Effect of Dietary Intervention With a High Intake of Vitamin K on Insulin, Osteocalcin, Leptin, Vitamin K Serum Levels and Cardiovascular Risk in Young Adults With and Without Overweight or Obesity.
Phase: NA
Conditions: Overweight and Obesity
Sponsor: University of Guadalajara

Effect of Dietary Intervention with a High Intake of Vitamin K on Insulin, Osteocalcin, Leptin, 
Vitamin K Serum Levels and Cardiovascular Risk in Young Adults With and Without 
Overweight or Obesity 
 
February 2021 

 
STUDY PROTOCOL 
 
Overweight and obesity are public health problems worldwide and in Mexico, both related to the 
increase in weight and body fat that are risk factors for the development of chronic degenerative 
diseases such as type 2 diabetes mellitus (DM2) and cardiovascular diseases (CVD). The World Health 
Organization (WHO) defines obesity as the excessive accumulation of fat that can harm health and is 
diagnosed when the Body Mass Index (BMI) is greater than or equal to 30 kg/m2. 
In Mexico, according to figures from the National Health and Nutrition Survey (ENSANUT), at the 
national level, 75.2% of adults are overweight or obese (39.1% overweight and 36.1% obese), with 
respect to chronic degenerative diseases there is a prevalence of 10.3 % for diabetes, 18.4% for 
hypertension and it has been reported that 19.5% of the population has elevated cholesterol and 
triglycerides. 
 
Correct diet and a healthy lifestyle are factors that help to avoid overweight and obesity; However, 
the current diet of the adult Mexican population is not adequate, because there is low consumption 
of the recommended food groups. Fruits and vegetables are healthy foods that less than 50% of the 
country's population consume daily. Regarding other recommended food groups, eggs are consumed 
by 29.9% of the population, dairy products by 47%. and unprocessed meats by 64.6%. Which is 
inadequate, due to the vitamins and minerals that these food groups provide. 
 
Vitamins are considered micronutrients and they play an essential role in metabolism. Vitamins are 
classified according to their nature as fat-soluble or water-soluble. Water-soluble vitamins include 
vitamin B1, B1, B2, B3, B5, B6, B7, B9, B12 and C, while fat-soluble vitamins are vitamins A, E, D and 
K. Vitamin K (VK) has two main structural forms, which are phylloquinone (K1) which is found naturally 
in plant foods, mainly in green leafy vegetables, and menaquinone (K2) which is produced in the 
intestine and is found in some fermented foods. VK acts as a cofactor for the carboxylation of glutamic 
acid residues in proteins involved in blood coagulation and bone metabolism, facilitating calcium 
uptake through the carboxylation of the protein osteocalcin. This last protein, osteocalcin in its 
carboxylated form (OCc), serves as an indicator of the level of VK, the more OCc exists in the blood it 
is considered that there is a good level of VK, the osteocalcin that is not carboxylated thanks to the 
VK, circulates in the blood and is known as undercarboxylated osteocalcin (OCuc). VK requirements 
are 120mcg/day for men and 90mcg/day for women; Although VK is fat-soluble, the body stores small 
amounts that are quickly depleted without regular daily ingestion. 
 
Various studies mention that the high intake of VK has positive effects on insulin sensitivity and 
cardiovascular risk; likewise, VK deficiency is negatively associated with the same factors. Yoshida et 
al. suggest that the increase of VK intake to through diet can reduce the progression of insulin 
resistance. The mechanism through which VK improves insulin sensitivity is unknown. In animals, 
OCuc has been shown to stimulate beta cell proliferation and increase insulin secretion; However, 
Yoshida et al. found that in subjects receiving VK supplements the OCuc levels were lower and the 
insulin resistance was reduced, compared to the control group without supplements. This suggests 

that VK may intervene in glucose metabolism through other mechanisms not fully described, such as 
suppression of inflammation. Also, it has been reported that VK improves insulin sensitivity in 
peripheral tissues without intervening in insulin resistance. Rasekhi et al. analized the effects of 
administering 100 mcg of VK through supplements on glucose metabolism in premenopausal 
prediabetic women. They found that in the group with VK ingestion, OCc levels increased and OCuc 
levels decreased and insulin sensitivity showed to improve. There were no alterations in total 
osteocalcin or in the HOMA-IR index. Which suggests that VK could improve insulin action in 
peripheral tissues. 
 
Dietary supplementation with VK has been associated with an increase in insulin sensitivity and 
improvement in glycemic status. Yoshida et al. studied vitamin K supplementation for 36 months in 
men and women, administering 500 mcg/day of VK, 600 mg of calcium in the form of calcium 
carbonate and 10 mcg of vitamin D. They found that VK in supplements produces a protective effect 
on the progression of insulin resistance; Hussein et al. showed in animal models with type 2 diabetes 
the effect on glycemic status after the administration of VK supplements for 8 weeks. Those rats type 
showed a decrease in fasting serum glucose, HbA1c and insulin resistance, in addition to improving 
insulin sensitivity. When comparing the control group with the diabetes group without supplements, 
total OC levels were lower in the diabetes group; however, there was a dose-dependent relationship 
and the group with VK supplements showed to increase OC levels. In Mexico, the numbers of obesity 
and overweight increase considerably, the dietary factor has high relevance in the generation of these 
conditions, currently, studies on vitamin supplementation have been carried out, however in our 
country there is no evidence of the effect of dietary intervention with a high intake of VK on people 
with metabolic disorders such as overweight and obesity, so carrying out this study may contribute 
to explaining the participation of a high intake of VK on the improvement and prevention of 
overweight and obesity, in addition to evaluating the possibility to improve metabolic and bone 
health from a diet calculated in the Mexican population. 
 
