipid responsiveness to dietary interventions . Proc Nutr
Soc. 1999, 58:171-187. 10.1079/pns19990023
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https://dx.doi.org/10.3390/nu9050517
https://dx.doi.org/10.1093/jn/135.6.1339
https://dx.doi.org/10.1093/jn/135.6.1339
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https://dx.doi.org/10.1079/pns19990023
Ketogenic Diet-induced Elevated Cholesterol, Elevated Liver Enzymes and Potential Non-alcoholic Fatty Liver Disease
Abstract
Introduction
Case Presentation
TABLE 1: Patients laboratory values before, during and after ketogenic diet
FIGURE 1: Left transverse view of liver
FIGURE 2: Right sagittal view of liver
Discussion
Conclusions
Additional Information
Disclosures
References
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You read about blockchain ledger technology. Now do some additional research out on the Internet and share your URL with the rest of the class
be aware of which features their competitors are opting to include so the product development teams can design similar or enhanced features to attract more of the market. The more unique
low (The Top Health Industry Trends to Watch in 2015) to assist you with this discussion.
https://youtu.be/fRym_jyuBc0
Next year the $2.8 trillion U.S. healthcare industry will finally begin to look and feel more like the rest of the business wo
evidence-based primary care curriculum. Throughout your nurse practitioner program
Vignette
Understanding Gender Fluidity
Providing Inclusive Quality Care
Affirming Clinical Encounters
Conclusion
References
Nurse Practitioner Knowledge
Mechanics
and word limit is unit as a guide only.
The assessment may be re-attempted on two further occasions (maximum three attempts in total). All assessments must be resubmitted 3 days within receiving your unsatisfactory grade. You must clearly indicate “Re-su
Trigonometry
Article writing
Other
5. June 29
After the components sending to the manufacturing house
1. In 1972 the Furman v. Georgia case resulted in a decision that would put action into motion. Furman was originally sentenced to death because of a murder he committed in Georgia but the court debated whether or not this was a violation of his 8th amend
One of the first conflicts that would need to be investigated would be whether the human service professional followed the responsibility to client ethical standard. While developing a relationship with client it is important to clarify that if danger or
Ethical behavior is a critical topic in the workplace because the impact of it can make or break a business
No matter which type of health care organization
With a direct sale
During the pandemic
Computers are being used to monitor the spread of outbreaks in different areas of the world and with this record
3. Furman v. Georgia is a U.S Supreme Court case that resolves around the Eighth Amendments ban on cruel and unsual punishment in death penalty cases. The Furman v. Georgia case was based on Furman being convicted of murder in Georgia. Furman was caught i
One major ethical conflict that may arise in my investigation is the Responsibility to Client in both Standard 3 and Standard 4 of the Ethical Standards for Human Service Professionals (2015). Making sure we do not disclose information without consent ev
4. Identify two examples of real world problems that you have observed in your personal
Summary & Evaluation: Reference & 188. Academic Search Ultimate
Ethics
We can mention at least one example of how the violation of ethical standards can be prevented. Many organizations promote ethical self-regulation by creating moral codes to help direct their business activities
*DDB is used for the first three years
For example
The inbound logistics for William Instrument refer to purchase components from various electronic firms. During the purchase process William need to consider the quality and price of the components. In this case
4. A U.S. Supreme Court case known as Furman v. Georgia (1972) is a landmark case that involved Eighth Amendment’s ban of unusual and cruel punishment in death penalty cases (Furman v. Georgia (1972)
With covid coming into place
In my opinion
with
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The ability to view ourselves from an unbiased perspective allows us to critically assess our personal strengths and weaknesses. This is an important step in the process of finding the right resources for our personal learning style. Ego and pride can be
· By Day 1 of this week
While you must form your answers to the questions below from our assigned reading material
CliftonLarsonAllen LLP (2013)
5 The family dynamic is awkward at first since the most outgoing and straight forward person in the family in Linda
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The most important benefit of my statistical analysis would be the accuracy with which I interpret the data. The greatest obstacle
From a similar but larger point of view
4 In order to get the entire family to come back for another session I would suggest coming in on a day the restaurant is not open
When seeking to identify a patient’s health condition
After viewing the you tube videos on prayer
Your paper must be at least two pages in length (not counting the title and reference pages)
The word assimilate is negative to me. I believe everyone should learn about a country that they are going to live in. It doesnt mean that they have to believe that everything in America is better than where they came from. It means that they care enough
Data collection
Single Subject Chris is a social worker in a geriatric case management program located in a midsize Northeastern town. She has an MSW and is part of a team of case managers that likes to continuously improve on its practice. The team is currently using an
I would start off with Linda on repeating her options for the child and going over what she is feeling with each option. I would want to find out what she is afraid of. I would avoid asking her any “why” questions because I want her to be in the here an
Summarize the advantages and disadvantages of using an Internet site as means of collecting data for psychological research (Comp 2.1) 25.0\% Summarization of the advantages and disadvantages of using an Internet site as means of collecting data for psych
Identify the type of research used in a chosen study
Compose a 1
Optics
effect relationship becomes more difficult—as the researcher cannot enact total control of another person even in an experimental environment. Social workers serve clients in highly complex real-world environments. Clients often implement recommended inte
I think knowing more about you will allow you to be able to choose the right resources
Be 4 pages in length
soft MB-920 dumps review and documentation and high-quality listing pdf MB-920 braindumps also recommended and approved by Microsoft experts. The practical test
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One thing you will need to do in college is learn how to find and use references. References support your ideas. College-level work must be supported by research. You are expected to do that for this paper. You will research
Elaborate on any potential confounds or ethical concerns while participating in the psychological study 20.0\% Elaboration on any potential confounds or ethical concerns while participating in the psychological study is missing. Elaboration on any potenti
3 The first thing I would do in the family’s first session is develop a genogram of the family to get an idea of all the individuals who play a major role in Linda’s life. After establishing where each member is in relation to the family
A Health in All Policies approach
Note: The requirements outlined below correspond to the grading criteria in the scoring guide. At a minimum
Chen
Read Connecting Communities and Complexity: A Case Study in Creating the Conditions for Transformational Change
Read Reflections on Cultural Humility
Read A Basic Guide to ABCD Community Organizing
Use the bolded black section and sub-section titles below to organize your paper. For each section
Losinski forwarded the article on a priority basis to Mary Scott
Losinksi wanted details on use of the ED at CGH. He asked the administrative resident