Work hypothesis 
 
Dietary intervention with a high intake of vitamin K of 500mcg for 6 weeks, increases VK and OC 
serum levels, decreases insulin and leptin levels and reduces cardiovascular risk in young adults, 
with and without overweight or obesity. 
 
Aims 
 
General aim 
To evaluate the effect of dietary intervention with a high intake of vitamin K of 500mcg on serum 
levels of vitamin K, insulin, osteocalcin, leptin and on cardiovascular risk in young adults with and 
without overweight or obesity. 
 
Specific aims 
- Design a dietary intervention with a 500mcg of vitamin K 

- Compare serum levels of vitamin K, insulin, osteocalcin, leptin and cardiovascular risk markers 
before and after the intervention with high vitamin K intake in subjects with and without overweigh 
or obesity. 
- Analyze the relation between vitamin K serum levels and body composition characteristics, 
metabolic markers and insulin, osteocalcin, leptin and cardiovascular risk percentage in subjects 
with and without overweigh or obesity. 
 
Ethical considerations: 
 
The study will adhere to the regulations established by the Mexican government in the regulations 
of the general health law regarding health research. 
It will be carried out in accordance with the ethical standards and principles for medical research on 
human subjects established in the Declaration of Helsinki in Helsinki, Finland in 1964, updated in 2008 
in Seoul, South Korea. 
The subjects who will participate in the study will voluntarily sign the informed consent letter. The 
project aims to contribute to the study of metabolic diseases in adults and young people with the aim 
of finding strategies for prevention or use as early dietary treatment and the consideration of foods with 
a high supply of vitamin K. The participants in the study will be assigned an identification key to 
guarantee the reliability of their clinical data. If they decide to withdraw from the study, the clinical 
history and biological samples associated with the individual will be deleted. All individuals who 
participate in the study will benefit from obtaining and explaining their clinical data, information 
about the cardiovascular risk they have at the time of the intervention and after it, in addition to 
being provided with nutritional care and intervention by professionals of nutrition, all this without 
costs. 
 
Biosafety aspects: 
 
This is an investigation with minimal risk, the laboratory has the necessary infrastructure and 
materials for the management of RPBI according to the Official Mexican STANDARD NOM-087-ECOL- 
SSA1-2002, Environmental protection - Environmental health - Biological hazardous waste - infectious 
- Classification and handling specifications. Waste from the use of reagents for ELISA and wet 
chemistry will be discarded according to the manufacturer's criteria. The reagents will be stored 
according to what is stipulated in the manufacturer's sheets and according to the provisions of NOM- 
054-SEMARNAT1993 and NOM-052-SEMARNAT-2005, no toxic, radioactive or explosive reagents will 
be worked with. The implementation of the project will be subject to what is described in the General 
Health Law on health research, DOF reform 04-02-2014 Art. 13 to 17. The main researcher has been 
trained by the CUCS Biosafety Committee. 

 
 
METHODOLOGY 
 
Technical methods and instruments for collection, recording, processing and analysis: 
 
- Peripheral blood samples will be taken by venipuncture using the Vacutainer® system. 
- The serum variables of glucose, triglycerides and lipids will be obtained by means of colorimetric 
enzymatic reagents. Vitamin K and insulin will be obtained through ELISA Kits. 
- Anthropometric measurements will be carried out using a TANITA brand bioimpedance scale, SECA 
brand measuring tape and Slim Guide brand plycometers. All measurements will be taken according 
to ISAK. 
- Blood pressure will be measured with an OMRON brand baumanometer. 
- Patient data will be recorded in a log and in an Excel database. For the statistical processing of the 
data, the Excel, SPSS Statistics version 20.0 and GraphPad Prism 8 programs will be used. 
 
 
Research organization: 
 
The study will be carried out at the University of Guadalajara, Department of Molecular Biology and 
Genomics, Biochemistry Research Laboratory of the University Center for Health Sciences. 
 
1. Once the patients are included, an initial evaluation will be carried out that consists of: 
- Evaluation of nutritional status: Dietary analysis by 24-hour recall, anthropometric analysis (with 
bioimpedance, measurement of skin folds and circumferences), clinical history. 
- Blood sample collection: determination of glucose and serum lipids, vitamin K, insulin and 
osteocalcin. 
- Blood pressure measurement. 
- Dietary intervention: Explanation of the study and dietary indications. 
 
2. Dietary intervention with a high intake of vitamin K for 6 weeks 
- Weekly monitoring of dietary adherence 
- Nutritional advice: resolve doubts, evaluation of dietary adherence. 
 
3. Final evaluation 
- Evaluation of nutritional status: anthropometric analysis. 
- Blood sample collection: determination of glucose and serum lipids, vitamin K, insulin, osteocalcin 
and leptin. 
- Blood pressure measurement. 
- Final evaluation of dietary adherence through a survey. 
 
4. Data analysis, statistics. 

 
 
 
 
 
and Pearson for parametric tests.